isoform-like proteins in marine euryhaline milkfish (Chanos

JOURNAL OF EXPERIMENTAL ZOOLOGY 311A:522–530 (2009)A Journal of Integrative BiologyRelative Changes in the Abundance of BranchialNa 1 /K 1 -ATPase a-Isoform-Like Proteins in MarineEuryhaline Milkfish (Chanos chanos) Acclimatedto Environments of Different SalinitiesCHENG-HAO TANG 1 , YU-HUEI CHIU 1 , SHU-CHUAN TSAI 2 , ANDTSUNG-HAN LEE 11 Department of Life Sciences, National Chung Hsing University,Taichung, Taiwan2 Institute of Life Sciences, Central Taiwan University of Science and Technology,Taichung, TaiwanABSTRACT Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos),the euryhaline teleost, exhibited adaptive changes in branchial Na 1 /K 1 -ATPase (NKA) activity withdifferent Na 1 and K 1 affinities. Since alteration of activity and ion-affinity may be influenced bychanges in different isoforms of NKA a-subunit (i.e., the catalytic subunit), it is, thus, intriguing tocompare the patterns of protein abundance of three major NKA a-isoform-like proteins (i.e., a1, a2,and a3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance ofthree NKA a-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), aswell as hypersaline water (HSW, 60%) were analyzed by immunoblotting. In the acclimationexperiments, the SW group revealed significantly higher levels of NKA a1- and a3-like proteins thanthe FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSWrevealed the abundance of branchial NKA a1-like and a3-like proteins decreased significantly after96 and 12 hr, respectively, and no significant difference was found in NKA a2-like protein.Furthermore, when fish were transferred from SW to FW, the amounts of NKA a1- and a3-likeproteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in theabundance of branchial NKA a1- and a3-like proteins may fulfill the requirements of altering NKAactivity with different Na 1 or K 1 affinity for euryhaline milkfish acclimated to environments ofvarious salinities. J. Exp. Zool. 311A:522–530, 2009. r 2009 Wiley-Liss, Inc.How to cite this article: Tang C-H, Chiu Y-H, Tsai S-C, Lee T-H. 2009. Relative changes inthe abundance of branchial Na 1 /K 1 -ATPase a-isoform-like proteins in marine euryhalinemilkfish (Chanos chanos) acclimated to environments of different salinities. J. Exp. Zool.311A:522–530.Maintaining a stable internal environment isimportant for vertebrates to survive in varioushabitats. Compared to terrestrial animals, fishmust manage more challenging osmotic and ionicgradients from aquatic environments with diversesalinities, ion compositions, and pH values(Hwang and Lee, 2007). Salinity adaptation ineuryhaline teleosts is a complex process involvinga set of physiological responses to environmentswith differing ionoregulatory requirements.Although ionoregulation in fish is mediated by agroup of structures including the intestine andkidney, the gill is the major organ for the balanceof ion movement between gains and losses (Hiroseet al., 2003; Evans et al., 2005). To maintainosmolality and ionic balance, marine teleostsdrink seawater (SW) to compensate for passivewater loss and actively secrete salt via the gills aswell as kidneys. On the contrary, freshwater (FW)teleosts do not drink (or drink very little) water,Grant sponsor: National Science Council of Taiwan NSC 95-2313-B-005-040-MY3. Correspondence to: Tsung-Han Lee, Department of Life Sciences,National Chung Hsing University, 250 Kuo Kuang Road, Taichung402, Taiwan. E-mail: thlee@dragon.nchu.edu.twReceived 24 December 2008; Revised 3 April 2009; Accepted 22April 2009Published online 29 May 2009 in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/jez.547r 2009 WILEY-LISS, INC.

