Wen-Kai Yang

Tse-Lih Chao

Hsin-Ju Chuang

Yao-Chung Hu

Catherine Lorin-Nebel

Eva Blondeau-Bidet

Wen-Yi Wu

Cheng-Hao Tang

Shu-Chuan C Tsai
email: sctsai@ctust.edu.tw

Tsung-Han H Lee
email: thlee@email.nchu.edu.tw

Gene expression of Na+/K+-ATPase α-isoforms and FXYD proteins and potential modulatory mechanisms in euryhaline milkfish kidneys upon hypoosmotic challenges

Keywords: Cc, Chanos chanos, FW, freshwater, PCR, polymerase chain reaction, PK, protein kinase, QPCR, quantitative realtime PCR, SE, standard error, SW, seawater, TM, transmembrane domain

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Milkfish (Chanos chanos), a marine teleost that resides in tropical regions from the Pacific Ocean to the Indian Ocean, is a commercially important aquaculture species in Southeast Asian countries, including Taiwan [START_REF] Martinez | Milkfish (Chanos chanos) culture: situations and trends[END_REF]. Milkfish can survive in hypoosmotic (i.e., fresh water [FW]) or hyperosmotic (e.g., seawater [SW]) environments [START_REF] Bagarinao | Systematics, distribution, genetics and life history of milkfish, Chanos chanos[END_REF][START_REF] Martinez | Milkfish (Chanos chanos) culture: situations and trends[END_REF]. In Taiwan, milkfish are cultured in ponds with water of various salinities including FW, brackish water, or SW [START_REF] Martinez | Milkfish (Chanos chanos) culture: situations and trends[END_REF]. Hence, for elucidating the effects of environmental factors (such as salinity) on physiological mechanisms as the background knowledge to improve aquaculture practice of milkfish farm, further investigation of their molecular mechanisms of osmoregulation in response to ambient salinity changes is warranted [START_REF] Lin | The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water[END_REF]. There is evidence that the milkfish is able to maintain internal homeostasis, such as muscle water content, osmolality of the plasma and urine, and concentrations of plasma Na + and Cl -, within narrow physiological ranges under different environmental salinity conditions [START_REF] Kang | Seawater-acclimation abates cold effects on Na + ,K + -ATPase activity in gills of the juvenile milkfish, Chanos chanos[END_REF][START_REF] Lin | The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water[END_REF][START_REF] Tang | Elevated Na + /K + -ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water[END_REF]. Thus, milkfish is a good experimental species for studies on osmoregulation.

Teleosts maintain relatively stable internal environments by the activation of osmoregulatory organs, including the gill, kidney, and gut [START_REF] Marshall | Ion transport, osmoregulation, and acid-base balance[END_REF][START_REF] Takei | Diverse mechanisms for body fluid regulation in teleost fishes[END_REF][START_REF] Whittamore | Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish[END_REF]. Among these organs, the kidney plays an important role in osmoregulation in both SW and FW teleosts, performing completely different functions in these two environments [START_REF] Madsen | Water transport and functional dynamics of aquaporins in osmoregulatory organs of fishes[END_REF][START_REF] Marshall | Ion transport, osmoregulation, and acid-base balance[END_REF][START_REF] Takei | Diverse mechanisms for body fluid regulation in teleost fishes[END_REF]. In SW residents, the hypertonic environment results in passive loss of water and influx of excess ions. They thus drink SW to prevent dehydration and actively excrete ions through the kidneys and gills. In contrast, FW teleosts gain excess water loading and lose ions to their surrounding hypotonic environment. For compensation, the primary function of fish kidneys is to excrete excess water and reabsorb filtered solutes [START_REF] Madsen | Water transport and functional dynamics of aquaporins in osmoregulatory organs of fishes[END_REF][START_REF] Marshall | Ion transport, osmoregulation, and acid-base balance[END_REF]. In these processes, renal Na + /K + -ATPase (NKA) plays a crucial role in ionoregulation/osmoregulation for cellular homeostasis of ions and water [START_REF] Marshall | Ion transport, osmoregulation, and acid-base balance[END_REF][START_REF] Whittamore | Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish[END_REF].

NKA is a primary driving force for other ion-transporting systems in the osmoregulatory organs, including the kidneys [START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Whittamore | Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish[END_REF]. It is a P-type ATPase and consists of catalytic αsubunits and smaller glycosylated β-subunits [START_REF] Morth | A structural overview of the plasma membrane Na + ,K + -ATPase and H + -ATPase ion pumps[END_REF][START_REF] Toyoshima | First crystal structures of Na + ,K + -ATPase: new light on the oldest ion pump[END_REF]). Among them, the catalytic α-subunit, containing the binding sites of Na + ,K + , and ATP, plays a crucial role in NKA function, including osmoregulation [START_REF] Evans | Osmotic and ionic regulation in fishes[END_REF][START_REF] Tang | Relative changes in the abundance of branchial Na + /K + -ATPase α-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities[END_REF]. In birds and mammals, four α-isoforms (α1, α2, α3, and α4) of NKA with tissue-specific distribution have been reported [START_REF] Blanco | Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation[END_REF][START_REF] Morth | A structural overview of the plasma membrane Na + ,K + -ATPase and H + -ATPase ion pumps[END_REF][START_REF] Suhail | Na + ,K + -ATPase: ubiquitous multifunctional transmembrane protein and its relevance to various pathophysiological conditions[END_REF]. Among these isoforms, α1 is expressed ubiquitously in transporting epithelia for maintaining osmotic balance and cell volume regulation. Meanwhile, α2 is mainly found in the brain, muscle, and heart; α3 is present primarily in the neurons and ovaries; and α4 is expressed only in the testis. The switch of these α-isoforms is considered to meet specific requirements for differentiated cell-specific functions of cation transport [START_REF] Blanco | Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation[END_REF][START_REF] Blanco | Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function[END_REF][START_REF] Suhail | Na + ,K + -ATPase: ubiquitous multifunctional transmembrane protein and its relevance to various pathophysiological conditions[END_REF]. In teleosts, only three α-isoforms (α1, α2, and α3) but numerous paralogs have been identified [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change?[END_REF][START_REF] Ching | Na + /K + -ATPase α-subunit (nkaα) isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. As in mammals, tissue-specific expression of these teleostean α-subunits has been demonstrated. Moreover, in the osmoregulatory organs, the expression (mRNA and/or protein) of the predominant NKA α-isoforms (especially α1 and its paralogs) is salinity-dependent, indicating their roles in fish osmoregulation [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change?[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF].

FXYD proteins identified in some teleosts are considered to be important modulatory mechanisms for NKA activity [START_REF] Saito | Identification of zebrafish Fxyd11a protein that is highly expressed in ion-transporting epithelium of the gill and skin and its possible role in ion homeostasis[END_REF][START_REF] Tipsmark | Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change[END_REF][START_REF] Yang | Expression profiles of branchial FXYD proteins in the brackish medaka Oryzias dancena: a potential saltwater fish model for studies of osmoregulation[END_REF]. At present, at least 12 members of the FXYD protein family (i.e., FXYD1-12) have been reported in mammals and fish, and most FXYD proteins exhibit distinct tissue distribution [START_REF] Geering | Functional roles of Na,K-ATPase subunits[END_REF][START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Yang | Expression profiles of branchial FXYD proteins in the brackish medaka Oryzias dancena: a potential saltwater fish model for studies of osmoregulation[END_REF]. The FXYD protein family has been more widely studied in mammals and elasmobranchs compared to teleosts [START_REF] Crambert | FXYD Proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase[END_REF][START_REF] Geering | Functional roles of Na,K-ATPase subunits[END_REF]. Recently, salinity effects on expression of FXYD proteins were investigated in teleostean osmoregulatory ograns [START_REF] Tipsmark | Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change[END_REF][START_REF] Wang | FXYD8, a novel regulator of renal Na + /K + -ATPase in the euryhaline teleost, Tetraodon nigroviridis[END_REF][START_REF] Yang | Intestinal FXYD12 and sodium-potassium ATPase: a comparative study on two euryhaline medakas in response to salinity changes[END_REF]. The potential function of some FXYD proteins upon osmotic challenge has been demonstrated in a limited number of teleostean species [START_REF] Chang | FXYD11 mediated modulation of Na + /K + -ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments[END_REF][START_REF] Saito | Identification of zebrafish Fxyd11a protein that is highly expressed in ion-transporting epithelium of the gill and skin and its possible role in ion homeostasis[END_REF][START_REF] Wang | FXYD8, a novel regulator of renal Na + /K + -ATPase in the euryhaline teleost, Tetraodon nigroviridis[END_REF]Yang et al., 2016b). To date, however, the roles of most FXYD proteins are not yet clear in teleosts.

