Determinants of incubation period: do reptilian embryos hatch after a fixed total number of heart beats?

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1 1302 The Journl of Experimentl Biology 212, Pulished y The Compny of Biologists 2009 doi: /je Determinnts of inution period: do reptilin emryos hth fter fixed totl numer of hert ets? Wei-Guo Du 1,2, *, Rjkumr S. Rdder 1,, Bo Sun 2 nd Rihrd Shine 1 1 Shool of Biologil Sienes A08, University of Sydney, NSW, 2006 Austrli nd 2 Hngzhou Key Lortory for Animl Siene nd Tehnology, College of Biologil nd Environmentl Sienes, Hngzhou Norml University, , Hngzhou, People s Repuli of Chin *Author for orrespondene (e-mil: dwghz@126.om) Deesed Aepted 9 Ferury 2009 SUMMARY The eggs of irds typilly hth fter fixed (ut linege-speifi) umultive numer of hert ets sine the initition of inution. Is the sme true for non-vin reptiles, despite wide intrspeifi vrition in inution period generted y vrile nest tempertures? Non-invsive monitoring of emryo hert et rtes in one turtle speies (Pelodisus sinensis) nd two lizrds (Bssin duperreyi nd Tkydromus septentrionlis) show tht the totl numer of hert ets during emryogenesis is reltively onstnt over wide rnge of wrm inution onditions. However, inution t low tempertures inreses the totl numer of hert ets required to omplete emryogenesis, euse the emryo spends muh of its time t tempertures tht require mintenne funtions ut tht do not llow emryoni growth or differentition. Thus, ool-inuted emryos llote dditionl metoli effort to mintenne osts. Under wrm onditions, totl numer of hert ets thus predits inution period in non-vin reptiles s well s in irds (the totl numer of hert ets re lso similr); however, under the older nest onditions often experiened y non-vin reptiles, mintenne osts dd signifintly to totl emryoni metoli expenditure. Key words: emryoni development, hert rte, metoli rte, therml dependene, reptile, therml time. INTRODUCTION Rtes of development omprise one of the most importnt xes of life-history vrition nd spn n enormous rnge. Genertion times rnge from minutes or hours in some teri, through to dedes in mny vertertes (Clder, 1984). In mny oviprous (egg-lying) orgnisms, the durtion of inution of the eggs my e ritil life-history vrile. The durtion of inution reltive to the rest of the lifespn n vry even within single phylogeneti linege; for exmple, some hmeleons spend more thn 50% of their totl lifespn within the egg (Krsten et l., 2008) wheres in other lizrds, the egg stge omprises <2% of the mximum lifespn [e.g. Vrnus komodoensis (Cogger nd Zweifel, 1992)]. The egg stge differs from lter life-history stges in mny distintive eologil hrteristis. Most notly, emryos of most speies (exept those with dispersive eggs) re immoile nd thus re unle to disperse, thermoregulte or feed or protet themselves ginst predtors. We might thus expet strong seletion on the durtion of the inution period nd on the timing of hthing (Shine, 1978; Shine nd Olsson, 2003). Despite the strong fitness onsequenes of intrspeifi vrition in the durtion of inution [nd thus the timing of hthing, e.g. Amphiolurus muritus (Wrner nd Shine, 2007)], field studies often report extensive environmentlly indued vrition in these trits, even within single popultion (Perrins, 1967; Olsson nd Shine, 1997). Some of this vrition is under prentl ontrol; for exmple, mny irds dely rooding the eggs until ll hve een lid, nd the low tempertures of n unttended nest dely the onset of emryoni development (Stoleson nd Beissinger, 1995). More generlly, the therml regime tht n egg experienes n mssively lter its totl inution period, espeilly in speies without prentl ontrol of nest tempertures. For exmple, eggs of the lizrd Bssin duperreyi n hth in 26 dys if kept t 30 C ut