The amphibious fish Kryptolebias marmoratus uses different strategies to maintain oxygen delivery during aquatic hypoxia and air exposure

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1 214. Pulished y The ompny of iologists Ltd The Journl of Experimentl iology (214) 217, doi:1.1242/je.1161 RESERH RTILE The mphiious fish Kryptoleis mrmortus uses different strtegies to mintin oxygen delivery during qutic hypoxi nd ir exposure ndy J. Turko*, yleih E. Roertson, Kristin inchini, Megn Freemn nd Ptrici. Wright STRT Despite the undnce of oxygen in tmospheric ir reltive to wter, the initil loss of respirtory surfce re nd ccumultion of cron dioxide in the lood of mphiious fishes during emersion my result in hypoxemi. Given tht the ility to respond to low oxygen conditions predtes the verterte invsion of lnd, we hypothesized tht mphiious fishes mintin O 2 uptke nd trnsport while emersed y mounting co-opted hypoxi response. We cclimted the mphiious fish Kryptoleis mrmortus, which re le to remin ctive for weeks in oth ir nd wter, for 7 dys to normoxic rckish wter (15, ~21kP O 2 ; control), qutic hypoxi (~3.6kP), normoxic ir (~21 kp) or eril hypoxi (~13.6kP). ngiogenesis in the skin nd ucco-operculr chmer ws pronounced in ir- versus wter-cclimted fish, ut not in response to hypoxi. qutic hypoxi incresed the O 2 -crrying cpcity of lood vi lrge (4%) increse in red lood cell density nd smll increse in the ffinity of hemogloin for O 2 (P 5 decresed 11%). In contrst, ir exposure incresed the hemogloin O 2 ffinity (decresed P 5 ) y 25% without ffecting the numer of red lood cells. cclimtion to eril hypoxi oth incresed the O 2 -crrying cpcity nd decresed the hemogloin O 2 ffinity. These results suggest tht O 2 trnsport is regulted oth y O 2 vilility nd lso, independently, y ir exposure. The ility of the hemtologicl system to respond to ir exposure independent of O 2 vilility my llow extnt mphiious fishes, nd my lso hve llowed primitive tetrpods to cope with the complex chllenges of eril respirtion during the invsion of lnd. KEY WORDS: Hemogloin, Oxygen-crrying cpcity, Hemogloin oxygen ffinity, ir-rething orgn, ir-rething fish, Mngrove rivulus INTRODUTION The trnsition from qutic to terrestril life represents mjor step in verterte evolution ecuse the physicl conditions etween these environments re drmticlly different. Oxygen soluility is reltively low in wter nd even well-oxygented qutic environments contin only out 3% of the O 2 ville in tmospheric ir (Dejours, 1988). Low concentrtions of qutic O 2, prticulrly in hypoxic hitts, hve often een hypothesized to e one of the driving forces ehind the evolution of mphiious or terrestril life histories ecuse invsion of lnd would llow nimls to exploit the O 2 -rich eril environment (Grhm, 1997). Deprtment of Integrtive iology, University of Guelph, Guelph, ON N1G 2W1, nd. *uthor for correspondence (turko@uoguelph.c) Received 4 July 214; ccepted 15 Septemer 214 Tking dvntge of eril O 2 presents severl chllenges for fishes. -rething fishes exchnge respirtory gses cross the gills ut during emersion the gill lmelle typiclly collpse nd colesce, reducing the surfce re ville for respirtion. ccumultion of O 2 in the lood of emersed fishes, resulting from the low soluility of O 2 in ir versus wter, cn lso impir the ility of hemogloin (H) to ind nd trnsport O 2 (Rhn, 1966; Ultsch, 1987). High lood prtil pressure of O 2 (P O 2 ) my reduce intrerythrocytic ph nd reduce the ffinity of H for O 2 y the ohr effect, which prevents O 2 loding t the gs-exchnge surfce (ohr et l., 194). Impired O 2 trnsport cn lso occur vi the Root effect: phenomenon found only in fishes, which prevents H from ecoming fully O 2 sturted t high P O 2 (Root nd Irving, 1941; Gillen nd Riggs, 1971). It hs een hypothesized tht welldpted ir-rething nd mphiious fishes should possess reltively ph-insensitive H to reduce the consequences of ohr nd Root effects on O 2 trnsport (Grhm, 1997; Shrtu nd runer, 214); however, not ll ir-rething fishes follow this pttern (e.g. Frmer et l., 1979; Wells et l., 1997). Thus, despite the fct tht mphiious fishes re surrounded y O 2 -rich ir during emersion, high lood P O 2 coupled with reduced respirtory surfce re my cuse mphiious fishes to ecome hypoxemic until physiologicl nd morphologicl djustments to the O 2 trnsport system cn e mde. Fishes cn increse their cpcity for O 2 uptke, inding nd trnsport in response to hypoxi or hypoxemi. For exmple, lmellr perfusion cn e incresed during exposure to qutic hypoxi to increse the surfce re ville for O 2 uptke (ooth, 1979; Soivio nd Tuurl, 