In the milkfish, the effects of salinity on NKA expression, including NKA activity [START_REF] Kang | Seawater-acclimation abates cold effects on Na + ,K + -ATPase activity in gills of the juvenile milkfish, Chanos chanos[END_REF][START_REF] Lin | The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water[END_REF][START_REF] Tang | Elevated Na + /K + -ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water[END_REF] and potential isoform composition of NKA α-subunits (protein levels only) [START_REF] Tang | Relative changes in the abundance of branchial Na + /K + -ATPase α-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities[END_REF]Yang et al., 2016a), have been investigated. Molecular characteristics and modulatory mechanisms of NKA α-subunit isoforms, however, are not clear. Hence, this study focused on potential modulatory mechanisms, including isoform composition of NKA αsubunits and FXYD proteins, on NKA expression/activity in response to salinity changes. The goals of the present study were to (1) identify the isoforms of milkfish NKA α-subunits; and (2) investigate the gene expression of NKA α-subunits and FXYD protein in milkfish kidneys upon hypoosmotic challenge. This paper sheds a light on the molecular modulatory mechanisms of NKA expression of euryhaline teleosts following salinity challenge.

Materials and methods

Ethical approval

The protocols employed for the experimental fish were reviewed and approved by the Institutional Animal Care and Use Committee of the National Chung Hsing University (IACUC approval no. 105-130), and were carried out in accordance with the approved guidelines.

Experimental animals and environments

Juvenile milkfish (C. chanos) with a standard length of 9-11 cm were purchased from the Da-Shun milkfish aquafarm in Tainan, Taiwan. Fish were transported to the laboratory, where they were acclimated to aerated dechlorinated local tap water (i.e., FW) or SW (35‰) at 28 ± 1 °C under a 12-h light: 12-h dark cycle. SW was prepared from FW by adding standardized amounts of synthetic sea salt (Blue Treasure Synthetic Sea Salt; Blue Treasure, Surry Hills, Australia). The details of water parameters were reported in our previous study [START_REF] Yang | The ultrastructural characterization of mitochondria-rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus)[END_REF]. Water was continuously circulated through fabricfloss filters. Fish were fed twice a day with a diet of commercial pellets.

Acclimation and transfer experiments

For long-term acclimation, the milkfish were acclimated to SW or FW for at least 4 weeks. In the time-course experiments, after long-term acclimation, milkfish in the study groups were transferred directly from SW to FW, and vice versa. Moreover, fish in the control group were transferred from SW to SW or from FW to FW for their respective experiments. Milkfish were sampled at 1/2, 1, 2, 4, and 7 days posttransfer, based on previous studies [START_REF] Hu | Na + ,K + -ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos[END_REF][START_REF] Tang | Relative changes in the abundance of branchial Na + /K + -ATPase α-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities[END_REF]. Before sampling, the fish were not fed for one day and were then anesthetized using 39.8 μM phenoxyethanol. The tissues and organs were dissected and directly placed into liquid nitrogen and then stored immediately at -80 °C for subsequent analysis.

Total RNA extraction and reverse transcription

Total RNA from various milkfish tissues/organs was extracted with IsoI-RNA Lysis Reagent (2302700; 5 Prime, Gaithersburg, MD, USA) in accordance with the manufacturer's instructions. The integrity and concentration of extracted RNA was verified and measured by 1% agarose gel electrophoresis and a NanoDrop 2000 (Thermo, Wilmington, DE, USA), respectively. For reverse transcription, cDNAs were synthesized with the iScript™ cDNA Synthesis Kit (170-8891; Bio-Rad, Hercules, CA, USA) or SMART™ RACE cDNA Amplification Kit (634858; Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. The RNA and cDNA were stored at -80 °C and -20 °C, respectively.

Sequencing and identification of milkfish NKA α-subunits

Genes coding for NKA α-subunits were derived from the milkfish transcriptome database [START_REF] Hu | Na + ,K + -ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos[END_REF]. They were confirmed through RT-(reverse transcription) or RACE-(rapid amplification of cDNA ends) PCR (polymerase chain reaction) and then sequenced by the Tri-Biotech (Taipei, Taiwan).

To characterize NKA proteins, the amino acid sequences were deduced from sequenced genes using the NCBI Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Subsequently, the transmembrane domains were predicted using the MEMSAT-SVM provided by the PSIPRED protein structure prediction server (http://bioinf. cs.ucl.ac.uk/psipred/) [START_REF] Buchan | Scalable web services for the PSIPRED Protein Analysis Workbench[END_REF], and comparisons were made with transmembrane domains of teleostean NKA including the Senegalese sole (Solea senegalensis; [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF] and European sea bass (Dicentrarchus labrax; [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF]. Potential phosphorylation sites of protein kinase C (PKC) and A (PKA) were predicted via NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) [START_REF] Blom | Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence[END_REF]. The binding sites of Na + and K + were predicted according to the homology modeling of the cation binding sites of human NKA [START_REF] Ogawa | Homology modeling of the cation binding sites of Na + K + -ATPase[END_REF].

For identification of these protein sequences, multiple sequence alignments, phylogenetic analysis, and protein identity were performed. Amino acid sequences for NKA α-subunits from different organisms (Tables S1 andS2) were aligned by MUSCLE (multiple sequence comparison by log-expectation) using MEGA X [START_REF] Kumar | MEGA X: molecular evolutionary genetics analysis across computing platforms[END_REF] for the subsequent analyses. These aligned sequences were used to confirm the positions of α1a-like and α1b-like residues based on [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF] in order to distinguish the α1 paralogs by comparison with sequences of other teleostean species (Fig. S1). A phylogenetic tree was constructed by the maximum likelihood method with 1000 bootstraps using MEGA X. Moreover, amino acid identity among teleosts and mammals was assessed using BioEdit 7.2.5 with the Sequence Identity Matrix [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF].

Quantitative real-time PCR (QPCR)

The methods, including PCR protocol and data calculation, were described previously [START_REF] Chang | FXYD11 mediated modulation of Na + /K + -ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments[END_REF][START_REF] Hu | Na + ,K + -ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos[END_REF]. Expression levels of target genes were quantified using 2× FastStart Universal SYBR Green Master (04913850001; Roche, Mannheim, Germany) with the MiniOpticon real-time PCR system (Bio-Rad). Then, mRNA values for the target genes were normalized using the expression of β-actin mRNA from the same cDNA sample [START_REF] Tang | Elevated Na + /K + -ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water[END_REF]. Primer information used is listed in Table S2. The amplification efficiency was similar for all primer pairs used in this study (range: 0.93-1.08). To confirm the specificity of these amplifications from each test sample, sequencing, melting curve analysis, and electrophoresis were carried out. For each unknown sample, relative fxyd gene expression was obtained using the formula 2^-[(Ct target genes,n -Ct β-actin,n ) -(Ct target genes,c -Ct β-actin,c )], where Ct corresponds to the threshold cycle number [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2 -ΔΔCT method[END_REF]. The letter "n" indicates each cDNA sample used in these experiments and "c" indicates the control mixed with cDNAs from many organs.

Statistical analysis

All values are shown as the mean ± standard error (SE). The Shapiro-Wilk normality test was applied. Because some of the data were not normally distributed, non-parametric tests were used. Differences in the mRNA levels between groups were analyzed via the Mann-Whitney U test or Kruskal-Wallis test for pair-wise or multiple comparisons, respectively. All analyses were performed using SPSS 20 software (SPSS, Chicago, IL, USA), and P values of < 0.05 were considered to be significant.

Results

Characterization of milkfish NKA α-isoforms

Four NKA α-isoforms, CcNKA α1a, α1c, α2, and α3, were identified in milkfish (Fig. 1; Table S2). Among them, CcNKA α2 was a partial sequence because only 952 deduced amino acids were obtained. These amino acid sequences of four CcNKA α-isoforms shared similar characteristic features, including transmembrane domains (TMs), phosphorylation sites of PKC and PKA binding sites, binding sites for Na + and K + , and a conserved C-terminus (Fig. 1). For further confirmation, a multiple sequence alignment of teleostean NKA α1-isoforms was carried out (Fig. S1). The results revealed that CcNKA α1a belongs to one of the teleostean NKA α1a groups, because it has more α1a-like residues (blue triangles) as opposed to α1b-like residues (green triangles). On the other hand, in the phylogenetic analysis, amino acid sequences of NKA α-subunit paralogs from teleosts, chicken, and human, with the sea urchin as outgroup, were aligned and a phylogenetic tree was constructed (Fig. 2). These proteins were divided into four groups (α1a/b, α1c, α2, and α3), and all CcNKA α-isoforms of milkfish were classified into their respective groups. Among these clades, moreover, close relationships were noted among the α1a/b and α1c of teleosts and the α1 of human and chicken. Additionally, higher identities of both cDNA and amino acid sequences were found between CcNKA α1a or α1c and α3 rather than α2 (Table 1). Moreover, the sequence identities among all CcNKA α-isoforms were higher for protein than for cDNA (Table 1). The comparisons of amino acid sequences of CcNKA α1a, α1c (Table 2), α2, and α3 (Table 3) individually with the homologues from other animal species showed that they shared a higher degree of homology with teleosts than with bird and mammal. Among the sequences of NKA α-isoforms, CcNKA α2 revealed a lower identity with other teleostean NKA α2 (83.5-85.6%; Table 3).