require more thn 60 dys if kept t 22 C (Shine nd Hrlow, 1996). Wht ftors determine the reltionship etween inution tempertures nd totl developmentl periods? The simplest explntion is tht the trnsformtion from zygote to hthling is prolonged y ool onditions euse temperture regultes the rte of hemil tivities (inluding those ontriuting to overll metoli rte nd hene rtes of emryogenesis). Although mny dditionl phenomen [e.g. emryoni dipuse, preoil vs ltriil hthing (Ar nd Tzw, 1999; Andrews et l., 2008)] n lso ffet the durtion of the egg stge, muh of the vrition in totl inution periods ppers to e driven y vrition in rtes of emryogenesis. Consistently, emryos developing t high tempertures hve shorter development times thn those kept t low tempertures (Deeming nd Ferguson, 1991; Hoegh-Gulderg nd Perse, 1995). This phenomenon is so widespred tht it hs een formlised y the onept of therml time [the liner reltionship etween development rte nd temperture (Honek, 1996; Trudgill et l., 2005)]. The physiologil mehnisms tht link developmentl time to temperture re proly omplex ut the rdiovsulr system is known to ply ritil role in nutrient nd oxygen delivery during emryoni development (Birhrd nd Reier, 1996; Birhrd nd Deeming, 2004; Tzw, 2005). When emryoni growth is elerted y higher temperture, the rdiovsulr system must deliver nutrients t n inresed rte to fuel the fster growth. Therefore, the reltionship etween temperture nd inution period my e driven y the therml dependene of rdiovsulr funtion. In keeping with this ide, the rte of emryoni hert et (n inditor of rdiovsulr tivity) is positively orrelted with developmentl rte in irds (Ar nd Tzw, 1999; Tzw, 2005).

2 Totl numer of hert ets in reptiles 1303 Within n vin linege, the umultive numer of hert ets etween oviposition nd hthing is reltively onstnt, onsistent with the generl onstny in hert ets per life spn for dult endotherms (Ar nd Tzw, 1999). Non-vin reptiles present n dditionl omplition in this respet euse emryoni development ours over muh wider rnge of tempertures thn in irds (Deeming nd Ferguson, 1991). So, does the umultive numer of hert ets predit inution durtion in lizrds nd turtles (s it does in irds) despite the enormous vrition in inution periods generted y flututions in nest tempertures? Although previous ttempts to monitor hert rtes in reptile emryos hve hd to overome mjor tehnil ostles (Birhrd nd Deeming, 2004), reent methodologil dvnes filitte non-invsive monitoring (Rdder nd Shine, 2006; Du nd Shine, 2008) nd thus provide n opportunity to lrify the issues rised ove. In the present study, we mesured the therml dependene of emryoni hert et rtes in three speies of non-vin reptiles. With dt on the durtion of inution t eh of these tempertures, we ould then lulte the totl numer of hert ets of emryos during inution. The results of these lultions llowed us to ssess whether (s in irds) we n predit the durtion of reptilin inution from the umultive totl numer of hert ets sine oviposition. MATERIALS AND METHODS Egg olletion nd inution We used three speies of oviprous non-vin reptiles from divergent phylogeneti lineges: the Austrlin three-lined skink Bssin duperreyi Wiegmnn (Sinide), the Chinese northern grss lizrd Tkydromus septentrionlis Gunther (Lertide) nd the Chinese soft-shelled turtle Pelodisus sinensis Gry (Trionyhide). In Novemer 2007, we olleted 26 freshly lid eggs of B. duperreyi from field nests ner Cnerr in southestern Austrli [see Shine nd Hrlow nd Shine et l. (Shine nd Hrlow, 1996; Shine et l., 1997) for detils of study re nd speies iology]. In My 2008, we olleted 14 T. septentrionlis eggs lid in ptivity y femles reently field-olleted in Zhejing, estern Chin, nd otined 35 P. sinensis eggs from privte turtle frm, lso in Zhejing. The men developmentl