1981). Fish dpted or cclimted to hypoxi often hve lood with high O 2 -crrying cpcity nd possess H with reltively high O 2 -inding ffinity (Weer nd Fgo, 24; runer nd Vl, 26; Wells, 29). ir-rething nd mphiious fishes similrly tend to possess lood with high O 2 - crrying cpcity, which hs een hypothesized to compenste for the impired O 2 uptke nd trnsport cused y elevted lood P O 2 during emersion (Frmer, 1979; Frmer et l., 1979; Grhm, 1997). During the evolution of mphiious lifestyles y erly tetrpods or extnt mphiious fishes, the hypoxi response could hve een used to mintin O 2 trnsport in the fce of high P O 2 during emersion. The ility to sense nd respond to hypoxi likely predtes the invsion of lnd y nimls, ecuse even the sl enorhditis elegns is known to ccumulte the trnscription fctor hypoxi inducile fctor-1 during hypoxic exposure (Rytkӧnen et l., 211). fter 24h of emersion the mphiious fish Gillichthys mirilis lters gene expression in similr pttern to fish tht experienced qutic hypoxi, suggesting tht similr strtegies my e used to mintin metolism (Grcey, 28). However, it is unknown whether these trnscriptionl chnges result in ltered protein synthesis or hve functionl consequences. The The Journl of Experimentl iology 3988

2 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 response of O 2 -trnsport systems in mphiious fishes tht spend longer periods (dys to weeks) out of wter is similrly not well understood. Given the reduced cpcity for trnsport of lood O 2 s result of high P O 2 nd the loss of respirtory surfce re from the gills during emersion, we hypothesized tht mphiious fishes mintin O 2 uptke nd trnsport while out of wter y co-opting their hypoxi response. This hypothesis predicts tht mphiious fishes cclimted to either qutic hypoxi or terrestril environment would use conserved suite of phenotypic chnges to increse the O 2 -crrying cpcity, the ffinity of H for O 2 nd the flow of lood to extrrnchil sites of gs exchnge. lterntively, mphiious fishes my use different responses to tolerte qutic hypoxi nd ir exposure. The mngrove rivulus Kryptoleis mrmortus (Poey 188) is n idel model for studying the dpttions used y mphiious fishes to mintin O 2 uptke nd trnsport. In the wild, mngrove rivulus typiclly inhit extremely hypoxic (P O 2 <2.5kP) puddles nd cr urrows filled with wter, ut these fish lso frequently emerse nd find refuge in terrestril hitts such s dmp lef litter or inside rotting logs tht my lso e hypoxic (Tylor et l., 28, Ellison et l., 212; Tylor, 212). Emersions cn lst upwrds of 2months (Tylor, 199), during which time mngrove rivulus remin ctive nd re thought to otin O 2 y cutneous respirtion (Grizzle nd Thiygrjh, 1987; ooper et l., 212; Wright, 212). To test the hypothesis tht ir-exposed fish mount hypoxi response, we exposed K. mrmortus to 7 dys of qutic normoxi, qutic hypoxi (P O 2 ~3.6kP), eril normoxi or eril hypoxi (P O 2 ~13.6kP). We mesured the O 2-crrying cpcity of lood (numer of red lood cells, hemtocrit nd [H]), H O 2 ffinity nd extr-rnchil ngiogenesis, predicting tht similr phenotypes should occur in fish exposed to qutic hypoxi nd ir exposure. RESULTS lood nlysis oth hypoxi nd ir exposure significntly incresed lood [H] (two-wy NOV: oxygen, P<.1; medium, P<.5; interction, P=.92; Fig. 1). Hemtocrit ws significntly incresed in ll three tretment groups reltive to normoxic control fish (two-wy NOV: oxygen, P<.1; medium, P=.23; interction, P<.5; Fig. 1). Hypoxi significntly incresed the numer of red lood cells (Rs), ut ir exposure hd no effect (two-wy NOV: oxygen, P<.1; medium, P=.46; interction, P=.52; Fig. 1). s result of the smll lood volumes used in this study, repeted smpling ws impossile nd therefore men corpusculr volume (MV), men corpusculr hemogloin (MH) nd men corpusculr hemogloin concentrtion (MH) could only e clculted from the men vlues for ech tretment group, precluding sttisticl nlysis. None of the vlues vried drmticlly etween tretment groups (Tle 1). ir exposure significntly decresed P 5, the lood P O 2 t which H is 5% sturted with O 2 nd P 5 ws incresed y elevted in vitro O 2, ut there ws no overll effect of oxygen vilility (three-wy NOV: medium, P<.1; O 2, P<.1; O 2, P=.6; Fig. 2). significnt interction etween respirtory medium nd O 2 vilility ws found (P<.1, ll other interctions P>.5) nd post hoc tests reveled tht ir exposure significntly incresed H O 2 ffinity (reduced P 5 ) to greter extent in normoxic reltive to eril hypoxic conditions (Fig. 2). The Root effect ws significntly lrger etween.3kp nd 4.kP P O 2 thn etween.3kp nd.6kp P O 2, ut ws not influenced y ny tretment