Research Article Interspecific Variation in Temperature Effects on Embryonic Metabolism and Development in Turtles

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1 Interntionl Scholrly Reserch Network ISRN Zoology Volume 212, Article ID , 13 pges doi:1.542/212/ Reserch Article Interspecific Vrition in Temperture Effects on Emryonic Metolism nd Development in Turtles Dy B. Ligon 1, 2 nd Mtthew B. Lovern 1 1 Deprtment of Zoology, Oklhom Stte University, Stillwter, OK 7478, USA 2 Deprtment of Biology, Missouri Stte University, 91 South Ntionl, Springfield, MO 65897, USA Correspondence should e ddressed to Dy B. Ligon, dyligon@missouristte.edu Received 22 Novemer 211; Accepted 25 Decemer 211 Acdemic Editors: P. V. Lindemn nd P. Scps Copyright 212 D. B. Ligon nd M. B. Lovern. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. We mesured temperture-induced differences in metolic rtes nd growth y emryos of three turtle species, Mcrochelys temminckii, Trchemys script, ndaplone spinifer, tdifferent, constnt, tempertures. Oxygen consumption rte (VO 2 )ws mesured during development nd used to chrcterize chnges in metolism nd clculte totl O 2 consumption. Results from eggs incuted t different tempertures were used to clculte Q 1 stdifferent stges of development nd to look for evidence of metolic compenstion. Totl O 2 consumption over the course of incution ws lowest t high incution tempertures, nd lte-term metolic rte Q 1 swere<2 in ll three species. Both results were consistent with positive metolic compenstion. However, incution temperture effects on egg mss-corrected htchling size vried mong species. Aplone spinifer htchling mss ws unffected y temperture, wheres T. script mss ws gretest t high tempertures nd M. temminckii mss ws lowest t high tempertures. Htchling mss : length reltionships tended to correlte negtively with temperture in ll three species. Although we cnnot reject positive metolic compenstion s contriutor to the oserved VO 2 ptterns, there is precedence for drwing the more prsimonious conclusion tht differences in yolk-free size lone produced the oserved incution temperture differences without energetic cnliztion y temperture cclimtion during incution. 1. Introduction Although the suite of iochemicl ctivities tht contriutes to n orgnism s metolism is complex nd therefore chllenging to model [1], strong reltionships exist etween ody temperture nd whole-orgnism metolic rte [2]. Vrious thermoregultory mechnisms re employed y nimls to dissocite ody temperture from mient temperture. Endothermy hs evolved repetedly s physiologicl mens of surviving suoptiml therml conditions, ut ectothermic species often rely primrily on ehviorl strtegies to regulte ody temperture. In ddition to ehvior, however, ectotherms my exhiit physiologicl mechnisms to ddress therml constrints of their environment. Solutions for surviving inhospitle tempertures my include mnipulting iochemicl rection rtes y vrying enzyme concentrtions or receptor densities, production of chperone proteins to increse the rnge of tempertures over which trget enzymes remin functionl [3], chnging the composition of cell memrnes to ffect permeility nd (rrely) producing temperture-specific isozymes [4 6]. In comprison to other life stges when ehvior cn ply n importnt role in overcoming therml constrints, mens for mintining suitle ody tempertures of oviprous nimls during emryonic development re limited. Three fctors contriute minimizing n emryo s exposure to or effects of suoptiml therml conditions: (1) indirect ehviorl temperture selection vi mternl thermoregultion or nest-site selection; (2) developmentl dipuse during periods when therml conditions re unsuitle; (3) utiliztion of one or more of the physiologicl strtegies listed ove. These strtegies my function lone or in comintion to circumvent therml limittions on development. For exmple, ecuse the efficcy of mternl nest-site choice my e limited y the stochstic nture of environmentl tempertures [7], mternl nest-site selection seems likely to

2 2 ISRN Zoology function in comintion with complementry physiologicl compenstory strtegies. Much ttention hs een pid to the effects of temperture on htchling trits, prticulrly mong tx tht exhiit temperture-dependent sex determintion. In ddition to effects on popultion sex rtios in mny species [8 1], temperture hs een demonstrted to influence htchling size, posthtching growth, locomotor performnce, metolic rte, gility, nd crypsis [11 28]. Thewysin whichtempertures during emryonic development ffect postemryonic endpoints hve received more ttention mong reptiles thn hve endpoints mesured during emryonic development [29]. Thus, some spects of how differences in the therml environment experienced y emryos led to morphologicl nd performnce differences remin poorly understood. The finite quntity of energy nd mterils pckged in n egg must e udgeted to meet development nd