Tissue distribution of milkfish Ccnka α genes

The QPCR results revealed tissue-specific expression for most of the Ccnka α genes (Fig. 3). Ccnka α1a was present predominantly in the intestine and kidney, meanwhile Ccnka α1c was mainly expressed in the brain, heart, and liver. Ccnka α2 was expressed pervasively in all detected organs, while Ccnka α3 was primarily found in the brain and liver.

Salinity effects on Ccnka α and Ccfxyd genes in kidneys of SW and FW milkfish

In milkfish kidneys, Ccnka α1a was most predominantly expressed among Ccnka α genes at both salinities, followed by Ccnka α1c (Fig. 4A). Meanwhile, renal Ccfxyd2 mRNA abundance was the highest in both SW and FW groups, followed by Ccfxyd8 and Ccfxyd9 (Fig. 4B). Low mRNA levels of Ccnka α2, Ccnka α3, Ccfxyd7a, Ccfxyd7b, and Ccfxyd11 were observed in milkfish kidneys. Moreover, the expression of Ccnka α1a, Ccfxyd2, Ccfxyd7b, and Ccfxyd9 genes in milkfish kidneys was salinity-dependent (Fig. 4). Renal mRNA levels of Ccnka α1a, Ccfxyd2, and Ccfxyd9 were significantly higher in the FW group than in the SW group. In contrast, a higher mRNA abundance of renal Ccfxyd7b was found in the SW-acclimated milkfish.

Time-course changes in the Ccnka α and Ccfxyd mRNA levels of milkfish kidneys upon salinity challenge

In milkfish kidneys, after transfer from SW to FW, the mRNA profiles of Ccnka α and Ccfxyd genes were distinct, divided roughly into three patterns (Fig. 5). (1) mRNA expression of Ccnka α1a, Ccfxyd2, and Ccfxyd8 first changed (increased/decreased) and then recovered, compared to either the initial time-point (day 0) or the control group at the same time-point (Fig. 5A,C,D). ( 2) mRNA levels of Ccfxyd9 quickly increased and then slightly decreased. Expression of Ccfxyd9 mRNA was not significantly different from the initial time-point but was significantly higher than that of the control group at 7 days post-transfer (Fig. 5E). (3) No difference was observed in Ccnka α1c mRNA expression among different time-points or between the study and control group of each time-point after transfer (Fig. 5B). Among these profiles, most of the peak values were observed within 2 days post-transfer. On the other hand, after transfer from FW to SW, mRNA levels of renal Ccnka α1a were clearly downregulated (Fig. 6A), while mRNA expression of renal Ccnka α1c and Ccfxyd2 was first changed (increased/decreased) and then recovered (Fig. 6B,C). In renal Ccfxyd8 mRNA abundance, a significant change was found between 1 and 2 days post-transfer, and there was no difference among the other time-points (Fig. 6D). Among these profiles, mRNA levels of renal Ccnka α1a and Ccfxyd2 clearly decreased at 1 day post-transfer, and the peak value of Ccnka α1c mRNA was observed at 4 days post-transfer. Moreover, no difference was observed in Ccfxyd9 mRNA expression of milkfish kidneys among time-points or between the study and control group of each time-point after transfer (Fig. 6E).

Meanwhile, in both control groups (transfer from SW to SW and from FW to FW), no difference was found among time-points after transfer.

Fig. 2. Phylogenetic analysis of putative amino acid sequences of NKA α-isoforms using the maximum likelihood method. The numbers at the forks indicate the bootstrap values (%). Sea urchin NKA α was used as the outgroup for the root tree. Accession numbers are listed in Tables S1 andS2.

Table 1 Percentage of cDNA (open reading frame only; above diagonal) and amino acid (below diagonal) sequence identity among milkfish Na + /K + -ATPase (CcNKA) α-subunits. S2.

Discussion

Milkfish NKA α-isoforms

To further verify the modulatory mechanisms and molecular responses of milkfish NKA in response to salinity changes, we identified the cDNA sequences of nka α-subunits of milkfish for the first time. Four isoforms of the NKA α-subunits in milkfish, i.e., CcNKA α1a, α1c, α2, and α3, were identified. Among them, the amino acid sequences of CcNKA α1a and α1c were the most similar compared to α2 and α3. Similar results (highly similar α1 paralogs) were found in other teleosts [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF]. A previous study indicated that fish α1 paralogs are derived from an ancestral α1. The ancestral α1 was first duplicated into α1a/b and α1c, and then α1a/b was duplicated into α1a and α1b [START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. The grouping of α1a and α1b in the phylogenetic trees of previous studies was sometimes confusing because of high similarities between their sequences [START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change?[END_REF][START_REF] Ip | Roles of three branchial Na + -K + -ATPase αsubunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. In this study, only one gene of the α1a/b paralogs was cloned from milkfish. However, it cannot be clearly identified from α1a or α1b via phylogenetic analysis and protein identity as compared with other homologues. Therefore, according to [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF], a multiple sequence alignment was performed. Subsequently, the gene was named Ccnka α1a because of its higher similarity of amino acid sequences with other teleostean NKA α1a. In addition to CcNKA α1a, other CcNKA α-subunits could be named via the results of phylogenetic analysis and protein identity, referring to the nomenclature from previous studies [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF].

Previous studies indicated that amino acid sequences of NKA αsubunits are highly conserved across animal species [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Martin | Structure-function relationships in the Na + ,K + -pump[END_REF]. In this study, higher similarities (over 80 and 90% for α2 and others, respectively) of amino acid sequences among CcNKA α-isoforms and their respective homologues from other animal species were obtained. Likewise, similar results were found in other fish, including the Senegalese sole [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF], electric eel (Electrophorus electricus; [START_REF] Ching | Na + /K + -ATPase α-subunit (nkaα) isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus[END_REF], and European seabass [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF]. However, in the climbing perch (Anabas testudineus), NKA α1a showed 70-80% homology to most NKA α1isoforms from other teleosts [START_REF] Ip | Roles of three branchial Na + -K + -ATPase αsubunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus[END_REF]. These results revealed that there might still be certain differences among NKA α homologues. On the other hand, in a protein structure analysis, four CcNKA α-isoforms showed the typical topology of P-type ATPase, including 10 TMs with ion-binding sites, five extracellular domains, and four intracellular domains containing phosphorylation sites of PKC and PKA, as seen in mammalian NKA α-subunits [START_REF] Blanco | Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation[END_REF][START_REF] Martin | Structure-function relationships in the Na + ,K + -pump[END_REF]. These characteristic features have also been found in other teleosts [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF][START_REF] Ching | Na + /K + -ATPase α-subunit (nkaα) isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus[END_REF]. Moreover, the lysine (K)-rich sequences in the N-terminus of CcNKA α-isoforms (except CcNKA α2) present in many teleostean NKA α-subunits are considered to play a key role in forming a cation-selective gate via an excess of positively charged amino acids during certain stages of the transport process [START_REF] Blanco | Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function[END_REF][START_REF] Daly | Functional consequences of amino-terminal diversity of the catalytic subunit of the Na,K-ATPase[END_REF][START_REF] Vasilets | Structure-function relationships of cation binding in the Na + /K + -ATPase[END_REF] and/or in interacting with other proteins [START_REF] Toyoshima | First crystal structures of Na + ,K + -ATPase: new light on the oldest ion pump[END_REF]. The KETYY motif in the tails of CcNKA αisoforms is well-conserved except for lysine (K), indicating that it is crucial for Na + binding [START_REF] Morth | A structural overview of the plasma membrane Na + ,K + -ATPase and H + -ATPase ion pumps[END_REF]. The conserved degrees of KETYY motif differ among teleostean NKA α-subunits, and these differences might cause a difference in Na + affinity [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF][START_REF] Ching | Na + /K + -ATPase α-subunit (nkaα) isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus[END_REF]. Taken together, these conserved motifs and domains of the CcNKA α-isoforms play an important role in membrane localization and in ionoregulation via phosphorylation and dephosphorylation, as do other NKA α-subunits.