condition (three-wy NOV: medium, P=.21; O 2, P=.6; O 2, lood [H] (g dl 1 ) Hemtocrit (%) Red lood cells (1 6 ) mm (15) (14) (16) (9) Normoxi * ir Hypoxi P<.5; ll interctions, P>.5; Tle 1). H O 2 inding coopertivity (Hill coefficient, n H ) ws significntly incresed y hypoxi nd high P O 2, ut not ir exposure (three-wy NOV: medium, P=.52; O 2, P<.1; O 2, P<.1; ll interctions, P>.5; Tle 1). ngiogenesis nd eril ventiltion Under conditions of qutic normoxi (control), the mrker for ngiogenesis D31 ws visile in the dorsl nd ventrl cutneous epithelium, s well s within the uccl nd operculr chmers * ir (7) (8) (5) (5) Normoxi ir Hypoxi ir (9) (9) (7) (6) Normoxi ir Hypoxi Fig. 1. qutic hypoxi nd ir exposure cuses chnges in the lood of Kryptoleis mrmortus. () H protein concentrtion, () hemtocrit nd () red lood cell counts. Kryptoleis mrmortus were cclimted for 7 dys to one of four tretment groups: normoxic wter, normoxic ir, hypoxic wter or hypoxic ir. Hypoxi in wter (P O 2 =3.59±.6kP) nd ir (P O 2 =13.57±.6kP) ws chieved y mixing N 2 gs with tmospheric ir. Different lower cse letters indicte significnt overll differences etween normoxi nd hypoxi (two-wy NOV, P<.5). Different upper cse letters indicte significnt differences etween the four tretment groups if significnt interction etween oxygen vilility nd respirtory medium ws found (two-wy NOV, P<.5). sterisks denote significnt overll difference etween fish in wter versus ir (two-wy NOV, P<.5). Smple sizes re given in prentheses. Error rs represent s.e.m. ir The Journl of Experimentl iology 3989

3 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 Tle1. Hemtologicl properties of Kryptoleis mrmortus cclimted for 7dys to different O 2 conditions Tretment qutic normoxi eril normoxi qutic hypoxi eril hypoxi Root effect (%).3kP P O2 shift 7.7±3.72 (9) 11.41±3.66 (9) 4.96±2.72 (1) 6.55±1.69 (8) 3.7kP P O2 shift 14.18±3.9 (9)* 19.54±6.92 (9)* 9.78±4.6 (1)* 7.82±3.61 (8)* Hill coefficient.3kp O ±.3 (9) 1.35±.3 (9) 1.44±.7 (1) 1.48±.9 (8).6kP O ±.6 (9) 1.45±.6 (9) 1.4±.9 (1) 1.5±.5 (8) 4.kP O ±.7 (9), * 1.45±.7 (9), * 1.49±.5 (1), * 1.7±.17 (8), * Men corpusculr volume (fl) 59.7± ± ± ±5.47 Men corpusculr hemogloin (pgcell 1 ) 114.6± ± ± ±21.72 Men corpusculr hemogloin concentrtion (gdl 1 ) 19.4± ± ± ±3.95 Hypoxi in wter (P O2 =3.59±.6kP) nd ir (P O2 =13.57±.6kP) ws chieved y mixing N 2 gs with tmospheric ir. Vlues re expressed s mens ± s.e.m. Smple sizes re given in prentheses. Superscript letters within row represent significnt overll differences (P<.5) etween normoxi nd hypoxi. sterisks denote significnt overll difference (P<.5) from ll other O 2 concentrtions. (Fig. 3). fter 7 dys of ir exposure, there ws striking increse in fluorescence of the uccl nd operculr chmers (Fig. 3). cross ll four ody regions, D31 fluorescent intensity ws significntly incresed fter ir exposure, independent of O 2 vilility (three-wy NOV: medium, P<.1; O 2, P=.94; ll interctions, P>.5; Fig. 3). Significntly reduced fluorescence ws oserved in the ventrl epithelium compred with the other ody regions mesured (three-wy NOV: region, P<.1; Fig. 3). We oserved ir-exposed mngrove rivulus opening their mouths nd gulping ir when out of wter. Rtes of eril ventiltion fter 1 h of ir-rething were more thn doule the rtes oserved fter 1 dy or 7 dys of ir exposure (P<.5; Fig. 4). The ctivity of ir-exposed fish (ody movementsh 1 ) did not significntly chnge over the 7dy ir-exposure period, lthough there ws trend towrds decresed movement fter 1dy (P=.6; Fig. 4). The mjority of eril ventiltions occurred within 3s of ody movement, lthough some ventiltions were seprted from ody movements y more thn 1min (Fig. 4). DISUSSION Our results do not support the hypothesis tht mphiious fishes mintin O 2 uptke nd trnsport while out of wter y co-opting the hypoxi response. Insted, we found tht K. mrmortus hve flexile O 2 uptke nd delivery strtegies tht respond to chnges in the respirtory environment. ehviorl nd immunohistochemicl dt suggest tht, during ir exposure, the mouth nd uccooperculr chmer provide supplementry gs exchnge surfce to the cutneous epithelium. The hemtologicl system lso displys flexiility in response to chnges in the respirtory environment. In contrst to our predictions, different responses were oserved in fish exposed to ir nd hypoxi. ir exposure incresed H O 2 ffinity nd incresed the concentrtion of H in the lood without incresing the numer of circulting Rs. In contrst, hypoxi in oth qutic nd eril environments sustntilly incresed the O 2 - crrying cpcity of the lood vi n increse in the density of Rs, without