growth requirements. Therefore, temperture-induced differences in energy utiliztion could trickle down to ffect posthtching condition of offspring y directly or indirectly influencing morphology, physiologicl performnce, or postemryonic energy reserves. The effects of temperture on emryo metolism hve een studied in vriety of reptiles [3 36]. Ptterns in the reltionship etween temperture nd emryo energy expenditure hve een firly consistent. For exmple, Angillett et l. [33] found tht, in the lizrd Sceloporus undultus, energy expenditure over the course of incution ws similr t 3,32,nd34 C, ut 1 15% lower compred to emryos tht developed t 28 C. Similrly, Crocodylus johnstoni emryos consumed 1% less oxygen t 31 C thn t 29 C[37], nd emryonic Nile soft shell turtles (Trionyx triunguis) incuted t 27 nd 3 C exhiited similr energetic expenditure, ut used 5% less oxygen t 33 C [38]. In contrst to this pttern of lower oxygen consumption t higher tempertures, no effect of incution temperture ws oserved mong Emydur signt emryos incuted t 24 nd 31 C[39]orCheloni myds incuted t 26 nd 3 C[4]. In comintion, these studies suggest tht emryos of mny reptiles utilize less energy during development t higher incution tempertures, ut some species my exhiit cpcity to physiologiclly correct for suoptimlly high or low ody tempertures, commonly referred to s positive compenstion [4, 41].Thegolofthisstudyws to investigte differences in incution temperture effects on emryonic development nd metolism mong symptric ut phylogeneticlly diverse ssemlge of freshwter turtles [42]: Trchemys script; Aplone spinifer; Mcrochelys temminckii. Our ojectives were threefold. The first ws to mesure the cpcity for metolic compenstion during emryonic development y compring stge-specific VO 2 (oxygen consumption rte) of emryos exposed to different constnt incution tempertures. These mesurements were then used to clculte differences in Q 1 (the rte of chnge of physiologicl process s consequence of incresing the temperture y 1 C) t different stges of development. Second, we evluted temperture effects on energetic cost of development y clculting the totl volume of oxygen (O 2totl ) used over the course of incution, nd mss-conversion efficiency sed on differences in htchling mss fter correcting for vrition in initil egg mss. Third, we qulittively ssessed the degree of vrition in different species responses to temperture. 2. Mterils nd Methods All procedures for this reserch were pproved y the Oklhom Stte University Institutionl Animl Cre nd Use Committee (Protocol AS23), gurnteeing complince with niml cre guidelines descried in The Guide for the Cre nd Use of Lortory Animls, 7th edition (1996). Trchemys script nd M. temminckii htchling mss dt were used in other nlyses relted to posthtching effects of T inc (incution temperture) [43] Study Species. The three turtle species included in this study represent three different fmilies nd, lthough symptric in prts of their rnge, they exhiit sustntil ecologicl differences. Trchemys script (Fmily Emydide) is medium in size nd primrily qutic ut engges in frequent sking nd terrestril migrtions etween wter odies nd conforms to the Type I pttern of temperture-dependent sex determintion [44], wherein mles re produced t low tempertures nd femles t high tempertures. Aplone spinifer (Fmily Trionychide) exhiits mny dpttions to pelgic lifestyle, including hydrodynmic form nd sustntil cpcity for qutic respirtion. In contrst to most turtles, sex is determined geneticlly in memers of this fmily [45]. Finlly, Mcrochelys temminckii (Fmily Chelydride) is very lrge-odied, primrily ottomdwelling species tht seldom leves wter except to nest. It follows type II pttern of temperture-dependent sex determintion, chrcterized y development of femles t low nd high tempertures, nd mles or mixed sex rtio t intermedite tempertures [44] Egg Collection. Eggs were otined for ll three species in My nd June 24. A single A. spinifer nest ws excvted from snd r t Sequoyh Ntionl Wildlife Refuge (SNWR) in estern Oklhom, nd trnsported to Oklhom Stte University (OSU) within 15 h fter deposition. Grvid T. script weretrppedinnoxowtsnwr using ited hoop nets. These turtles were trnsported to OSU where oviposition ws induced using oxytocin (.1 IU/kg IM) [46]. Turtles were plced individully in plstic tus contining pproximtely 15 cm of wter to minimize ccidentl destruction of eggs y the turtles. After eggs were otined, the dult femles were relesed t SNWR. Finlly, M. temminckii eggs were otined from cptive group mintined t Tishomingo Ntionl Fish Htchery s prt of cptive reeding/reintroduction progrm. The dult turtles which produced the eggs for this study originted from SNWR. Eggs were lid nturlly nd excvted from nests within two d following oviposition. No eggs of ny of the three species showed signs of the white nding chrcteristic of erly development [47] prior to rriving t OSU.