RELATIVE CHANGES IN THE ABUNDANCE OF BRANCHIAL 523produce diluted urine via the kidneys to balancethe passive water gain, and actively absorb saltsthrough the gills from the environment (Marshalland Grosell, 2006). Effective ionoregulatory mechanismsthus enable teleosts to retain an osmoticand ionic constancy in their internal milieus andsurvive in hypertonic or hypotonic environments.Na 1 /K 1 -ATPase (NKA) is a membrane-spanningprotein that couples the exchange of twoextracellular K 1 ions for three intracellular Na 1ions to the hydrolysis of one molecule of ATP (Postand Jolly, ’57). It is crucial not only for sustainingintracellular homeostasis, but also for providing adriving force for many transporting systemsincluding fish gills (Hwang and Lee, 2007). Thea-subunit of NKA containing the binding sites forATP, Na 1 , and K 1 is considered to be the catalyticsubunit. The b-subunit is a type II glycosylatedpolypeptide that is thought to assist in the foldingand placement of the a-subunit into the cellmembrane (Blanco and Mercer, ’98). A third,nonessential g-subunit has been identified inmammals (Reeves et al., ’80) and is thought toplay roles in modulating Na 1 , K 1 , and ATPbinding affinities to the NKA ab complex (Therienet al., ’99). Immunocytochemical studies demonstratedthat NKA was localized mainly in epithelialmitochondrion-rich cells of teleostean gills.The current model depicted that mitochondrionrichcells in gill epithelia of teleosts used NKA asthe primary driving force for operation of thedifferent ion transporters depending on theenvironmental salinity (Hwang and Lee, 2007;Evans, 2008).NKA exhibits four a (a1-4) and three b (b1-3)isoforms. Sequence variations in the amino terminusand 11 amino acids in the large intracellularloop between transmembrane domains 4 and 5distinguished four isoforms of the NKA a-subunit(Takeyasu et al., ’90; Pressley, ’92). Thesea-subunit isoforms, which showed tissue-specificand developmentally dependent patterns of expressionin birds and mammals, have beensuggested to be presented in all vertebrate classes,including teleosts (Takeyasu et al., ’90; Pressley,’92). Comparing the sequences of one orthologousisoform from many different species yields anamino acid identity of 90% or greater (Blanco andMercer, ’98). In teleosts, full length sequence ofNKA a-isoforms have been cloned in the whitesucker (a1; Catostomus commersoni; Schönrocket al., ’91), the European eel (a; Anguilla anguilla;Cutler et al., ’95), tilapia (a1 and a3; Oreochromismossambicus; Feng et al., 2002), killifish (a1 anda2; Fundulus heteroclitus; Semple et al., 2002),and rainbow trout (a1a, a1b, a1c, a2, and a3;Oncorhynchus mykiss; Richards et al., 2003). Inaddition, nine distinct NKA a-subunit genes havebeen identified in zebrafish (Danio rerio) genomicdatabases. Among them, four (a1a.1, a1a.2, a1a.4,and a1a.5) of the seven a1 genes were found to beexpressed in the mucous cells of skin (Blasioleet al., 2002; Canfield et al., 2002). In addition,partial sequences of a-isoforms were reported inthe spiny dogfish (a3; Squalus acanthias; Hansen,’99) and the Antarctic nototheniid (a1, a2, and a3;Trematomus bernachii; Guynn et al., 2002).Milkfish (Chanos chanos) is a marine teleostwidely distributed throughout the tropical andsubtropical Indo-Pacific. The euryhalinity of milkfishwas demonstrated throughout its life history,although it did not appear to require the freshwater (FW) for any part of its life cycle (Bagrinao,’94). Milkfish are found naturally or culturedcommercially in FW, SW, and hypersaline water(HSW) (Crear, ’80). These characteristics makemilkfish a good experimental species for studies onosmoregulation. Previous studies revealed thatmilkfish, like other euryhaline teleosts, exhibitedadaptive changes in branchial NKA responses (i.e.,mRNA, protein, activity, immunoreactive cellnumber) upon salinity challenge (Lin et al., 2003,2006). Moreover, Pagliarani et al. (’91) demonstratedthat NKA proteins isolated from FW- andSW-acclimated fish gills had different biochemicalproperties and suggested that different NKAisoforms might correlate with ion regulation indifferent environmental salinities. Since a-subunitis the catalytic subunit of NKA, alteration of NKAa-isoforms protein expression was supposed to be acrucial mechanism for euryhaline teleosts to adaptto environments of various salinities. Feng et al.(2002) provided evidence of enhanced transcriptionof branchial NKA a1 and a3 genes inMozambique tilapia following transfer from FWto SW. Recent studies focused on mRNA expressionpatterns of two NKA a-isoforms (a1a and a1b)revealed their different responses to salinitychanges in salmonid fishes (Richards et al., 2003;Shrimpton et al., 2005; Bystriansky et al., 2006,2007a,b; Nilsen et al., 2007). However, Morrisonet al. (2006) reported that changes in the protein,but not mRNA levels of NKA a-subunit isoforms(i.e., a1, a2, and a3), occurred in the gills of warmacclimated Antarctic teleost (Trematomus bernacchii).This motivates this study to investigate theprotein expression patterns of gill NKA a-subunitisoforms in euryhaline milkfish following salinity-J. Exp. Zool.