In NKA α-subunits, the form and number of isoforms vary among teleostean species. Some teleosts, including zebrafish (Danio rerio; [START_REF] Liao | Expression regulation of Na + -K + -ATPase α1subunit subtypes in zebrafish gill ionocytes[END_REF], rainbow trout (Oncorhynchus mykiss; [START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF], Senegalese sole [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF], Japanese medaka (Oryzias latipes; [START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change?[END_REF], and Japanese eel (Anguilla japonica; [START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF], have more NKA α-subunit isoforms than milkfish. In teleosts, moreover, most of these subunits belong to the highly diversified α1-isoforms, with tissue-specific distribution [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. These two findings were also observed in milkfish, which showed two α1-isoforms and distinct tissue distribution patterns of four Ccnka α genes. As in other teleosts, Ccnka α1a is present primarily in the osmoregulatory organs, while Ccnka α1c is expressed ubiquitously in milkfish. However, their distribution patterns and expression levels vary by species [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF][START_REF] Ip | Roles of three branchial Na + -K + -ATPase αsubunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. In the Senegalese sole, the nka α1a is mainly expressed in the gill, kidney, and intestine [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF], while in the rainbow trout nka α1a is present primarily in the gill [START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF]. On the other hand, Ccnka α2 mRNA are expressed ubiquitously in all detected organs, yet their abundance is unexpectedly low as compared to that of other Ccnka α genes. In mammals and other teleosts, nka α2 is expressed predominantly in the muscle [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF][START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change?[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF][START_REF] Suhail | Na + ,K + -ATPase: ubiquitous multifunctional transmembrane protein and its relevance to various pathophysiological conditions[END_REF]. Similar to results found in other animal species, Ccnka α2 mRNA was present primarily in milkfish Accession numbers are listed in Tables S1 andS2.

Table 3 Amino acid identity (%) among NKA α2-and α3-isoforms. Accession numbers are listed in Tables S1 andS2.

muscle in our preliminary study (data not shown). In particular, the tissue distribution patterns of Ccnka α3, which was found mainly in the brain and liver, were different from those of other teleosts. Some teleostean nka α3 are expressed predominantly in the brain alone, as in the Senegalese sole (both α3a and α3b; [START_REF] Armesto | Molecular characterization and transcriptional regulation of the Na + /K + ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis)[END_REF]. Other teleostean nka α3 are ubiquitously expressed in various organs, as in the Mozambique tilapia (Oreochromis mossambicus; α3; [START_REF] Feng | Gene expression of Na + -K + -ATPase α1 and α3 subunits in gills of the teleost Oreochromis mossambicus, adapted to different environmental salinities[END_REF] and rainbow trout (α3; [START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF]. Meanwhile, in the swamp eel (Monopterus albus), α3a is found only in the brain and α3b is widely distributed in various organs [START_REF] Chen | Properties and expression of Na + /K + -ATPase α-subunit isoforms in the brain of the swamp eel, Monopterus albus, which has unusually high brain ammonia tolerance[END_REF]. Overall, these distinct tissue distribution patterns of four Ccnka α genes might reveal that they play different roles in diverse organs as other NKA α-subunits in mammals and other teleosts.

Salinity-dependent expression of renal Ccnka α and Ccfxyd genes in milkfish

Both switching of NKA α-subunits (the catalytic subunit) and FXYD proteins are modulatory mechanisms for NKA activity [START_REF] Geering | Functional roles of Na,K-ATPase subunits[END_REF][START_REF] Toyoshima | First crystal structures of Na + ,K + -ATPase: new light on the oldest ion pump[END_REF]. Recently, the relative abundance of multiple NKA α-isoforms and FXYD proteins was found to be altered with ambient salinity changes in several species of teleosts [START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF][START_REF] Yang | Intestinal FXYD12 and sodium-potassium ATPase: a comparative study on two euryhaline medakas in response to salinity changes[END_REF]. Most of these studies, however, focused on the gills. Moreover, no study investigated the potential modulatory mechanisms for NKA expression/activity by analyzing isoform compositions of NKA α-subunits and FXYD proteins simultaneously in teleostean kidneys. In milkfish kidneys, Ccnka α1a and Ccfxyd2 might play major roles, since they were highly expressed. Conversely, the low levels of renal Ccnka α2, α3, Ccfxyd7a, Ccfxyd7b, and Ccfxyd11 implied that they might be functional in other organs, like those of mammals and other teleosts [START_REF] Blanco | Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation[END_REF][START_REF] Geering | Functional roles of Na,K-ATPase subunits[END_REF][START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Yang | Expression profiles of branchial FXYD proteins in the brackish medaka Oryzias dancena: a potential saltwater fish model for studies of osmoregulation[END_REF]. In mammalian NKA α-subunits, NKA α-isoforms possessed different affinities for substrate ions, ouabain, and ATP [START_REF] Blanco | Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation[END_REF]. Furthermore, in salmonid gills, NKA α1a and α1b were considered to be the FW-and SW-type isoforms, respectively [START_REF] Dalziel | Origins and functional diversification of salinity-responsive Na + ,K + ATPase α1 paralogs in salmonids[END_REF][START_REF] Mccormick | Distinct freshwater and seawater isoforms of Na + /K + -ATPase in gill chloride cells of Atlantic salmon[END_REF][START_REF] Richards | Na + /K + -ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer[END_REF]. In the kidneys of SW or FW milkfish, the mRNA abundance of Ccnka α1a was higher than that of other Ccnka α genes, indicating that there might be no switching of the NKA α-isoforms, unlike the results in fish gills.

Upon salinity challenge, in this study, parallel expression patterns were found in renal Ccnka α1a and Ccfxyd2, showing an increase in FWacclimated milkfish. Their expression profiles were similar to those found in previous studies focused on NKA expression of milkfish kidneys, including mRNA (pan-α1), protein (both pan-α-and pan-β-subunits), and enzyme activity [START_REF] Tang | Elevated Na + /K + -ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water[END_REF]Yang et al., 2016a). A previous study also reported that in milkfish kidneys, protein abundance of the NKA α1-isoform was increased in the FW-acclimated group, while no difference was found in NKA α2-and α3-isoforms between the SW and FW groups (Yang et al., 2016a), which is in accordance with our data. Moreover, there was no difference in Ccnka α1c mRNA levels between FW and SW. Therefore, in FW-acclimated milkfish, induction of renal Ccnka α1a transcripts rather than Ccnka α1c might contribute to increased protein abundance of the NKA α1-isoform and then enhance NKA activity. On the other hand, a few studies have investigated the renal FXYD2 of teleosts, including the Atlantic salmon (Salmo salar; [START_REF] Tipsmark | Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change[END_REF] and zebrafish [START_REF] Saito | Identification of zebrafish Fxyd11a protein that is highly expressed in ion-transporting epithelium of the gill and skin and its possible role in ion homeostasis[END_REF]. In kidneys of Atlantic salmon, fxyd2 mRNA levels were higher in FW fish than in the SW group [START_REF] Tipsmark | Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change[END_REF]. Although some studies have focused on other FXYD proteins of teleostean kidneys in response to salinity changes [START_REF] Hu | Identification of fxyd genes from the spotted scat (Scatophagus argus): molecular cloning, tissue-specific expression, and response to acute hyposaline stress[END_REF][START_REF] Liang | Na + /K + -ATPase response to salinity change and its correlation with FXYD11 expression in Anguilla marmorata[END_REF][START_REF] Tang | Effects of salinity acclimation on Na + /K + -ATPase responses and FXYD11 expression in the gills and kidneys of the Japanese eel (Anguilla japonica)[END_REF][START_REF] Wang | FXYD8, a novel regulator of renal Na + /K + -ATPase in the euryhaline teleost, Tetraodon nigroviridis[END_REF]Yang et al., 2016b), the role of renal FXYD2 in salinity challenge is not yet clear. Mammalian FXYD2 is also expressed in the kidney and inhibits NKA activity via binding with NKA [START_REF] Arystarkhova | Beneficial renal and pancreatic phenotypes in a mouse deficient in FXYD2 regulatory subunit of Na,K-ATPase[END_REF][START_REF] Geering | Functional roles of Na,K-ATPase subunits[END_REF]. In the present study, however, we have no evidence regarding the potential inhibitory role of CcFXYD2 on NKA activity. In summary, when milkfish were acclimated to FW, high levels of renal Ccnka α1a and Ccfxyd2 might cooperate to maintain a balance of high NKA activity for ionoregulation. More studies should be performed in the future to clarify their roles in osmoregulation.

To further investigate the potential modulatory mechanisms, timecourse experiments were performed. The levels of renal Ccnka α1a and Ccfxyd2, the predominant participators, changed quickly within 1 day after both transfer treatments. Similar results (quick changes) were also found in the gill NKA expression (both mRNA and protein of pan-α1) of milkfish since 1 day after transfer from SW to FW [START_REF] Hu | Na + ,K + -ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos[END_REF]. Unlike the gill, changes in renal Ccnka α1a and Ccfxyd2 expression were recovered subsequently. Similar patterns (recovered or unchanged expression) were found in renal Ccnka α1c, Ccfxyd8, and Ccfxyd9 after transfer. Furthermore, in milkfish kidneys, at 7 days post-transfer, significant changes in mRNA abundance were only observed in Ccnka α1a (from FW to SW) and Ccfxyd9 (from SW to FW). These findings of recovered and stable expression in nka α genes have also been reported in kidneys of other teleosts [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF][START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF][START_REF] Zhu | Characterization and expression of Na + /K + -ATPase in gills and kidneys of the Teleost fish Oreochromis mossambicus, Oreochromis urolepis hornorum and their hybrids in response to salinity challenge[END_REF]. In kidneys of the European seabass, mRNA levels of both nka α1a and α1b increased quickly and then recovered after transfer from SW to FW [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF]. However, no difference in expression was found in most renal nka α genes of the Fig. 5. Time-course changes in mRNA expression of renal Ccnka α genes in the milkfish after transfer from seawater (SW) to fresh water (FW) (the study group) or to SW (the control group). The values are means ± SE (N = 4 or 6 in the initial time-point or others, respectively). Different letters indicate significant differences among time-points in the study group (from SW to FW; Kruskal-Wallis test; P < .05). No difference was found among time-points in the control group (from SW to SW). The asterisks (*) indicate significant differences between the study and control groups at the same time-point post transfer (Mann-Whitney U test; P < .05).