3 ISRN Zoology Incution. Eggs from ech species were mesured (±.1mm) nd weighed (±.1 g), nd then ssigned to n incution tretment in rndomized lock design (lock = clutch). Within ech incution tretment, eggs were distriuted mong 1 5 plstic shoeoxes (1.5 L) hlffilled with dmp vermiculite (1 : 1 vermiculite : wter y mss; 15 kp wter tension; [48]). Shoeoxes were then ssigned to one of three constnt-temperture incutors set t 26.5, 28.5, nd 3.5 C. Boxes were rotted within ech incutor dily to eliminte the possiility of position effects, nd ech ox ws weighed weekly nd rehydrted s necessry to mintin its initil mss. Eggs were cndled every 2-3 dys during erly development nd eggs tht filed to develop were discrded to eliminte sustrte for invsion of mold Htchlings. Upon pipping, ech egg ws plced in plstic jr lined with dmpened pper towels so tht the identity of individuls could e determined fter htching. After emerging from the egg shell, htchlings were kept in the plstic jrs until residul yolks were completely internlized, period tht lsted 9 d nd vried mong species. Aplone spinifer were photogrphed nd T. script nd M. temminckii received unique mrkings to ensure future identifiction. Cuticle scissors were used to cut notches in unique comintions of mrginl scutes on T. script. A smll sewing needle ws used to tie smll loops of dentl floss through unique comintions of scutes on M. temminckii [27]. Htchlings were removed from the incutor to flowthrough rcewys Metolic Rte. Metolic rtes were estimted y mesuring chnges in oxygen concentrtion in chmers vi closed system respirometry [49] nd clculting VO 2 [5]. Eggs were plced individully in metolic chmers constructed from 169 ml plstic jrs with screw-top lids. A stopcock ws inserted through ech lid nd seled in plce with silicon. Initil ir smples were drwn from ech chmer into stopcock-equipped 3 cc syringes, nd then seled. The chmers were then plced into the incutors for h (longer during erly development when VO 2 ws expected to e low). Chmers were then removed from the incutor, nd finl ir smples were drwn into second set of syringes. Eggs were weighed t the conclusion of ech mesurement nd returned to the plstic shoeoxes. Oxygen concentrtions of ll ir smples were nlyzed in 1 ml liquots with Sle Systems FC-1 oxygen nlyzer. A strem of ir ws drwn from outside the uilding t regulted flow rte of 1 ml/min. It pssed through seril columns of Drierite nd Ascrite to remove wter nd CO 2, respectively. Ech liquot ws injected into the ir strem, which pssed through smll column of Drierite nd Ascrite nd then through the oxygen nlyzer. VO 2 ws clcultedfor echturtle s the difference etween the initil nd finl volumes of oxygen fter correcting for chmer volume [5]. VO 2 of T. script nd M. temminckii emryos ws mesured t 7 d intervls strting 7 d fter oviposition. A. spinifer emryo VO 2 ws mesured t 2-3 d intervls erly nd lte in development, nd on 7 d schedule during the middle third of incution. The volume of oxygen used over the course of incution ws clculted for ech turtle tht successfully htched y summing the trpezoidl res creted y djcent VO 2 mesurements using the eqution: i 24(VO O 2n +VO 2n 1 )(T n T n 1 ) 2totl =, (1) 2 where O 2totl ws the oxygen consumed over the durtion of incution, VO 2 ws the rte of oxygen consumption t ge n mesured in ml/h, T ws the numer of dys since the eginning of incution, n ws the dys on which VO 2 ws mesured, nd p ws dy on which ech turtle pipped. Becuse turtles htched 7 d fter the lst emryo VO 2 mesurement, VO 2p t the time of pipping ws estimted sed on the difference etween VO 2 mesurements prior to nd fter htching. These estimtes were clculted s: [ (VO2htchling )( ) 1 VO 2p = VO 2finl Thtchling T finl ( Tpip T finl ) ] +VO 2finl, where VO 2htchling ws mesured t ech htchling s ssigned incution temperture following internliztion of the residul yolk nd VO 