ny mjor effect on H O 2 ffinity. These results indicte tht the chnges in ngiogenesis nd inding ffinity of H for O 2 oserved during ir exposure re not the result of ltered O 2 vilility, ut insted my e controlled y secondry pthwy tht cn e ctivted concurrently with the hypoxi-ctivted pthwy in the cse of eril hypoxi. ir-exposed mngrove rivulus might, for exmple, respond to chnges in lood O 2 or ph rther thn O 2 (Putnm et l., 24; sey et l., 21; Tresguerres et l., 21). If the inding ffinity of H for O 2 reflects lnce etween O 2 uptke nd delivery, the undnce of O 2 in tmospheric ir compred with wter is predicted to result in reltively lower H O 2 P 5 (Torr) P O2 =.3 kp P O2 =.6 kp P O2 =4. kp (9) (9) (1) (13) (9) (9) (9) (8) (9) (9) (8) (1) ir ir ir ir ir ir Normoxi Hypoxi Normoxi Hypoxi Normoxi Hypoxi Fig. 2. ir exposure increses the ffinity of hemogloin for O 2 in Kryptoleis mrmortus. Fish were cclimted for 7 dys to one of four tretment groups: normoxic wter, normoxic ir, hypoxic wter or hypoxic ir. Hypoxi in wter (P O 2 =3.59±.6kP) nd ir (P O2 =13.57±.6kP) ws chieved y mixing N 2 gs with tmospheric ir. Different upper cse letters indicte significnt overll differences in P 5 (i.e. cross ll concentrtions of O 2, not within one P O 2 ) etween the four tretment groups when significnt interction etween oxygen vilility nd respirtory medium ws found (three-wy NOV, P<.5). P 5 is presented in Torr; 1Torr=133P. There ws significnt overll difference in P 5 etween ech concentrtion of O 2 used (three-wy NOV, P<.5), ut there ws no significnt interction etween O 2 nd oxygen vilility (P=.71) or etween O 2 nd respirtory medium (P=.82). Smple sizes re given in prentheses. Error rs represent s.e.m. The Journl of Experimentl iology 399

4 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 Fluorescent intensity (.u.) Mouth Dorsl epithelium rnchil cvity Ventrl epithelium Mouth Dorsl epithelium rnchil cvity Ventrl epithelium 1 mm * * * * (4) (4) (4) (4) (5) (5) (5) (5) (5) (5) (4) (4) (5) (4) (5) (5) ir ir Normoxi Hypoxi Fig. 3. ir exposure induces ngiogenesis in the cutneous epithelium nd ucco-operculr chmer of Kryptoleis mrmortus. Representtive composite imges re presented of immunohistochemicl stining for the ngiogenesis mrker D31 (green) in sgittl section through the hed of Kryptoleis mrmortus cclimted to normoxic wter () or ir () for 7 dys. omposite imges were creted from low-mgnifiction ( 4) fluorescent nd differentil interference contrst photomicrogrphs stitched together nd overlid using doe Photoshop. () Quntifiction of D31 fluorescence (ritrry units;.u.) in fish from one of four tretment groups: normoxic wter, hypoxic wter, normoxic ir or hypoxic ir. Different letters indicte significnt overll differences etween fish in wter versus ir (three-wy NOV, P<.5). sterisks denote significnt overll difference (three-wy NOV, P<.5) etween the ventrl epithelium nd ll other ody regions. Error rs represent s.e.m. ffinity in ir-rething nimls. onsistent with this hypothesis, the ir-rething fishes Protopterus nnectens, Polypterus seneglus nd lris lzer ll reduce H O 2 ffinity during the ontogenetic shift from rething in wter to rething in ir (Grhm, 1997). fter 3weeks out of wter, the mphiious nterury mudfish Neochnn urrowsius lso decreses H O 2 ffinity (Wells et l., 1984). onversely, hypoxi-tolernt orgnisms re generlly chrcterized y high H O 2 ffinities to mximize O 2 uptke (Grhm, 1997; Wells, 29). Why do mngrove rivulus contrdict this pttern nd increse ffinity of H for O 2 fter 7 dys of ir exposure? The lungfish P. mphiius increses H O 2 ffinity while ir-rething during estivtion, proly to enle reduced lung tidl volumes while inhiting hypoxic suterrnen environments (Johnsen et l., 1976). In the non-estivting mngrove rivulus, however, there ws no significnt effect of O 2 vilility on H O 2 ffinity, suggesting tht these fish re using different strtegy to estivting lungfish. We propose tht the elevted ffinity of H for O 2 oserved in irexposed mngrove rivulus serves to mintin reltively stle effective P 5 cross respirtory medi y counterlncing the ohr effect in emersed fish resulting from incresed lood P O 2 nd/or decresed lood ph. The P 5 of control (qutic normoxi) mngrove rivulus t P O 2 of.6kp (P 5 =24.7±1.3Torr; 1 Torr=133P) typicl prtil pressure in venous lood of qutic tropicl fishes ws pproximtely equl to tht of lood from ircclimted fish t P O 2 of 4.kP (P 5=24.3±1.5Torr), which is typicl level for emersed mphiious fishes. Thus, H O 2 ffinity my e mintined in K. mrmortus under vriety of environmentl conditions, regrdless of widely vrile