Japanese eel after transfer from FW to SW [START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF]. Previous studies reported that renal nka α expression might be modulated in a short time [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF] and that expression patterns of temporal changes might vary with species [START_REF] Wong | Flexible selection of diversified Na + /K + -ATPase α-subunit isoforms for osmoregulation in teleosts[END_REF][START_REF] Zhu | Characterization and expression of Na + /K + -ATPase in gills and kidneys of the Teleost fish Oreochromis mossambicus, Oreochromis urolepis hornorum and their hybrids in response to salinity challenge[END_REF]. Moreover, the osmoregulatory capabilities of teleostean kidneys might be correlated with their evolutionary histories or natural habitats (Yang et al., 2016a,b). In the milkfish, which has excellent osmoregulatory capacity [START_REF] Tang | Elevated Na + /K + -ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water[END_REF]Yang et al., 2016a), our results showed that genes expressed at the kidney level can be effectively regulated. The findings also revealed that the regulatory process might be divided into two periods, a short-term rapid modulation period (within 7 days) and a subsequent moderate regulation period, in response to salinity challenge. Other modulatory mechanisms of NKA α-subunits might be involved during this period, such as endocrine regulation and protein phosphorylation [START_REF] Crambert | FXYD Proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase[END_REF][START_REF] Toyoshima | First crystal structures of Na + ,K + -ATPase: new light on the oldest ion pump[END_REF]. Notably, when the SW-residing milkfish were transferred to a hypoosmotic environment (FW), changes in renal Ccnka and Ccfxyd genes were more dramatic and more irregular compared to those in the group transferred from FW to SW, particularly for Ccnka α1a and Ccfxyd2. Expression of these proteins was not assessed in this study and thus remains to be elucidated.

Conclusions

This study revealed the potential modulatory mechanisms for NKA expression from isoform compositions of NKA α-subunits and FXYD proteins in kidneys of the milkfish upon hypoosmotic (i.e. FW) challenges. Four isoforms of milkfish NKA α-subunits with tissue-specific distribution were identified: CcNKA α1a, α1c, α2, and α3. The highly conserved amino acid sequences and similar characteristic features to homologues from other animal species revealed their potential function. Notably, in milkfish kidneys, CcNKA α1a and CcFXYD2 were predominantly expressed. Increases in renal Ccnka α1a and Ccfxyd2 genes were found after exposure of milkfish to FW. After transfer, most renal Ccnka and Ccfxyd genes (particularly Ccnka α1a and Ccfxyd2) clearly changed their expression, although most of these changes were recovered subsequently. Our results suggest that the NKA activity of milkfish kidneys might be modulated via CcNKA α1a and CcFXYD2 for Fig. 6. Time-course changes in mRNA expression of renal Ccnka α genes in the milkfish after transfer from fresh water (FW) to seawater (SW) (the study group) or to FW (the control group). The values are means ± SE (N = 4 or 6 in the initial time-point or others, respectively). Different letters indicate significant differences among time-points in the study group (from FW to SW; Kruskal-Wallis test; P < .05). No difference was found among time-points in the control group (from FW to FW). The asterisks (*) indicate significant differences between the study and control groups at the same time-point post transfer (Mann-Whitney U test; P < .05).

osmoregulation in response to salinity challenge. To our knowledge, this study provides the first evidence of the potential modulatory mechanisms for NKA expression/activity from isoform compositions of NKA α-subunits and FXYD proteins in kidneys of euryhaline teleosts. Further studies are necessary to illustrate more regulatory mechanisms of milkfish upon salinity challenge and the results are expected to benefit and improve its aquaculture practice.

Fig. 1 .

 1 Fig. 1. Alignment of putative amino acid sequences of milkfish NKA (CcNKA) α-isoforms. Dashes represent gaps, and the predicted transmembrane domains (TMs) are shown in green underlines. Vertical boxes represent coordinating residues for Na + (Na) or K + (K) binding and for either Na + or K + binding (Na/K). Potential phosphorylation sites are shown with crosses (+), asterisks (*), and hash tags (#) for different kinases, including protein kinase C, protein kinase A, and both, respectively. Triangles indicate the KETYY motif. ECD, extracellular domain; ICD, intracellular domain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig. 3. Tissue distribution of four milkfish NKA (Ccnka) α genes. The values are means ± SE (N = 8 or 6 for gill/kidney or other organs; combined with equal sample size from all salinity groups). Different letters indicate significant differences among Ccnka α genes (Kruskal-Wallis test; P < .05). Int., intestine.

Fig. 4 .

 4 Fig. 4. Comparisons of renal Ccnka α mRNA abundance in the milkfish. The values are means ± SE (N = 4). Different letters indicate significant differences among Ccnka α genes in seawater (SW; letter: a, b) or freshwater groups (FW; letter: x, y) (Kruskal-Wallis test; P < .05). The asterisks (*) indicate significant differences between the SW and FW groups for the same gene (Mann-Whitney U test; P < .05).

Table 2

 2 Amino acid identity (%) among NKA α1-isoforms.

	Classification	Species	Milkfish
			α1a (%)	α1c (%)
	Teleost	Killifish α1a	92.2	90.7
		European seabass α1a	91.5	90.4
		European seabass α1b	89.2	92.5
		Senegalese sole α1a	91.9	90.5
		Senegalese sole α1b	89.6	93.1
		Stickleback α1a	89.1	88.2
		Stickleback α1b	88.6	91.8
		Japanese medaka α1a	88.5	87.5
		Japanese medaka α1b	90.1	88.9
		Japanese medaka α1c	88.8	92.3
		Rainbow trout α1a	82.7	82.9
		Rainbow trout α1b	90.5	88.7
		Rainbow trout α1c	87.7	91.8
		Climbing perch α1a	81.2	80.2
		Climbing perch α1b	86.0	85.9
		Climbing perch α1c	90.5	89.8
		Electric eel α1c1	91.0	96.0
	Bird	Chicken α1	87.5	88.6
	Mammal	Human α1	88.5	89.4

Acknowledgements

This study was financially supported in part by the Ministry of Science and Technology (MOST), Taiwan, through the MOST Research Project (MOST-106-2313-B-005-038-MY3) and the MOST-CNRS (France) International Project for Scientific Cooperation (MOST-105-2911-I-005-501) grants to T.H.L. This work was also financially supported in part by the iEGG and Animal Biotechnology Center from The Feature Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan (MOE-107-S-0023-F) to T.H.L. W.K.Y. was supported by postdoctoral fellowships from MOST (MOST-106-2811-B-005-004) and MOE (MOE-107-S-0023-F).

Conflict of interest

The authors have no competing interests to declare.


Claire Richards

Roland Cahen
email: roland.cahen@ensci.com

Nicolas Misdariis
email: misdariis@ircam.fr

Actronika Sas

DESIGNING THE BALANCE BETWEEN SOUND AND TOUCH: METHODS FOR MULTIMODAL COMPOSITION

HAL is

INTRODUCTION

The experiences of grazing one's hand across a soft surface and listening to a concerto may not seem to have much in common. However, in both cases, the body is detecting and interpreting vibratory information, whether that vibration is a result of the friction produced by the movement of skin on a surface, by the movement of performers' bows on the strings of violins, or by touching the surface of a speaker playing music. In our research, we seek to illuminate the physical aspect of sound perception, by bringing the vibrations directly in contact with the body. In parallel to the development of our multimodal wearable device, we conducted a psychophysical study on extra-tympanic hearing thresholds of the human torso, with the aim to begin a definition of audio-haptic signal design guidelines for the wearable [START_REF] Richards | Vibratory Detection Thresholds for the Spine, Clavicle and Sternum[END_REF]. In this study, instead of taking a psychophysical approach, we decided to start by applying the basic parameters obtained from the first study's results within a more creative developmental framework: an intensive workshop.

Our intention in organizing this workshop was not to produce data, but to gain more clarity on the research questions we can ask. In the next sections, we first present the project context of exploring a set of audio-haptic forms of expression, followed by basic technical details of the multimodal harness. Then, we describe functionalities of authoring tools we developed, trial feedback, and the research questions generated by the sensory experience they permit (or not). Finally, we discuss our perspectives on future development, for both experimental and compositional purposes.