524C.-H. TANG ET AL.transfer, to illustrate the potential role of NKAa-isoforms in this marine euryhaline teleost uponsalinity challenge.MATERIALS AND METHODSExperimental animalsJuvenile milkfish (C. chanos) with 13.670.2 cmtotal length and 22.175.9 g body weight wereobtained from a local fish farm. Values of total lengthand body weight were expressed as the mean7SEM.SW (35%) orHSW(60%) were prepared from localtap water (i.e., FW) added with proper amounts ofsynthetic sea salt (Instant Ocean, Aquarium Systems,Mentor, OH). The water was continuously circulatedthrough fabric-floss filters. Milkfish were kept in SWat 28711C with a daily 12 hr photoperiod for at leasttwo weeks before experiments. Fish were fed a dailydiet of commercial fodder.Acclimation experimentsMilkfish kept in the laboratory for at least twoweeks were transferred into experimental tankswith HSW, SW, and FW, respectively, and theculture conditions were identical to thosedescribed above. Milkfish were held in experimentaltanks for two weeks prior to sampling.Time-course environmentsFor time-course experiments, milkfish of theexperimental group were transferred directly fromstock SW to FW or HSW, while fish in the controlgroup were transferred to SW simultaneously.Because fish were transferred by nets, the controlgroup was used to monitor the effect of handlingfish alone. After transfer, the fish were caught bynetting at 12, 24, 48, 96, and 168 hr and sampledfor immunoblotting.Preparation of gill homogenatesGills were excised and blotted dry. After removingthe gill arches, gills were immediately cut intosmall pieces. All subsequent procedures wereperformed on ice. Gill scrapings were suspendedin the mixture of homogenization solution (100 mMimidazole-HCl buffer, pH 7.6; 5 mM Na 2 EDTA;200 mM sucrose; and 0.1% sodium deoxycholate)and proteinase inhibitors (10 mg antipain, 5 mgleupeptin, and 50 mg benzamidine dissolved in5 mL aprotinin) (v/v: 100/1). Homogenization wasperformed with a homogenizer (Polytron PT1200E KINEMATICA, Lucerne, Switzerland) at amaximum speed for 25 strokes. The homogenateswere then centrifuged at 13,000g for 20 min at 41C.The supernatants were used to determine proteinconcentrations and for immunoblotting. Proteinconcentrations were identified by BCA proteinassay reagents (PIERCE, Rockford, IL) usingbovine serum albumin (Sigma, St. Louis, MO) asa standard. All gill lysates were stored at 801Cbefore immunoblotting.AntibodiesNKA a1-isoform antibody, mouse monoclonalantibody (a6F) raised against the a1-isoform of theavian NKA was purchased from the DevelopmentalStudies Hybridoma Bank (DSHB; Iowa City,IA). The dilution rate is 1:5,000 for immunoblotting.NKA a2-isoform antibody was the commercialgoat polyclonal antibody (sc-16049), raisedagainst a peptide mapping within an internalregion of the NKA a2-subunit of human origin(Santa Cruz Biotechnology, Santa Cruz, CA). Thedilution rate is 1:500 for immunoblotting. NKAa3-isoform antibody was the commercial goatpolyclonal antibody (sc-16052), raised against apeptide mapping within an internal region of NKAa3 of human origin (Santa Cruz Biotechnology).The dilution rate is 1:100 for immunoblotting.Alkaline phosphatase-conjugated goat anti-mouseor rabbit anti-goat antibodies (Jackson ImmunoResearch, West Grove, PA) were used as secondaryantibodies for immunoblotting.Immunoblotting analysisImmunoblotting procedures were performed accordingto Tang et al. (2008) with little modification.Seventy-five microgram of protein from gillhomogenates was heated with sample buffer at371C for 30 min and fractionated