lood O 2 levels. Like H in mngrove rivulus, protein function in other nimls is often highly conserved during cclimtion to chnging environmentl conditions (Somero, 1995). For exmple, expression of lterntive troponin isoforms help to mintin crdic function in Oncorhynchus mykiss nd Dnio rerio cclimted to vrying eril reths h 1 Frequency (8) (8) (8) 1 h 1 d 7 d Durtion of ir exposure >6 Movements h 1 1 h ir exposure 1 d ir exposure 7 d ir exposure Time etween reth nd movement (s) P=.6 (8) (8) (8) 1 h 1 d 7 d Durtion of ir exposure Fig. 4. eril ventiltion nd ody movements in Kryptoleis mrmortus exposed to ir. Frequency of eril ventiltion () nd numer of ody movements () ws mesured over 7 dys. Fish (N=8) were cclimted to ir for 1 h, 1 dy or 7 dys. () umultive frequency of eril ventiltions in reltion to whole ody movements in 1 h recording periods fter 1 h, 1 dy nd 7 dys of ir exposure. Significnt differences (one-wy NOV, P<.5) etween time points re indicted y different letters. Smple sizes re given in prentheses. Error rs represent s.e.m. The Journl of Experimentl iology 3991

5 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 tempertures (ldermn et l., 212; Genge et l., 213). onservtion of H O 2 ffinity might llow mngrove rivulus to mintin consistent O 2 delivery in oth ir nd wter without requiring modifictions to O 2 -ccepting tissues such s skeletl muscle (Hoppeler nd Vogt, 21; Frser et l., 26). Fishes re le to modify H O 2 ffinity primrily y modifying the R intrcellulr environment or y chnging the reltive expression of H isoforms with different O 2 -inding ffinities (Grigg, 1969; Mrinsky et l., 199; Jensen, 1991; Vl, 2; Weer, 2; Rutjes et l., 27). Most commonly, ir-rething fishes increse orgnic phosphte concentrtions reltive to [H] during ehviorl or ontogenetic shifts to ir-rething in order to fcilitte O 2 unloding t the tissues (iker, 1984; Grhm, 1997; Vl, 2). onversely, hypoxi-tolernt fish species often mximize O 2 uptke using low rtios of orgnic phosphte to H compred with fishes tht inhit well-oxygented wter (Wells, 29). If ir-exposed mngrove rivulus re le to decrese orgnic phosphte concentrtions to increse H O 2 ffinity, it is puzzling why fish cclimted to hypoxi did not increse ffinity to the sme extent. t P O 2 of.3kp, which is typicl of rteril lood in wter-rething fish, the P 5 of hypoxi-cclimted fish ws 2.2±1.3Torr, compred with 21.±.7 Torr in control fish. When environmentl O 2 is limited there is often trde-off etween optimizing O 2 uptke to Rs nd O 2 unloding t the tissues. Perhps hypoxi-cclimted mngrove rivulus re constrined from incresing H O 2 ffinity y the need to fcilitte O 2 delivery to the tissues nd insted rely on other hypoxi-tolernce strtegies such s incresing the density of red cells in the lood or enlrging gill surfce re (Richrds, 211; Turko et l., 212; ook et l., 213). t high prtil pressures of O 2 there ws trend towrds incresed H O 2 ffinity in hypoxi-cclimted fish; this my result from the significntly incresed [H], which would provide extr phuffering cpcity nd thus lessen the mgnitude of the ohr effect t high P O 2 (Weer, 2; Wells, 29). Oxygen-crrying cpcity ws significntly incresed, vi incresed R density, y hypoxic cclimtion in either wter or ir ut not during ir exposure lone. Incresing the numer of circulting Rs in response to hypoxi to enhnce O 2 trnsport, vi erythropoiesis or splenic relese, is strtegy used y mny fishes (e.g. Tetens nd Lykkeoe, 1981; Murd et l., 199; Soldtov, 1996; Li et l., 26), ut there re exceptions (e.g. McLeod et l., 1978; Routley et l., 22). Incresed O 2 -crrying cpcity llows fish to increse O 2 uptke from the environment without compromising unloding t the tissues nd my therefore e more efficient strtegy for coping with hypoxi thn djusting H O 2 ffinity (runer nd Wng, 1997). It is curious, then, why ir exposure lone incresed H O 2 ffinity ut did not induce incresed R density. Extr Rs my increse lood viscosity nd impede flow through the epiderml cpillry eds used for eril gs exchnge (ldwin nd Wells, 199). Overll, we oserved reltively minor Root shift (12.2±2.1% men reduction in mximum O 2 sturtion in ll tretment groups under normoxic conditions; P O 2 =22kP) cross lrge (3.7kP) O 2 grdient. Neither ir-rething nor hypoxi exposure ffected the mgnitude of this shift, which ws generlly smll compred with other teleosts (Pelster nd Rndll, 1998). This suggests tht mngrove rivulus H is well dpted to mintining ner-mximl O 2 sturtion t high P O 2 during emersion, even immeditely fter leving wter. ngiogenesis ws oserved in the skin nd ucco-operculr region of ir-exposed