CONTEXT AND MOTIVATIONS

The perceptual ranges and discriminatory limits of hearing and touch are compatible and overlapping. Understanding the body's ability to interpret vibrotactile information can be broken down into several key characteristics of the presented stimulus: its frequency, intensity, duration, waveform and position on the skin's surface. The elements of vibrotactile signal design have much in common with those of auditory signal design (pitch, amplitude, duration, timbre, distance), but the two senses do not process the sensory information in the same way [START_REF] Gunther | Cutaneous grooves: Composing for the sense of touch[END_REF]. Opposed to hearing, which is centralized to the head, we perceive vibrations all over our body [START_REF] Merchel | Psychophysical Comparison of the Auditory and Vibrotactile Perception-Absolute Sensitivity[END_REF]. The perception of vibrations in the frequency range between approximately 10-1000 Hz overlaps the detection capacities of the two senses.

Though there are distinct differences in processing capacities between the two sensory organs, researchers have found that auditory stimulation can affect tactile perception [START_REF] Bresciani | Feeling what you hear: auditory signals can modulate tactile tap perception[END_REF][START_REF] Crommett | Auditory adaptation improves tactile frequency perception[END_REF], and vice versa [START_REF] Schürmann | Touch activates human auditory cortex[END_REF][START_REF] Gillmeister | Tactile enhancement of auditory detection and perceived loudness[END_REF]. Beyond influencing the perception of presented auditory stimuli, tactile stimuli alone have been found to activate the auditory cortex, both in normal-hearing and congenitally deaf adults. Researchers have found this effect to be greater for deaf versus hearing participants [START_REF] Auer | Vibrotactile activation of the auditory cortices in deaf versus hearing adults[END_REF]. The mutual facilitation of one sense on the perception of the other can be attributed to processes of multimodal integration and cross-modal enhancement [START_REF] Shimojo | Sensory modalities are not separate modalities: plasticity and interactions[END_REF].

The perception of hearing and touch can therefore be influenced separately and simultaneously, by using the same source of stimulation: vibration. In this paper, we discuss the excitation of the two modalities by means of extratympanic conduction of sound and vibrotactile stimulation of the skin. The existing literature on extra-tympanic conduction provides an essential foundation towards understanding how we are able to hear sounds that do not stimulate the outer ear. However, notable past investigations of these mechanisms are primarily based on the anatomy of the skull [START_REF] Stenfelt | Bone-conducted sound: physiological and clinical aspects[END_REF]. Commonly described as bone conduction, this paper refers to contact-based sound transmission as "extra-tympanic", because there is speculation that soft tissue is involved in transmission mechanisms, and researchers have found that sound can be conducted from other parts of the body [START_REF] Adelman | Relation between body structure and hearing during soft tissue auditory stimulation[END_REF].

Many researchers have therefore investigated the use of vibration technology to augment auditory experiences by creating interfaces for the body. A large portion of these interfaces were specifically created for the improvement of musical experiences, and within this category, many devices have been developed specifically for the back and upper body. The model human cochlea [START_REF]Composing vibrotactile music: A multi-sensory experience with the emoti-chair[END_REF] is a tactile device that separates audio signals into frequency bands from 20-1000 Hz, displayed to the back via 16 voice-coil actuators, arranged on the back of an armchair. The Hapbeat and Vibeat both integrate motors that vibrate in time with music, worn as a necklace or bracelet. The Soundshirt integrates 30 micro-actuators (type unspecified) in a thin, flexible garment created for hearing-impaired individuals to have a tactile experience of philharmonic music. Users feel lower frequencies in the lower abdomen, while higher frequencies are displayed across the upper body. This frequency-based organization of tactile stimulations draws comparison to the model human cochlea while it differs from the Subpac, a backpack-style vest that enhances bass frequencies via two large voice coil actuators located on the back.

Our research differentiates itself from the prior art through our approach to mixed-modality effect design. The resulting experience of sound, perceived either by means of extra-tympanic hearing, tactile stimulation of the skin or a mixture of both modalities, is accessible to all hearing profiles. Hearing loss comes in many shapes and sizes, differing based on the frequencies affected, severity, symmetry, and type of lesion (conductive, sensorineural or mixed) [START_REF] Margolis | Toward a standard description of hearing loss[END_REF]. Someone with severe sensorineural hearing loss could appreciate tactile sensations, while an individual with conductive hearing loss could hear the wide range of acoustic frequencies displayed via extra-tympanic conduction. Beyond catering to diverse hearing profiles, our project addresses normal hearing profiles as well, and is inspired by the principles of universal design: conception whose aim is to include all users, regardless of ability. There are nine actuators integrated on the structure: two on the clavicles, two on the ribs, and five along the spine. The straps are velcro-adjustable, two of which go around the legs to secure the last module against the body. The multichannel audio-haptics card is connected to the harness, situated below the last module on the spine.

THE MULTIMODAL HARNESS

Conception and Functionality

Before beginning the design process1 , we defined two key constraints for the multimodal harness: adhesion of the module against the body, and user comfort. In order to achieve effective sound transmission and avoid signal attenuation, the vibrating module should be made of hard material, secured tightly against the body. Balancing tightness and comfort, two contradictory qualities, became a principal design challenge which we addressed with the flexibility of the modules and the ergonomic aspects of the harness structure (fig. 1).

The technical aspects of the module (fig. 2) allow them to adapt to the body's form at the different points of stimulation (spine, clavicles, and ribs). The hard plastic parts in direct contact with the user are ergonomically shaped (wide and flat on the ribs, extruded on the lower spine, rounded on the clavicles) in order to maximize the ratio of comfort to auditory signal transmission. The harness structure (fig. 1) has adjustable Velcro straps attached to all corners of the modules, pulling them against the body. Nine of Actronika's proprietary voice coil actuators are connected to the Mk-1 20-channel audio-haptic card. The Max environment interfaces with the USB Audio output on the Mk-1 board, which is detected as a sound card. Each of the nine actuators on the multimodal harness has its own channel on the multichannel audio-haptic card. In its current wired design, users' movements are restricted to the length of the 9V power supply and audio signal transmission cables.

DESIGN WORKSHOP

The two-week workshop, organized by the co-authors, took place at IRCAM in Paris, France in August 2021. During the workshop, five participants (P.01, P.02, etc.) tested our tools based on their own interest or direct involvement in the project: they were neither specifically selected nor remunerated for their participation. During every trial of the multimodal harness, users wear ear plugs and a noisecanceling headset in order to mask the external noise created by the modules' vibrations and focus on their hearing via extra-tympanic conduction.

The main objective of the design workshop was to create first drafts of audio-haptic signal design interfaces. Creating these tools would allow us to refine our assumptions about the sensory experiences afforded by the multimodal harness, informed by our previous psychophysical study, haptic illusions of signal movement [START_REF] Geldard | The cutaneous 'rabbit': A perceptual illusion[END_REF], and existing research on vibrotactile musical experiences [START_REF] Gunther | Cutaneous grooves: Composing for the sense of touch[END_REF][START_REF]Composing vibrotactile music: A multi-sensory experience with the emoti-chair[END_REF].

We hypothesize that the vibratory activation of tactile, auditory and bi-modal perception enables myriad possibilities for signal design: sensations that can travel in all directions to create illusions of movement, sound perception by vibratory transmission through musculoskeletal structures, and novel effects based on the multimodal integration of both sensations. While it is possible to approximately localize tactile stimuli on the body's surface [START_REF] Cholewiak | The perception of tactile distance: Influences of body site, space, and time[END_REF], auditory perception by means of extra-tympanic conduc- A graphic equalizer and a mute button is available for each actuator, with different colored bars for 7 frequency bands spanning 50-4000 Hz. The user can save their equalizer settings in the grid below the playlist object, recorded and saved for future use. To the right of the preset grid, a high shelf filter object, applied to all channels, attenuates the bass frequencies with respect to the high frequencies due to the resonant frequency of the actuator (70 Hz).

tion on the torso is essentially mono, since all sounds are entering the inner ear from the same pathway: the cervical spine. In other words, spatial audio information is lost, but we hypothesize that the localization of tactile stimuli could remain strong enough to transmit impressions of spatialized auditory source positions.

AUTHORING TOOLS FOR THE MULTIMODAL HARNESS

Each of the following authoring tools offers a different possibility for designing audio-haptic effects. In this section, we describe each of the authoring tools in three parts: the interface design, feedback from trial sessions, and finally key questions and limitations related to interface functionalities and perceptive experiences.

Inner.Music: Multimodal Music Player

Inner.Music is a music player, designed to test the perception of filtered source files transmitted to the multimodal harness. This tool features a body point-based equalizer tool, which allows the designer to calibrate the signals to each position of stimulation.