2finl ws the lst mesurement prior to pipping. These vlues were used to clculte the finl trpezoidl re of ech turtle s O 2totl to produce precise estimte of the volume of oxygen used etween oviposition nd htching Sttistics. Sttisticl nlyses were conducted seprtely for ech of the three species. Incution temperture effects on three chrcteristics of VO 2 during emryonic development were nlyzed: timing of initil VO 2 divergence; mximum VO 2 (VO 2mx ); developmentl stge-specific Q 1. The ge t which VO 2 of emryos t different tempertures first diverged ws ssessed y performing post hoc comprisons using differences of lest-squres mens from repeted mesures ANCOVA cross ll mesurements, with individul emryo s VO 2 srepetedcrossmesurement intervls nd initil egg mss s covrite. VO 2mx ws likely ffected y timing of mesurements; therefore, timing of VO 2mx, expressed s percentge of totl incution time, ws included s covrite in nlyses compring VO 2mx t different incution tempertures. Q 1 vlues were clculted t four different stges of development nd were sed on VO 2 of emryos t 26.5 nd 3.5 C. Developmentl stges were expressed s percent of incution time nd were distriuted such tht Q 1 ws compred ner the eginning, first nd second thirds, nd end of incution. Htchling size ws nlyzed in two different wys: (1) mss ws nlyzed in n ANCOVA with initil egg mss s covrite to ssess incution temperture effects on eggto-tissue mss conversion efficiency; (2) differences in ody (2)

4 4 ISRN Zoology Tle 1: Clutch, egg, nd htchling dt for turtles included in this study (men ± SE). Men incution periods re reported for eggs incuted t 26.5, 28.5, nd 3.5 C, respectively. Species Numer of Clutches Numer of eggs Htchling success (%) Incution period (d) Egg mss (g) Htchling mss (g) Htchling length (mm) A. spinifer 1 18 (18) 89 77, 61, ± ± ±.25 M. temminckii 4 19 (17 33) 66 93, 82, ± ± ±.17 T. script 5 58 (1 14) 93 71, 59, ± ± ±.11 Numer of eggs of ech species used in this study, with clutch size rnge in prentheses. Plstron length reported for A. spinifer, crpce length reported for M. temminckii nd T. script. Metolic rte (ml O2 h 1 ) Time (dys) () Aplone spinifer Metolic rte (ml O2 h 1 ) Time (dys) () Trchemys script Metolic rte (ml O2 h 1 ) C 28.5 C 26.5 C Time (dys) (c) Mcrochelys temminckii Figure 1: Chnges in VO 2 during emryonic development t three constnt tempertures. Asterisks indicte first mesurement t which VO 2 diverged cross ll three incution tempertures. Error rs = ±1SE. length (crpce length ws used for T. script nd M. temminckii nd plstron length ws used for A. spinifer) were compred cross incution tempertures with htchling mss s covrite to ssess differences in ody composition (herefter Body Condition Index, BCI). In ll of the ove nlyses, homogeneity of slopes mong tretments ws tested y compring interction terms tht included the covrite. In ll cses homogeneity ws confirmed, nd interction terms were removed prior to finl nlyses. Also, nonsignificnt covrites (P >.5) were removed to increse degrees of freedom for the error term. All sttisticl tests were conducted using SAS v. 9.1 Proc Mixed fter testing the homogeneity of vrince ssumption using Proc GLM (SAS Institute 22). All metolism,

5 ISRN Zoology 5 Metolic rte (ml O2 h 1 ) Metolic rte (ml O2 h 1 ) Stge of development (% incution) () Aplone spinifer Stge of development (% incution) () Trchemys script Metolic rte (ml O2 h 1 ) C 28.5 C 26.5 C Stge of development (% incution) (c) Mcrochelys temminckii Figure 2: Comprison of chnges in VO 2 of emryos mintined t different tempertures, with time expressed s percentge of totl incution durtion. Ovls superimposed on ech figure indicte smples used to clculte developmentl stge-specific Q 1 vlues (see text). mss,ndlengthvlueswerelog 1 trnsformed prior to nlysis to improve dt distriution. Results re expressed s men ± 1SE. 3. Results 3.1. Species