mngrove rivulus, ut not in fish exposed to qutic hypoxi. Insted of recruiting dditionl sites of gs exchnge to tolerte hypoxi, mngrove rivulus pper to increse their cpcity for rnchil O 2 uptke y incresing gill surfce re y reduction of the inter-lmellr cell mss (Turko et l., 212). During emersion, mphiious fishes often rely on ir-rething orgns such s lungs, uccl pouches or cpillry-rich cutneous surfces (Johnsen, 197; Stchell, 1976; Olson, 1994; Grhm, 1997). For exmple, ucco-phryngel lood vessels in G. mirilis re filled with lood fter 1 2min of emersion (Todd nd Eeling, 1966). Phylogenetic nlyses of mudskippers suggest tht species with n incresed relince on ir-rething possess incresed cpillry density on the tongue nd within the ucco-operculr chmer (Zhng et l., 2; Gonzles et l., 211). We lso oserved, for the first time, gulping ctivity y emersed mngrove rivulus, strongly implicting this region s site of gs exchnge. These eril ventiltions were usully ssocited with ody movements, suggesting tht trnsient increses in O 2 demnd might provide stimulus to refresh the ir in the ucco-operculr chmer. eril ventiltion frequency significntly decresed etween 1 h nd 1 dy of ir exposure nd ws proly correlted with n increse in lood flow to extrrnchil respirtory surfces. ugmented cutneous lood flow increses O 2 uptke nd proly llowed mngrove rivulus to reduce evportive wter loss y decresing eril ventiltion frequency (urggren nd Mwlukom, 1983; urggren nd Molli, 1984). ir exposure lso induces enlrgement of the inter-lmellr cell mss, reducing the effective gill surfce re, which my further limit wter loss during ucco-operculr ventiltion (Ong et l., 27). Oserving ucco-operculr respirtion ws highly unexpected, considering gill surfce re is reduced during ir exposure nd the rnchil region ws presumed to e non-functionl (Ong et l., 27; ooper et l., 212). Insted, eril O 2 uptke in this species ws thought to e chieved entirely cross the cutneous epithelium (Wright, 212). The dorsl surfce of mngrove rivulus hs long een known to e covered with dense network of epithelil cpillries (Grizzle nd Thiygrjh, 1987) nd recent pper from our lortory demonstrted ngiogenesis in the cudl fin of irexposed fish (ooper et l., 212). However, given the smll size (~1 mg) of mngrove rivulus nd the infrequent nture of gulping (1 reth every ~12min), it is not surprising tht this ehviour hs een overlooked, especilly considering tht disturnces typiclly cuse fish in oth ir nd wter to cese ventiltory ctivity (.T., unpulished oservtion). Overll, these results provide the first evidence tht mngrove rivulus hve phenotypiclly flexile respirtory system tht ppers to e independently regulted y oth O 2 vilility nd the respirtory medium. lthough it initilly ppers tht irexposed fish increse the ffinity of H for O 2, in contrst to most other mphiious fishes, we propose tht this chnge represents n effort to mintin stle P 5 in the fce of lrge chnges in lood P O 2 or ph when out of wter. We lso hve shown for the first time tht ir exposure promotes ngiogenesis in the ucco-operculr chmer, which mngrove rivulus ventilte y gulping ir. These results contrdict the hypothesis tht the hypoxi response ws coopted y fishes during the invsion of lnd nd insted suggest tht novel dpttions to the O 2 trnsport system hve evolved. MTERILS ND METHODS Experimentl nimls Kryptoleis mrmortus hermphrodites t lest 6 months of ge (Slc8E strin) (Ttrenkov et l., 21) were otined from the reeding colony mintined in the Hgen qul, University of Guelph. Fish were rered individully in 12ml semi-trnsprent plstic continers (Fisherrnd The Journl of Experimentl iology 3992

6 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 ollection ontiners; Fisher Scientific) under constnt conditions (25, 15 18, ph 8.1, 12h:12h light:drk cycle) (Frick nd Wright, 22). chnges were performed weekly using rtificil sewter (rystl Se Mrinemix; Mrine Enterprises Interntionl, ltimore, M, US) mde with reverse osmosis wter. Fish were fed rtemi nuplii three to four times per week. The experiments in this study were pproved y the University of Guelph niml re ommittee (niml Utiliztion Protocol 1G8). Experimentl fish were cclimted for 7 dys (25 ) to one of four tretment groups: rckish wter (control), ir, qutic hypoxi or eril hypoxi. ll fish were fsted for the durtion of the cclimtion period. ontrol fish were mintined under stndrd rering conditions (~21kP O 2 ). ir-cclimted fish were mintined on moist filter pper (15 rckish wter, ~21kP O 2 ) in plstic rering continers (Ong et l., 27). Hypoxi in oth wter nd ir ws chieved y introducing finely