by electrophoresison sodium dodecyl sulfate-containing 7.5% polyacrylamidegels. The prestained protein molecularweight marker was purchased form Fermentas(SM0671; Hanover, MD). Separated proteins werethen electro-ransferred to PVDF membranes(Millipore, Bedford, MA). Blots were preincubatedin PBST (phosphate buffer saline with Tween 20)buffer (137 mmol L 1 NaCl; 3 mmol L 1 KCl;10 mmol L 1 Na 2 HPO 4 ;2mmolL 1 KH 2 PO 4 ;0.2%Tween 20; pH 7.4) containing 5% w/v nonfat driedmilk to minimize nonspecific binding, then incubatedat 41C overnight, with the primary antibodiesdiluted in PBST containing 1% BSA and 0.05%sodium azide. The blots were washed in PBST andincubated at room temperature for 1 hr withJ. Exp. Zool.

RELATIVE CHANGES IN THE ABUNDANCE OF BRANCHIAL 525secondary antibodies. Blots were developed afterincubation with BCIP/NBT kit (Zymed, South SanFrancisco, CA). Immunoblots were scanned andimages were imported as TIF files into thecommercial image analysis software package(MCID software; Imaging Research, Ontario,Canada). Results were converted to numericalvalues in order to compare the relative intensitiesof the immunoreactive bands.Statistical analysisValues of relative intensities of immunoreactivebands were expressed as the mean7SEM andcompared using the one-way analysis of variance(ANOVA) followed by Tukey’s pair-wise methodfor acclimation experiments or Dunnett’s test fortime-course experiments.RESULTSLong-term acclimation of milkfish toenvironments with different salinitiesThe antibodies corresponding to different NKAa-isoforms (a1, a2, and a3) were used to determinethe protein expression patterns in milkfish gills.An approximately 105 kDa NKA a1-like immunoreactiveprotein was detected in gill homogenatespreparations from milkfish acclimated todifferent salinities (Fig. 1A). The intensity ofimmunoreactive bands in the SW-acclimatedgroup was about 4-fold higher than the FWacclimatedfish or HSW-acclimated fish (Fig. 1B).Immunoblots of NKA a2-like protein resulted in asingle immunoreactive band of about 114 kDa(Fig. 2A). The immunoreactive bands were similaramong all groups. Relative protein abundance inthe immunoreactive bands revealed the proteinabundance of NKA a2-like protein in the gills wereunaffected by environmental salinity (Fig. 2B).Immunoblots of NKA a3-like protein also resultedin a single immunoreactive band of about 105 kDa(Fig. 3A). Similar to the pattern of a1-like protein,milkfish exposed to SW environment exhibiteda significant increase (about 1.5-folds) in NKAa3-like protein expression compared with individualsexposed to FW and HSW (Fig. 3B).Abrupt exposure of milkfish from SW toHSWLike the experiment of long-term acclimation, a105 kDa NKA a1-like immunoreactive protein wasdetected in the gill of milkfish (Fig. 4A). Comparedto the 0 hr group, the levels of NKA a1-like proteinFig. 1. The protein expression of Na 1 /K 1 -ATPase a1-likeprotein in the gills of milkfish acclimated to environments ofdifferent salinities for two weeks. (A) Representative immunoblotprobed with a monoclonal antibody. The indicatedimmunoreactive band is approximately at 105 kDa (arrow).The immunoreactive bands of SW-acclimated fish were moreintensive than FW- or HSW-acclimated individuals. (B) Therelative protein abundance of immunoreactive bands of theSW group was 4-fold higher than those of FW or HSW groups.Values were mean7SEM (n 5 6). The asterisk indicated asignificant difference (Po0.05) using Tukey’s pair-wisecomparison test following a one-way ANOVA. M, marker;FW, fresh water; SW, seawater; HSW, hypersaline water.in gills