Interface Design and Composition Features

Inner.Music allows the designer to self set, adjust and balance the volumes of each actuator's filter bands, while listening to music. The interface is also a preset manager: individual sensory profiles, created by saving the nine filter band settings in presets, can vary each person's sensory preferences (see fig. 3). Our initial intention for Inner.Music was to create a tool that adjusts each actuator's spectrum in order to obtain a homogeneous audio-haptic sensation across all stimulated positions. However, we understood during the trial sessions that the ranges and nature of the two modalities' perceptions vary across positions, and according to different types of effect design or scenarios of use. We therefore chose to exploit these variations, creating an interface that allows the user to tweak their own sensory experience instead of imposing a generic predetermined sensory calibration.

Trial Feedback

While toying with the Inner.Music interface, participants employed various strategies: balancing the signal to achieve a perception of homogeneity across the positions, attributing levels and spectrum bands to each position in order to focus their perception on either the tactile or auditory modality, or muting positions that they did not appreciate. For example, some favored a stronger tactile sensation of the bass in the lower back, and routed the trebles to the upper back and clavicles to hear via extra-tympanic conduction. A common remark was that the music is audible, but appears to be coming from a distant place within their body, and in order to hear more details, they have to focus their attention. P.03 said, "It's possible to hear the sound, but to feel practically nothing." P.05 was especially sensitive to extra-tympanic auditory perception, saying: "I can hear the sound as if I were listening with headphones -maybe even better". On the other hand, P.01 was disappointed by the quality of the auditory experience and resorted to actively touching the modules to enhance his tactile perception (fig. 4). These differences in sound perception could be attributed to morphological differences affecting the transmission of the mechanical waves within the body [START_REF] Adelman | Relation between body structure and hearing during soft tissue auditory stimulation[END_REF]. A user's expectations may also impact how they judge their sensory experience: they might compare the quality of their auditory experience to other systems that were specifically designed for the ears, not the body.

Key Questions and Limitations

The strong morphological differences between each individual can be compared in a similar way to how differences in the structure of the outer ear can affect one's hearing. The Head Related Transfer Function (HRTF) is a physical transfer function that describes how a given sound coming from a specific point will reach the ear, in terms of spectral characteristics [START_REF] Wightman | Headphone simulation of free-field listening. I: stimulus synthesis[END_REF]. In comparison, we wonder if we could develop a Body Related Transfer Function (BRTF), a functionality capable of adjusting the multimodal harness's response to each individual in order for them to perceive the same signal, based on their morphological differences (height, weight, musculoskeletal characteristics).

Mp2p: Body Spatialization Interface

We created the Mp2p (Mono point-to-point) interface as a creative tool for spatialization of multimodal signals on the harness, by manipulating the signals' positions and trajectories.

Interface Design and Composition Features

Within the design space, simple mono signals can be played on any single actuator or grouped set of actuators and panned2 between them according to a set 1D path. Spatialization of the multimodal signals in the Mp2p system works based on configuration presets which organize the points of stimulation along different paths. The signal can be routed to each independent point, one channel after another, or to several points at once, by grouping channel outputs together. In fig. 5, we see three possible configuration presets: these allow the designer to create different illusions of movement (sporadic in the first preset, sideto-side in the middle preset, top-to-bottom in the right preset). The actuators' positions on the lateral and vertical axes of the body allow for directional and alternating effects. We defined a set of configurations, providing a Below each signal path is a graphic representation of the nine positions on the harness structure: the upper horizontal line is the clavicles, the lower is the ribs, and the vertical line is the spine. In the leftmost configuration, each position has its own independent channel. The middle configuration uses only two channels (light and dark green), with two positions paired to each channel -the five spine points are inactive. The right-most configuration groups the positions among four channels: the clavicles and top spine point (dark green), the second spine point (light green), the middle spine and ribs (cyan) and the lowest spine point (navy).

diverse selection of routing structures for the spatialized effects. The designer can manipulate the signal's position, duration, waveform (sine, saw tooth or noise), frequency (50 Hz to 4 kHz), and linear amplitude (0. to 1. float multiplier), and play their composition looped or one-shot (see fig. 6). By manipulating these variables, the sensory effects can be perceived as having continuous or discrete inter-position transitions: the signal can move gradually, or switch abruptly along its path.

Trial Feedback

The Mp2p interface is slightly more complex than the other two tools, so after learning about the basic parameters, participants generally chose to tweak our predefined patterns rather than create their own from scratch. P.03 was particularly impressed by the sensation created by the signal moving up and down the spine. If the signal moved quickly enough along the points, he felt a continuous sensation, like a "hand running up and down [his] back." The slower the movement of the signal along the points, the easier it was to localize from which position it came. The illusion we tried to create of a front-to-back shifting sensation between the front actuators (clavicles and ribs) and back (spine) was less convincing, perhaps due to the slightly central position of the actuators on the ribs, and because the sensation of something crossing one's body is a rather alien concept. The duration of the experience as a whole also impacted the sensory experience: after trying the patterns, P.02 said: To the right of the figure is the spatialization design, with three curves for position, frequency and amplitude. By modifying the curve, the designer can fine-tune the signal's duration at each group of positions, and its variations in amplitude and frequency across the composition.

I need more time to become familiar with the sensations I'm feeling on and in my body, since they are so new. Just a few minutes is not enough for my brain to really understand what is happening. I think with more time I'd be able to appreciate the sensations a bit more.

Participants made more comments on their "outer" body (tactile perception) than their "inner" body (extra-tympanic sound perception) during the spatialization experience. For most participants, the signal was only audible when displayed to certain positions (upper back and clavicles, i.e. those closest to the inner ear). The bi-modal aspect of the spatialization experience may therefore depend not only on a signal's position, intensity and spectral characteristics, but also on the user's conscious attention on either modality.

Key Questions and Limitations

When we started to design Mp2p, we tried to use an existing 3D spatializer tool: a vector-based amplitude panning (VBAP) algorithm [START_REF] Pulkki | Virtual sound source positioning using vector base amplitude panning[END_REF]. However, our configuration did not permit this because the listening point (the head) was off-center, i.e. not based on diffusion points (speakers) placed around the origin (listener). If we use a classic calculation of spatial decay (gain = 1/distance 2 ) according to airborne sound transmission, the signal disappears as soon as we move the point of diffusion away from the actuators' exact positions. Realistically, we would need to create a more specific spatialization model which takes into account the body's responses to extra-tympanic conduction and tactile stimulation of the skin, as described in section 4.1.3. The listening point (i.e. listener's position) would be the head, while the haptic sensations would be distributed: they would not refer to any central reference or origin point, since tactile perception is decentralized and perceived all over the surface of the skin (i.e. each actuator's position would have its own sensory reference).

One key limitation of Mp2p is the static nature of the configuration preset system. The signal path configurations are saved within presets and are static within a single composition. In other words, the designer cannot use multiple configuration presets in one spatialization time frame, limiting each composition to a single path that the signal can follow. One possible solution is to create a state-based programming interface which would include a configuration for each state, and a sequencer that allows the designer to edit each successive state and concatenate them with discrete or progressive transitions between states. In its current state, Mp2p does not integrate the equalizer profile function of Inner.Music, so in order to calibrate the signal intensity for the different sensitivities of each point of stimulation, designers must manually tweak and adjust the amplitude levels along the duration of the displayed signal. It is time-consuming to find the right nuances of intensity and frequency values for a given point in order to obtain the intended perceptive results. A final limitation of this tool is that the signal is mono: we cannot create "harmonies" of different sounds and movements. In section 6, we discuss some steps to address this constraint.

Drummer: Body-based Drum Machine

While we discussed musical metaphors during the workshop, we decided to create a basic nine-track step sequencer and drum machine.

Interface Design and Composition Features

Each track on the interface corresponds to one actuator and plays one selected sound file, imported from the user's library. The designer creates their drumming pattern by activating buttons on the sequencer, creating repetitive patterns of stimulation in which each actuator/position per-forms a sequence of an instrument (see fig. 7). The patterns can be saved as presets.

Trial Feedback

Participants described using the Drummer tool as a fun experience: the interface is simple and user-friendly, patterns can be quickly designed, and the ability to assign different instruments to the different points of stimulation is unique to this tool. Participants tended to move around while testing it, in sync with the rhythm. We heard several remarks about enhanced awareness of one's own body, and the presence of the wearable device. P.02 said, "I feel like the different instruments are playing directly on my body, or like I am the instrument!" She added that, since she heard the different sounds at the same time as she felt the stimuli on the different positions, the sound seemed to be coming directly from those points. This comment suggests that, even though the spatial audio information is lost in extra-tympanic conduction, the localized perception of tactile stimuli can contribute to impressions of auditory source positions. This comment also branched off into a discussion about sensory effects for physical rehabilitation, perhaps to help target the user's attention on a specific part of their body.