Comprisons. Eggs used in this study comprised one A. spinifer, five T. script, nd four M. temminckii clutches (Tle 1). Egg mss vried mong clutches in the ltter two species (P <.1), ut ecuse rndomized lock experimentl design ws employed, it did not vry mong incution tempertures (A. spinifer: P =.57; M. temminckii: P =.145; T. script: P =.953). Temperture ffected incution durtion (Tle 1), emryo VO 2 (Figures 1, 2)ndtotlO 2 consumed (Figure 3)in ll three species. However, temperture ffected incution durtion differently mong the three species. The 4 C difference experienced y eggs incuted t 26.5 compred to 3.5 C produced 31% nd 28% differences in incution time in A. spinifer nd T. script, respectively, compred to just 15% difference in M. temminckii. The effect of incution temperture on M. temminckii incution durtion ws n even smller 4% cross the 2 C spn etween C, compred to 19% nd 12% in A. spinifer nd T. script,respectively. Despite low VO 2 vlues during erly development, precision of mesurements ws sufficient to detect differences mong ll three incution tempertures t the first mesurement in A. spinifer nd T. script (dys 2 nd 5, resp.). Among M. temminckii emryos, men VO 2 t 26.5 differed from tht t 28.5 nd 3.5 C y dy 7 nd differed mong ll three tempertures y dy 14 (P <.1; Figure 1). Ptterns in the mgnitude of VO 2mx mong different incution tempertures vried mong species. Mcrochelys temminckii emryos mintined t 26.5 nd 28.5 hd similr VO 2mx (P =.353) nd were oth greter thn those incuted t 3.5 C(P <.1; Figure 4(c)). In contrst, VO 2mx mong T. script emryos ws lowest t 26.5 (P <.1) nd did not differ etween 28.5 nd 3.5 C(P =.54; Figure 4()). Finlly, A. spinifer VO 2mx ws gretest t the intermedite incution temperture (28.5 : P<.3) nd did not differ t the extremes ( C: P =.157; Figure 4()). The timing of VO 2mx (expressed s % totl incution durtion) ws unffectedy tempertureforll three species (M. temminckii: 87.5%, rnge = 82 97%, P =.521; T. script: 91%, rnge = 84 97%, P =.661; A. spinifer: 87%, rnge = 83 96%, P =.6). These vlues were no dout dependent on the timing of VO 2 mesurements, however. It should e noted tht wheres M. temminckii nd A. spinifer

6 6 ISRN Zoology Totl emryo oxygen consumption (ml O2) Totl emryo oxygen consumption (ml O2) c Incution temperture ( C) Incution temperture ( C) () Aplone spinifer () Trchemys script Totl emryo oxygen consumption (ml O2) Incution temperture ( C) (c) Mcrochelys temminckii Figure 3: Comprison of the totl volume of O 2 consumed over the durtion of incution y emryos incuted t different constnt tempertures. Lower cse letters indicte tretment differences (P <.5). Error rs =±1SE. exhiited distinctly peked emryo VO 2 ptterns, VO 2mx mong T. script tended to occur t the lst mesurement prior to htching (Figure 1).Theexceptionstothispttern were five out of 18 emryos incuted t 26.5 C tht peked t the penultimte VO 2 mesurement. Totl O 2 consumption over the course of emryonic development ws similr t 26.5 nd 28.5 C nd lower t 3.5 CinA. spinifer nd M. temminckii. By comprison, totl O 2 consumption in T. script ws highest t 28.5 C, intermedite t 26.5 C nd lowest t 3.5 C(Figure 3) Metolic Compenstion. VO 2 mesurements used for clculting Q 1 vlues were selected sed on their timing (trgets:, 33, 67, nd 1% of incution) nd degree of overlp in the timing of 26.5 nd 3.5 Cmesurements when expressed s proportion of incution durtion in order to closely mtch the developmentl stges compred mong emryos t different incution tempertures (Figure 2 nd Tle 2). Among A. spinifer,q 1 ws for the first three stges. However, t pproximtely 95% development Q 1 = 1.2, lower thn expected in the sence of positive compenstion. In comprison, T. script exhiited Q 1 s etween two nd three during the initil two-thirds of development, nd similr decrese close to htching (Q 1 = 1.4 t pproximtely 88% development). In