declined gradually within 48 hr posttransferand were decreased 4-fold at 96 hrand sustained to 168 hr after salinity transfer (Fig.4B). The representative immunoblot of NKA a2-likeprotein resulted in a single immunoreactive band ofabout 114 kDa (Fig. 5A). Compared to the 0 hrgroup, the abundance of branchial NKAa2-like protein was not significantly differentfollowing salinity transfer (Fig. 5B). Moreover,NKA a3-like immunoreactive proteins were detectedin gill protein preparations from milkfish.The NKA a3-like protein was approximately105kDa (Fig. 6A). Compared to the 0hr group,protein levels of gill NKA a3-like protein decreasedsignificantly since 12 hr post-transfer (Fig. 6B). Nosignificant difference was found in the levels of threeNKA a-isoform-like proteins during the time-coursein the control group fish (Figs. 4B, 5B, and 6B).J. Exp. Zool.

RELATIVE CHANGES IN THE ABUNDANCE OF BRANCHIAL 527Fig. 4. The summary of Na 1 /K 1 -ATPase (NKA) a1-likeprotein in the gills of milkfish sampled at a series of differenttimes after transfer from SW to HSW or FW. (A) Representativeimmunoblots of NKA a1-like protein probed with amonoclonal antibody revealed single immunoreactive bands atapproximately 105 kDa. (B) The relative protein abundancefollowing transfer from SW to HSW indicated that NKAa1-like protein levels declined gradually during the first 48 hrpost-transfer and decreased 4-fold at 96 and 168 hr aftertransfer. When fish were transferred from SW to FW, NKAa1-like protein levels decreased during the first 12 hr posttransfer,then increased gradually until 48 hr post-transfer,and finally decreased 3-fold at 96 and 168 hr post-transfer.Values were mean7SEM (n 5 6). The asterisks indicatedsignificant differences (Po0.05) compared with the 0 hr usingDunnett’s test following a one-way ANOVA. M, marker; SW,seawater; HSW, hypersaline water.in both absorptive (in FW) and secretory (in SW)modes (Marshall, 2002; Hwang and Lee, 2007).The branchial NKA expression (mRNA, protein,and activity) were higher in milkfish acclimated toFW (Lin et al., 2003, 2006) and HSW (unpublisheddata) than SW-acclimated individuals. This indicatesthat this marine species maintains a comparativelylow level of NKA expression in theirprimary natural habitats. Similarly, some euryhalineteleosts responded to FW with higher gillNKA expression, e.g., European seabass (Dicentrarchuslabrax; Lasserre, ’71), mullets (Mugilcephalus; Ciccotti et al., ’94), gilthead Sea Bream(Sparus auratusand; Laiz-Carrión et al., 2005),black porgy (Acanthopagrus schlegeli; Choi andAn, 2008). On the contrary, the other euryhalineteleosts responded with higher gill NKA expressionfollowing acclimation to SW, e.g., Atlanticsalmon (Salmo salar; D’Cotta et al., 2000), tilapiaFig. 5. The summary of Na 1 /K 1 -ATPase (NKA) a2-likeprotein in gills of milkfish sampled at a series of differenttimes after transfer from SW to HSW or FW. (A) Representativeimmunoblots of NKA a2-like protein probed with apolyclonal antibody revealed single immunoreactive bands atapproximately 114 kDa. (B) The relative protein abundancefollowing transfer from SW to HSW or FW indicated that NKAa2-like protein expression was not significantly differentfollowing salinity transfer compared to the 0 hr. Values weremean7SEM (n 5 6). The asterisks indicated significantdifferences (Po0.05) compared with the 0 hr using Dunnett’stest following a one-way ANOVA. M, marker; SW, seawater;HSW, hypersaline water.