Key Questions and Limitations

In the Drummer interface, the opportunities to create musical patterns remain basic: the designer can select the output channels, choose sound files, alter the tempo and number of columns, but cannot change relative signal intensity according to point of stimulation. The interface functionalities could be enriched with multichannel or MIDI sequences, an equalized distribution of the signals so that their intensities can be tailored to each point of stimulation, or a more dynamic variation of each track's intensity. One could also imagine guiding the user's movements by distributing different sensations to different parts of the body. However, body movements and changes in posture interfere with audio-haptic perception. The pressure of the modules against the body changes based on the way the user stretches their joints and limbs (see fig. 8). For example, when the user bends downwards, or stretches their arms in front of them, they pull the modules closer against their spine. The added pressure on the points of stimulation, along with the difference in the curvature of the spine, facilitates good contact of the modules and thus good transmission of the vibratory signals. However, if the user raises their arms above their head, the upper back arches inwards, creating more distance between the modules and the skin and reducing the vibratory transmission. This aspect of use of the multimodal harness could be seen as a limitation but also as an advantage: though the body's movements cause variations in audio-haptic perception, those variations are part of the physical interaction between the user and the wearable, and could be a source of perceptual cues for movement-related use cases. Modifying accents, rhythm or instrument positions according to the user's movements would be interesting to explore, but this implies the integration of motion and pressure sensors which is currently out of the project's scope.

DISCUSSION

During the two weeks of the workshop, we were able to address each theme of audio-haptic composition (multimodal music, sensory equalization, and signal spatialization). However, we experienced developmental setbacks due to time constraints and a lack of knowledge about user perception. Evidence of these current limitations can be found in several aspects of the interface functionalities: a sensory equalizer function is present in only one of the authoring tools (Inner.Music), the spatialized audio-haptic signals are mono and limited to basic wave types, and we are restrained to one-dimensional position configurations. Although they contain some similar elements, the authoring tools are distinct from one another, and the resulting sensory experiences are limited to each interface's distinct functionalities.

These constraints are temporary: we aim to streamline and optimize our authoring tools. For example, in future iterations, features could be consolidated into one interface: the sensory equalizer in Inner.Music, the curves in Mp2p for modulation of position, frequency and amplitude, and the position-specific sound selector in Drummer. For example, in a unified interface, the designer could use the sensory equalizer to first determine a baseline audiohaptic calibration that suits their perceptual preferences. They could then use Drummer's function to select specific sound files to display at each position, and then use Mp2p's curves for spatialization pattern design to map the signal display at each position, along with more fine-tuned variations in amplitude and frequency across the duration of the composition. Inspired by existing tools for audio spatialization [START_REF] Pottier | Holophon: Projet de spatialisation multisources pour une diffusion multi-hautparleurs[END_REF], we have already begun to optimize the mono-source design constraint of Mp2p in a separate interface (called "MC-Curv"). Instead of one single spatialized source, the designer could edit and spatialize up to four sources on the body at the same time, creating harmonies of movement across the stimulation points instead of single-path sensations.

Regarding the design of the multimodal harness, we hope to improve many aspects in the next iteration: sound transmission quality, comfort, and ease of adjustability. The second iteration of the harness involves changes for both the wearable structure and the module. The actuators will be integrated in an orthogonal position relative to the surface of the body, instead of parallel. Though they will protrude from the surface of the device, this will allow the voice-coil actuator's lengthwise-directed vibrations to penetrate the surface of the body rather than rubbing horizontally across the skin. We hypothesize the change in orientation will increase sound perception in the mid/high frequency range, bringing more clarity to certain instrumentals and vocal sounds, without impacting the quality of tactile stimulation.

CONCLUSIONS

Since there is no generic go-to tool for audio-haptic composition, we faced the same developmental challenges as past composers and researchers: we wanted to create and study multisensory effects, but we didn't have the tools to do so. In [START_REF] Gunther | Cutaneous grooves: Composing for the sense of touch[END_REF], the authors complain of the "awkwardness" of designing the vibrotactile sensory effects using Protools, a standard Digital Audio Workstation. Nine years later, in [START_REF] Geldard | The cutaneous 'rabbit': A perceptual illusion[END_REF], the authors used Premiere Pro to create "vibetracks" for a film, by placing clips of different sine waves one-by-one at precise points along the video track. Today, researchers acknowledge that tactile composition is still "largely unexplored", that composers need to "take into account haptic perceptual effects" and that they develop their own design tools for this purpose [START_REF] Turchet | Touching the audience: musical haptic wearables for augmented and participatory live music performances[END_REF]. Resorting to these painstaking methods drastically slows down the design process, and makes it difficult for progress to happen in the domain.

Another obstacle to overcome is the general lack of guidelines about variations in both auditory and tactile sensitivity across different sites on the body, and across individuals. Established ranges of sensitivity according to each position of stimulation could help provide compositional suggestions, or presets according to each modality. To this end, in an upcoming study we will follow up on our past psychophysical research [START_REF] Richards | Vibratory Detection Thresholds for the Spine, Clavicle and Sternum[END_REF] and evaluate participants' responses regarding both tactile and auditory perception while using the multimodal harness.

The authors call for smoother workflow design for audiohaptic composition, accessible to the research community. We hope that the products of our workshop can foster discussions about what features and guidelines might contribute to an ideal audio-haptic composition interface.

Figure 1 .

 1 Figure 1. Image of the multimodal harness. There are nine actuators integrated on the structure: two on the clavicles, two on the ribs, and five along the spine. The straps are velcro-adjustable, two of which go around the legs to secure the last module against the body. The multichannel audio-haptics card is connected to the harness, situated below the last module on the spine.

Figure 2 .

 2 Figure 2. Schema of the module, which integrates the actuator. The module snaps onto extended fabric so it can "breathe" up and down on its frame (B), attached via the PCB clip (C). The 3D-printed clip-on piece (A) attaches onto the indented edges of the actuator, and comes into contact with the body, transmitting the vibrations to the user.

Figure 3 .

 3 Figure 3. Inner.Music as seen in Max environment. Any sound file can be uploaded into the playlist object at the top of the interface. By default we propose a short list of rhythmic music with powerful bass content: electronic, disco-funk and pop. Left and right channels are routed to the left and right lateral actuators on the clavicles and ribs and mixed together on the vertical positions of the spine.A graphic equalizer and a mute button is available for each actuator, with different colored bars for 7 frequency bands spanning 50-4000 Hz. The user can save their equalizer settings in the grid below the playlist object, recorded and saved for future use. To the right of the preset grid, a high shelf filter object, applied to all channels, attenuates the bass frequencies with respect to the high frequencies due to the resonant frequency of the actuator (70 Hz).

Figure 4 .

 4 Figure 4. Photo of P.01, taken during the workshop. Since he wasn't satisfied with the quality of sound perception, he placed his fingers directly on the modules to enhance his tactile perception.

Figure 5 .

 5 Figure 5. Diagrams of three possible Mp2p spatialization path configurations. On the top of the figure, arrays of colored dots represent the signal path: each different color is a different channel.Below each signal path is a graphic representation of the nine positions on the harness structure: the upper horizontal line is the clavicles, the lower is the ribs, and the vertical line is the spine. In the leftmost configuration, each position has its own independent channel. The middle configuration uses only two channels (light and dark green), with two positions paired to each channel -the five spine points are inactive. The right-most configuration groups the positions among four channels: the clavicles and top spine point (dark green), the second spine point (light green), the middle spine and ribs (cyan) and the lowest spine point (navy).

Figure 6 .

 6 Figure 6. Graphic representation of the Mp2p interface functionality. A sine-wave signal follows a 1D path according to the chosen configuration preset (see fig.5). To the right of the figure is the spatialization design, with three curves for position, frequency and amplitude. By modifying the curve, the designer can fine-tune the signal's duration at each group of positions, and its variations in amplitude and frequency across the composition.

Figure 7 .

 7 Figure 7. The Drummer interface seen in the Max environment. The sequencer tracks each correspond to one actuator position on the harness. Above the sequencer, there is a preset grid for saving specific drumming patterns.

Figure 8 .

 8 Figure 8. Photo taken during the workshop. P.02 bends forward to change her perception of the transmitted vibrations.

A co-development organized with Actronika, industrial and research partner, and Les Vertugadins, a Parisian costume design studio.

Linear panning with Max [mc.mixdown @pancontrolmode 2] object gave the best results.

Acknowledgments

The authors extend a special thanks to Yann Boulet (les Vertugadins) for defining the fine details of the module and the harness structure, and Fanjun Jia (Actronika) for his contributions in 3D design. We also thank Emmanuel Flety from the Pôle Ingénierie et Prototypes (PIP) of the IRCAM STMS Lab, les Vertugadins for their continued collaboration, and Actronika for their essential advice and technical support. Finally, we thank all of our friends and colleagues who tried using the harness. Funding for this research comes from the Association Nationale de la Recherche et de la Technologie (ANRT) and Actronika, in the framework of a CIFRE contract.