contrst, M. temminckii registered Q 1 s less thn two throughout development. VO 2 mesurements t 89% development produced Q 1 = Cost of Development. Although htchling mss ws not significntly different mong incution tempertures (A. spinifer: P =.983; T. script: P =.65; M. temminckii: P =.2; Figure 5), temperture-induced differences were evident in mss conversion efficiency (Figure 6) nd htchling BCI (Figure 7) in T. script nd M. temminckii, ut not A. spinifer. Htchling mss scled positively to egg mss in ll three species. Trchemys script htchling mss fit the eqution M htchling =.4M egg.93 (r 2 =.32, P <.1), M. temminckii fit the eqution M htchling =.5M egg.93

7 ISRN Zoology 7 VO2mx (ml O2 h 1 ) VO2mx (ml O2 h 1 ) Incution temperture ( C) () Aplone spinifer 1.6 Incution temperture ( C) () Trchemys script VO2mx (ml O2 h 1 ) Incution temperture ( C) (c) Mcrochelys temminckii Figure 4: VO 2mx of turtle emryos incuted t three tempertures. Lower cse letters indicte tretment differences (P <.5). Error rs =±1SE. (r 2 =.5, P <.1), nd A. spinifer fit the eqution M htchling =.13M egg.92 (r 2 =.25, P =.4). Mss conversion efficiency ws unffected y incution temperture in A. spinifer (P =.93), ws reduced t high tempertures in M. temminckii (P =.3), nd ws elevted t high tempertures in T. script (P =.2; Figure 6). Htchling BCI, expressed s the reltionship etween log 1 -trnsformed length nd mss, did not differ cross incution tempertures in A. spinifer (Figure7()). BCI of htchlings ws not different t 26.5 nd 28.5 CinT. script, nd ws higher t oth these tempertures in comprison to htchlings from 3.5 C(Figure 7()). Among M. temminckii, BCI ws gretest t 26.5, intermedite t 28.5, nd lowest t 3.5 C(Figure 7(c)). 4. Discussion Interspecific vrition ws evident in the effects temperture hd on stge-specific Q 1 vlues, O 2totl,VO 2mx, htchling size, nd htchling composition (BCI). However, rodscle consistency in the direction nd timing of oserved ptterns suggest tht incution temperture influenced emryo energetics nd growth similrly mong the three species. Chemicl rection rtes typiclly exhiit Q 1 s of 2-3. Biologicl rtes tht fll elow this rnge re often interpreted to e indictive of temperture compenstion, wheres higher vlues indicte inverse compenstion, or hypersensitivity to temperture [51]. Progressively decresing Q 1 s over the course of emryonic development hve een oserved in severl poikilotherms, including turtle (Emys oriculris; [35]), nd two fishes (Dnio rerio nd Slmo girdneri; [52, 53]. This pttern frequently hs een proposed to reflect greter therml compenstion s development progresses. Q 1 vlues were highly vrile oth within nd mong species nd did not netly fit negtive correltion with emryonic development. However, s in previous studies, Q 1 ws consistently lower ner the conclusion of emryonic development thn during erlier stges in ll three species. Mcrochelys temminckii incuted t 3.5 Cconsumed less oxygen thn those t lower tempertures nd exhiited VO 2mx lower thn tht of turtles t 28.5 C nd similr to

8 8 ISRN Zoology Tle 2: VO 2 Q 1 vlues clculted t different stges of emryonic development in three turtle species. Stges re expressed s % of totl incution durtion, followed y the numer of dys since oviposition. Q 1 vlues were clculted from VO 2 s of turtles incuted t 26.5 nd 3.5 C. Stges (% incution durtion, dy of incution) Species T inc A. spinifer T. script M. temminckii %, 2 3.