(Oreochromis mossambicus; Lee et al., 2003), andspotted green pufferfish (Teteaodon nigroviridis;Lin et al., 2004). Different patterns of salinitydependentbranchial NKA expression may resultfrom diverse primary natural habitats of euryhalineteleosts with differing mechanisms to respondto ambient salinity changes. Furthermore, branchialNKA proteins isolated from FW- and SWacclimatedrainbow trout (Oncorhynchus mykiss)exhibited different biochemical properties. It wassuggested that diverse NKA isoforms might beinvolved in ionoregulation of environments withdifferent salinities (Pagliarani et al., ’91).Characterization of rat NKA a- and b-isoformsin insect cell lines demonstrated that Na 1 and K 1affinity varied among NKA isoform combinationswith a rank order of a2b14a1b14a3b1 anda1b14a2b14a3b1, respectively (Blanco andMercer, ’98). The biochemical or kinetic propertiesof NKA were thus strongly influenced by differentisoforms of catalytic a-subunit to match physiologicaldemands. Different affinities of gill NKAfor Na 1 and K 1 were reported in FW- andJ. Exp. Zool.

528C.-H. TANG ET AL.Fig. 6. The summary of Na 1 /K 1 -ATPase (NKA) a3-isoformin gills of milkfish sampled at a series of different timesafter transfer from SW to HSW or FW. (A) Representativeimmunoblots of NKA a3-isoform probed with a polyclonalantibody revealed single immunoreactive bands at approximately105 kDa. (B) The relative protein abundance followingtransfer from SW to HSW indicated that NKA a3-isoformdecreased significantly after 12 hr post-transfer. On the otherhand, following transfer from SW to FW, changes in therelative protein abundance of NKA a3-isoform levels declinedgradually during 48 hr post-transfer and significantly decreasedat 96 and 168 hr post-transfer. Values were mean7SEM (n 5 6). The asterisks indicated significant differences(Po0.05) compared with the 0 hr using Dunnett’s testfollowing a one-way ANOVA. M, marker; SW, seawater;HSW, hypersaline water.SW-acclimated milkfish (Lin and Lee, 2005).Therefore, alteration of NKA activity in gills offish following salinity changes may result fromdifferent NKA isoforms.There is accumulating evidence to presume thatNKA a-isoforms may change in teleosts to copewith the environmental fluctuations, e.g., salinityor temperature. The mRNA expression patterns ofgill NKA a1a and a1b examined in varioussalmonid species were found to express differentiallyin FW- and SW-acclimated individuals(Richards et al., 2003; Bystriansky et al., 2006,2007a,b; Nilsen et al., 2007). Changes in mRNAexpression are often assumed to parallel changes inprotein abundance. The lack of correlationbetween mRNA and protein of branchial NKAa-isoforms was reported (Morrison et al., 2006). Thislack of correlation between mRNA and proteinsuggested the correlation with posttranscriptionalregulation (Scott et al., 2004). This study,therefore, investigates the effect of environmentalsalinity on the protein expression of branchialNKA a-isoforms-like proteins in milkfish.The amino acid identities of individual isoformsacross species are higher than the identities ofdifferent isoforms within one species (Blanco andMercer, 1998). Therefore, the heterologous antibodiesto NKA a-isoforms (polyclonal antibodiesdirected against the rat a-specific isoforms) wereused in Atlantic salmon (Salmo salar) (D’Cottaet al., 2000) as well as in this study. Among theisoform-specific heterologous antibodies used inmilkfish, mouse monoclonal antibody a6F to theavian NKA a1-isoform and goat polyclonal antibodysc-16052 to human NKA a3-isoform had beenapplied in teleosts previously (tilapia, Oreochromismossambicus; Lee et al., 1998; Antarctic fish,Trematomus bernacchii; Brauer et al., 2005). Thisstudy