regulted flow of nitrogen into closed chmers contining experimentl fish (Regn et l., 211). Oxygen levels were mesured dily (Hch LDO11 electrode connected to Hch HQ3d meter, Hch ompny, Missisug, ON, nd) nd verged 3.59±.6kP (men ± s.e.m.; here nd throughout) in wter nd 13.57±.6kP in ir over the course of the experiments. The P O 2 chosen for qutic hypoxi hs een previously found to e slightly ove the criticl O 2 tension of mngrove rivulus (Turko et l., 212). The eril O 2 concentrtion ws selected fter preliminry experiments indicted tht more severe eril hypoxi ws lethl, wheres fish cclimted to less severe eril hypoxi (~17kP) did not show ny hemtologicl response tht differed reltive to fish cclimted to normoxic ir. lood nlysis To mesure hemtocrit, hemogloin concentrtion nd red lood cell counts, lood ws collected y cudl severnce using heprinised microhemtocrit tues (VWR, Missisug, ON, nd) (inchini nd Wright, 213). It should e noted tht stress induced y this method of lood smpling my hve cused the cute splenic relese of red lood cells nd thus our mesurements my e consistent overestimtes compred with the in vivo sitution. The red lood cell counts t O 2 -exchnging surfces (e.g. the gills or epithelil cpillries) my lso differ from the ortic lood we gthered (e.g. Klitzmn nd Duling, 1979; Hudetz et l., 1999). However, given the smll size of K. mrmortus, this ws the only prcticl method of lood collection. Hemtocrit (Hct) ws determined y centrifuging smples in seled microhemtocrit tues t 7 r.p.m. for 2min in n Interntionl linicl entrifuge (Model L, Interntionl Equipment, Needhm, M, US). To ccurtely mesure hemtocrit of smll lood volumes, imges were tken under dissecting microscope nd nlyzed using ImgeJ (inchini nd Wright, 213). Hemogloin concentrtion ws quntified using the cynmethemogloin method (lxhll nd Disley, 1973). Rs were counted in stndrd hemocytometer using 1:4 dilution of whole lood: ortlnd s isotonic sline (Wolf, 1963) further diluted 1:1 with.4% Trypn lue solution. The R indices, men R volume (MV), men cellulr H content (MH) nd men cellulr [H] (MH), were clculted from Hct/R count, ([H]/R count) 1 nd [H]/Hct, respectively. H O 2 equilirium curves were determined (25 ) t three different O 2 prtil pressures (.3,.6, 4.kP) using spectrophotometer (P wee 5, L Troe University, Melourne, ustrli) designed to utilize smll lood volumes (Reeves, 198; Henriksson et l., 28; inchini nd Wright, 213). The lower O 2 prtil pressures pproximted resting vlues for rteril (.3kP O 2 ) nd venous (.6kP O 2 ) lood in tropicl fishes (Heisler, 1982; Perry, 1986, Tufts nd Perry, 1998), while the higher P O 2 is typicl of tropicl mphiious fish during ir exposure (Johnsen, 197; Grhm, 1997). lood smples were otined vi cudl severnce nd were immeditely spred thinly etween two gs-permele memrnes (Gld Go-etween Freezer Film, Hort, Tsmni, ustrli) held in plce on circulr smple holder using n O-ring nd inserted into the P wee 5. Oxygen sturtion curves t ech P O 2 were creted y mesuring sornce t 39nm nd 435nm, respectively the isosestic point nd pek sornce of deoxygented H (Iuchi, 1973; Hoxter, 1979), t O 2 prtil pressures etween nd 22kP in 1kP increments. Ech O 2 nd O 2 comintion ws mixed from compressed O 2, O 2 nd N 2 y the P wee 5, humidified nd introduced to the smple within the temperture-controlled spectrophotometer. H-O 2 ffinity (P 5 ), Root effect nd coopertivity (Hill coefficient, n H ) were utomticlly clculted from the sturtion curves y mnufcturer-supplied spredsheet. The lood smpling method used my hve overestimted H O 2 ffinity y cusing the relese of ctecholmines, which ctivte NHE, increse red lood cell ph nd ultimtely reduce P 5 (Nikinm nd outilier, 1995; Reid et l., 1998). lood ws collected s quickly s possile to minimize the effects of ctecholminergic response, ut ecuse of the smll size of K. mrmortus, this ws the only prcticl method of collecting lood. If stress impcted lood prmeters, then the effect would e more or less consistent cross ll tretment groups. We considered mesuring the ph of whole lood t ech P O 2 used for the H O 2 ffinity mesurements to clculte the mgnitude of the ohr effect, ut the smll lood volumes nd tendency of the lood to clot mde this unfesile. ngiogenesis ngiogenesis ws determined y immunohistochemicl stining for the endothelil integrl memrne protein cluster of differentition 31 (D31), lso known s pltelet endothelil cell dhesion molecule (PEM-1) (leld et l., 1991; luk nd McDonld, 28), which hs previously een used to mesure ngiogenesis in Kryptoleis mrmortus (ooper et l., 212). Euthnized fish were fixed in 