%, %, %, 72 Q 1 = Q 1 = 3.98 Q 1 = %, 2 3.2%, %, %, %, %, %, %, 61 Q 1 = 2.14 Q 1 = 2.3 Q 1 = %, %, %, %, %, %, %, %, 84 Q 1 = 1.38 Q 1 = 1.72 Q 1 = %, %, %, %, 7 Q 1 = 1.24 Q 1 = 1.39 Q 1 =.67 5 Htchling mss (g) Htchling mss (g) Incution temperture ( C) () Aplone spinifer Incution temperture ( C) () Trchemys script Htchling mss (g) Incution temperture ( C) (c) Mcrochelys temminckii Figure 5: Mss of htchlings incuted t different constnt incution tempertures. Lower cse letters indicte tretment differences (P <.5). Error rs =±1 SE. those t 26.5 C. Such results could stem from metolic compenstion to temperture, ultimtely resulting in greter efficiency in the conversion of yolk to metoliclly ctive tissue. Although it ws infesile in this study to scrifice emryos or htchlings to directly mesure yolk : tissue rtios, evidence from Q 1 s nd htchling size support n lterntive conclusion: though incution temperture produced miniml differences in htchling mss, those from the highest temperture exhiited lower BCIs nd were, therefore, morphometriclly smller fter correcting for vrition in

9 ISRN Zoology 9 Mss conversion efficiency (egg mss: htchling mss residuls) slope =.917; r 2 =.25 Incution temperture ( C) Mss conversion efficiency (egg mss: htchling mss residuls) slope =.934; r 2 =.32 Incution temperture ( C) () Aplone spinifer () Trchemys script Mss conversion efficiency (egg mss: htchling mss residuls) slope =.868; r 2 =.46 Incution temperture ( C) (c) Mcrochelys temminckii Figure 6: Differences in htchling mss corrected for differences in initil egg mss. Vlues were clculted from L-S regression residuls from log-log plot of htchling mss ginst initil egg mss. Lower cse letters indicte tretment differences (P <.5). Error rs = ±1SE. mss, thn htchlings from lower tempertures. Thus, we conclude tht high tempertures negtively ffected emryo growth, ut tht those smller htchlings my hve emerged with lrge quntities of unmetolized yolk, thus ccounting for the lck of lrge differences in mss. The comintion of these fctors suggests tht less metoliclly ctive tissue ws present in high incution temperture htchlings, resulting in lower VO 2 even in the sence of metolic compenstion. The effects of high incution temperture on VO 2 nd O 2totl in T. script nd A. spinifer were similr to those in M. temminckii. At 3.5 C, VO 2mx ws lower thn t 28.5 C (though not significntly so mong T. script)nd htchlings t 3.5 C exhiited lower O 2totl thn conspecifics t lower tempertures. Wheres M. temminckii htchlings from 3.5 C incution temperture were slightly lighter thn from lower tempertures, no such negtive mssincution temperture reltionship ws evident in the two other species, nd T. script from the highest temperture were ctully slightly hevier. However, BCIs were lowest t 3.5 C in ll three species (though not significntly so in A. spinifer), suggesting tht, s ppers likely in M. temminckii, ody length ws smller nd the proportion of mss composed of metoliclly inctive yolk ws higher mong htchlings tht developed t high tempertures. Evidence supports such negtive correltion etween quntity of residul yolk nd the length-to-mss reltionship in nother turtle, Cheloni myds, in which posthtching yolk mss nd htchling yolk-free mss were mesured [4]. These results suggest, leit indirectly, tht incution temperture produced differences in yolk-free mss. This conclusion is consistent with numer of turtle nd squmte studies tht hve shown negtive correltion etween incution temperture nd yolk-free tissue mss [37, 4, 48, 54 56]. Although we cnnot reject positive metolic compenstion s n dditionl fctor contriuting to the oserved VO 2 ptterns, there is precedence for drwing the more prsimonious conclusion tht differences in yolk-free size lone produced the oserved incution temperture differences without compenstory response to temperture cclimtion during incution [55]. The differences in the effects of temperture on htchling size nd yolk reserves suggest tht the costs nd enefits of developing t reltively high tempertures differ mong the three species studied. It hs een demonstrted in severl lizrd species tht lrge htchling size increses survivl, presumly y incresing forging efficiency nd decresing susceptiility to predtion [57 64]. Studies suggest tht size ffects qutic turtle htchling survivl, s well [65 69]. However, htchling size my ffect fitness in sutler wys. It is lso generlly ssumed tht lrge postemryonic yolk