identified three NKA a-isoform-like proteinsin milkfish gills with these isoform-specific antibodiesby immunoblotting analysis (Figs. 1A, 2A,and 3A). Three NKA a-isoform-like proteins werealso found in the gills of Antarctic fish (Guynnet al., 2002), while only a1 anda3 werereportedintilapia gills (Lee et al., 1998; Feng et al., 2002). Theimmunoblots of milkfish gill lysates probed witheach isoform-specific antibody revealed single immunoreactivebands at approximately 105–115 kDa(Figs. 1A, 2A, and 3A), similar to those recognizedin tilapia (Lee et al., 1998) and Antarctic fish(Guynn et al., 2002; Brauer et al., 2005).This study revealed that the SW-acclimatedgroup was significantly higher than the FW- orHSW-acclimated groups in the levels of NKAa1- and a3-like proteins (Figs. 1 and 3). Expressionpattern of NKA a2-like protein in the gills were notsalinity-dependent (Fig. 2B). It was suggested thata1- and a3-like proteins might play the role forosmo- or ionoregulation but a2-like protein probablyperformed the other physiological function.The affinity of gill NKA for sodium increasedsignificantly in warm-acclimated Antarctic teleost(T. bernacchii; Morrison et al., 2006) that isopposite to milkfish, which has lower sodiumaffinity in SW than FW (Lin and Lee, 2005).Upregulation of gill a1- and a2-like proteinsinfluenced sodium affinities and fulfilled some ofthe requirements for the subtly moderated enzymebehaviors (e.g., elevated NKA activity) in warmacclimatedT. bernacchii (Morrison et al., 2006),while the changes in gill NKA a1- and a3-likeproteins may play the potential role for reductionof sodium affinities to decrease NKA activity inSW-exposed milkfish. In addition, the role of NKAJ. Exp. Zool.

KRITJA IN ZAVAROVANJANasprotne stranke imajo vzpostavljene postopke obvladovanja tveganja, za katere je potrebnapravočasna, natančna in ustrezno ločena izmenjava zavarovanja v zvezi s pogodbami oizvedenih finančnih instrumentih OTC, ki se sklenejo 16. avgusta 2012 ali pozneje (velja zaFNS) oziroma na datum prekoračitve praga kliringa ali po njem (velja za NNS+).FNS razpolagajo z ustreznim in sorazmernim deležem kapitala za upravljanje tveganja, kiga ustrezna izmenjava zavarovanja s premoženjem ne krije.Tehnični standardi v zvezi s kritji in zavarovanji so v obravnavi in še niso bili sprejeti.POROČANJE PRISTOJNIM ORGANOMFNS so dolžne pristojnemu organu poročati:- število nepotrjenih transakcij z IFI OTC, ki so odprte več kot pet delovnih dni, insicer mesečno (četrti odstavek 12. člena RTS 149/2013);- o vseh sporih med nasprotnimi strankami, ki se nanašajo na pogodbe o IFI OTC,njeno vrednotenje ali izmenjavo zavarovanja, za znesek ali vrednost, ki presega 15milijonov EUR, in trajajo najmanj 15 delovnih dni (drugi odstavek 15. člena RTS149/2013).Elektronski naslov za poročanje Agencijiemir@atvp.siv pripravi je poročanje preko elektronskega sistema za poročanje (NRS)IZJEMEUpravičencipokojninskeshemetransakcijeznotrajskupineIzjema od obveznosti kliringa(izjema do 15.8.2015)Izjema od obveznostiuporabe tehnik zmanjševanjatveganja(na podlagi pozitivne odločbeoziroma če pristojni organ neugovarja)Izjema od obveznostiporočanjaIzjeme, ki jih določa Uredba EMIR, veljajo za pokojninske sheme in transakcije znotrajskupine, in sicer v zvezi z obveznostjo kliringa in obveznostjo uporabe tehnik zmanjševanjatveganja. V zvezi z obveznostjo poročanja Uredba EMIR ne določa nikakršnih izjem;obveznost poročanja repozitorijem sklenjenih poslov velja za vse subjekte, ki sklepajopogodbe z IFI (tako na organiziranem kot izven organiziranega trga).stran 9/12