1% neutrl uffered formlin t 4 for 24h, declcified (Surgipth Declcifier II, Winnipeg, nd) for 1 h t 2 nd stored in 7% ethnol (4 ) until emedding. Gill rches were otined from the left side. Dissected tissues were routinely emedded in prffin, serilly sectioned in 5 μm increments, deprffinised in xylene nd rehydrted with grded ethnol series prior to stining. ll smples were processed t the sme time. Sections were incuted for 1h t room temperture in primry ntiody (1:4 Rt nti-mouse PEM/D31:PS; D Phrmingen), rinsed riefly with PS nd incuted overnight with lex-fluor-488-lelled secondry ntiody (1:1 got nti-rt IgG:PS; Invitrogen) in PS. Smples were then wshed five times with PS, mounted with Fluoromount (Sigm-ldrich) nd seled with nil polish. ll slides were viewed on the sme fternoon using Nikon epifluorescent microscope (Nikon Eclipse 9i microscope, Nikon, Melville, NY, US) nd imges were cptured ( 4) using identicl cmer settings (NIS Elements, Nikon). The ckground of ech imge ws sutrcted using the rolling ll sutrction tool in ImgeJ (US Ntionl Institutes of Helth, ethesd, MD, US) nd then the sme intensity threshold ws pplied (ooper et l., 212). ox ws then drwn round one of four ody regions (the mouth, rnchil cvity, dorsl epithelium or ventrl epithelium) nd the integrted density ws clculted using ImgeJ. eril ventiltion ontrry to our predictions, the immunohistochemicl dt suggested tht ir exposure incresed lood flow to the ucco-operculr cvity of mngrove rivulus. To confirm tht mngrove rivulus re le to use these surfces for gs exchnge while emersed, we performed study to mesure rtes of eril ventiltion. Mngrove rivulus were cclimted to ir s descried ove. fter 1 h, 1 dy or 7dys of ir exposure, fish were video recorded (Logitech Quickcm Pro, Fremont,, US) under dissecting microscope for 3 min nd ventiltory ctivity (defined s n opening of the mouth nd/or opercul) nd ody movements were recorded. Ech fish ws used only once. Sttisticl nlysis One-wy NOV nd post hoc Holm Sidk tests were used to test whether the durtion of ir exposure influenced ventiltion rte. Two-wy NOV with post hoc Holm Sidk tests were used to determine the overll effects of respirtory medium nd O 2 vilility on [H], Hct nd R counts. Threewy NOVs with post hoc Holm Sidk tests were used to determine the overll effects of respirtory medium, O 2 vilility nd O 2 on P 5 nd the mgnitude of the Root effect. three-wy NOV ws lso used to determine the effect of respirtory medium, O 2 vilility nd ody region on ngiogenesis (D31 fluorescence). Dt were log trnsformed when necessry to meet the ssumptions of norml distriution nd equl vrince. The Journl of Experimentl iology 3993

7 RESERH RTILE The Journl of Experimentl iology (214) doi:1.1242/je.1161 SigmPlot 11 (Systt Softwre, Sn Jose,, US) ws used for ll nlyses (criticl α=.5). Throughout the text vlues re given s mens ± s.e.m. cknowledgements The uthors with to thnk Dr D. Fudge for use of the microscope nd hemocytometer nd Dr G. Tttersll for help optimizing the P wee 5 equipment. We lso thnk Drs G. Scott, K. Gmperl nd K. Gilmour for thoughtful discussions regrding the dt. Thnk you to. Frnk, M. ornish, M. Dvies nd severl volunteers for help with fish mintennce. ompeting interests The uthors declre no competing finncil interests. uthor contriutions.j.t.,.r., K.., M.F. nd P..W. conceived nd designed the project..j.t.,.r., K.., M.F. executed the experiments..j.t. nlyzed the dt nd wrote the drft mnuscript..j.t.,.r., K.., M.F. nd P..W. revised the mnuscript. Funding Funding ws provided y the Nturl Sciences nd Engineering Reserch ouncil of nd Discovery grnts progrm to P..W. (RGPIN-12513) nd Nturl Sciences nd Engineering Reserch ouncil of nd grdute scholrships to.j.t.,.r. nd K.. References leld, S. M., Muller, W.., uck,.. nd Newmn, P. J. (1991). Moleculr nd cellulr properties of PEM-1 (endom/d31): novel vsculr cell-cell dhesion molecule. J. ell iol. 114, ldermn, S. L., Klimn, J. M., Deck,.. nd Gillis, T. E. (212). Effect of cold cclimtion on troponin I isoform expression in strited muscle of rinow trout. m. J. Physiol. 33, R168-R176. iker, M. M. (1984). dptive respirtory significnce of orgnophosphtes (TP & GTP) in ir-rething fishes. Hydroiologi 11, ldwin, J. nd Wells, R. M. G. (199). Oxygen trnsport potentil in tropicl elsmornchs from the Gret rrier Reef: reltionship etween hemtology nd lood viscosity. J. Exp. Mr. iol. Ecol. 144, luk, P. nd McDonld, D. M. (28). Mrkers for microscopic imging of lymphngiogenesis nd ngiogenesis. nn. N. Y. cd. Sci. 1131, inchini, K. nd Wright, P.. (213). 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