10 1 ISRN Zoology.2 Body condition (length: mss residuls) slope =.314; r 2 =.14 Incution temperture ( C) Body condition (length: mss residuls) slope =.27; r 2 =.58 Incution temperture ( C) () Aplone spinifer () Trchemys script Body condition (length: mss residuls) slope =.384; r 2 =.59 c Incution temperture ( C) (c) Mcrochelys temminckii Figure 7: Htchling ody composition indices clculted s residuls from L-S regression from log-log plot of ody length on mss. Lower cse letters indicte tretment differences (P <.5). Error rs = ±1SE. reserves increse fitness, either y incresing the time htchling cn survive efore eting or, similrly, incresing winter survivl in species tht dely emergence until spring [7, 71]. Trchemys script exhiited evidence of retining more residul yolk t 3.5 C thn t lower incution tempertures. Therefore, development t high incution temperture my confer the enefits of dditionl energy reserves, even t the cost of smller ody length. Becuse femles re produced exclusively t 3.5 C[42], nd femle T. script ttin sexul mturity t lrger size thn do mles [42], this difference in htchling composition lends support to the hypothesis tht temperture-dependent sex determintion evolved due to symmetricl enefits to the sexes of developing t different tempertures [9]. Similr ptterns were oserved in A. spinifer, nd so the enefits of developing t high incution tempertures re likely similr. However, mles nd femles do not differentilly enefit, s this species exhiits genetic sex determintion. As with T. script nd A. spinifer, evidence suggests tht M. temminckii tht were incuted t high tempertures retined reltively lrge yolk residuls. This potentil enefit to survivl ws offset y the fct tht htchlings were morphometriclly smller t 3.5 C. Additionlly, M. temminckii emryo mortlity ws high t 3.5 C[43], wheres htching success ws unffected y incution temperture in the two other species. Therefore, it is unlikely tht the phenotype expressed t high tempertures y M. temminckii htchlings enhnces fitness, ut is insted the result of limittions on growth t high tempertures. Future reserch investigting the wys tht incution temperture ffects emryo energetics nd htchling ody composition is needed to differentite costs ssocited with growth nd mintennce during development nd to ssess the reltive enefits to htchlings of mximizing ody size versus retining yolk for sustining posthtching metolism. A lrge ody of literture exists on the effects of incution temperture on htchling turtle size nd growth [11 15], ut more detiled nlyses of temperture s effect on the proportions of tissue nd yolk tht comprise htchling mss will lend greter insight into the role incution temperture plys in ffecting survivl nd, ultimtely, fitness.

11 ISRN Zoology 11 Acknowledgments The uthors thnk Sequoyh Ntionl Wildlife Refuge nd Tishomingo Ntionl Fish Htchery for providing turtle eggs for this reserch. S. Wtkins, P. Widder, nd E. Ligon ssisted with mesuring metolic rtes. The pper enefited gretly from comments y J. Bidwell, S. Fox, nd M. Pyton. Finncil support ws provided y the Oklhom Stte University Environmentl Institute, Assocition for the Study of Ichthyology nd Herpetology, Chicgo Herpetologicl Society, Chelonin Reserch Foundtion, Society of Integrtive nd Comprtive Biology, nd Sigm Xi. Experiments reported in this study comply with the current United Sttes lws on niml experimenttion. References [1] A. Clrke, Is there universl temperture dependence of metolism? Functionl Ecology, vol. 18, no. 2, pp , 24. [2] J. F. Gillooly, J. H. Brown, G. B. West, V. M. Svge, nd E. L. Chrnov, Effects of size nd temperture on metolic rte, Science, vol. 293, no. 5538, pp , 21. [3] M. E. Feder nd G. E. 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