stges of emryos t oviposition [sed on the lssifition shemes of Huert nd Tokit nd Kurtni (Huert, 1985; Tokit nd Kurtni, 2001)] were stge 31, 26 nd 5 for B. duperreyi, T. septentrionlis nd P. sinensis, respetively. All eggs were weighed (±0.001 g) nd individully inuted in 64 ml glss jrs filled with moist vermiulite ( 200 kp). The jrs were then inuted t 25 C (for B. duperreyi eggs) or 28 C (for T. septentrionlis nd P. sinensis eggs), lose to the men temperture of nturl nests in eh speies (Shine et l., 1997; Du, 2003; Du nd Feng, 2008). Hert rte detetion We mesured hert rtes of emryos pproximtely 25% through the totl inution period in ll speies. Previous studies on one of our study speies (Rdder nd Shine, 2006) nd our unpulished dt on oth of the other tx (W.-G.D. nd R.S., unpulished dt), revel no signifint ontogeneti shift in men hert rtes from <25% to >90% of inution (t ny given temperture); thus, the ext timing of our hert rte mesurements reltive to emryogenesis should hve little impt on the rte estimtes. Hert rtes [ets per minute (etsmin 1 )] were mesured using n infrred hert rte monitor (Buddy system; Avin Bioteh; vinioteh.om/uddy.htm) on dy 10 in B. duperreyi, dy 7 in T. septentrionlis nd dy 15 in P. sinensis, respetively. All eggs were pled in inutors set t 20, 25, 30, 33 or 35 C for two hour limtion period prior to mesurement nd were then pled individully on the monitor to reord hert rte. The Buddy system works y shining n infrred em onto the surfe of the egg, deteting minute distortions used y emryoni hert ets; reording hert rtes generlly tkes less thn minute nd does not ffet the egg or emryo. As our mesure of men hert et rte, we used the men rte in the first 30s fter the monitor first gve relile, onsistent reding (whih typilly ourred few seonds fter the mhine ws swithed on ut osionlly took 10 or 15s to stilise). The order of exposure of eh egg to test tempertures ws rndom. Clultions of totl hert ets nd totl effetive hert ets We used the hert rte dt to develop qudrti equtions tht desrie the reltionship etween test temperture nd hert et rtes of individul emryos. These equtions were then used to predit the hert et rte of emryos t eh inution temperture. Dt on the totl durtion of emryogenesis (=inution period, from oviposition to hthing) of these speies t different inution tempertures were otined from previous studies [B. duperreyi (Shine nd Hrlow, 1996); T. septentrionlis (Du, 2003); P. sinensis (Du nd Ji, 2003; Ji et l., 2003)]. We lulted reltive developmentl rte for given temperture y dividing the reported inution durtion t tht temperture y the shortest inution durtion reorded for tht speies in the lortory (25 dys t 30 C for B. duperreyi, 23.5dys t 33 C for T. septentrionlis nd 40dys t 33 C for P. sinensis) nd tking the inverse of this vlue (Shine nd Hrlow, 1996). The developmentl zero of eh speies (the ritil minimum temperture t whih the rte of emryogenesis fell to zero) ws then lulted from the liner reltionship etween developmentl rte nd inution temperture (Shine nd Hrlow, 1996). Eggs of mny reptile speies n tolerte tempertures well elow the developmentl zero [indeed, some n withstnd tempertures lose to freezing (Pkrd nd Pkrd, 1988)]. Although emryoni growth eses t suh tempertures (Georges et l., 2005), the rdiovsulr system of the nimls ontinues to funtion [e.g. we hve deteted hert et of B. duperreyi emryos t 11 C (W.- G.D. nd R.S., unpulished dt)] nd presumly this ontinued metoli effort plys n importnt role in mintining emryo viility. Aordingly, we defined two umultive hert et prmeters, sed on either inluding or exluding hert ets ourring t tempertures elow the developmentl zero. The first prmeter ws the totl numer of hert ets (THB) of n emryo throughout its emryoni development, lulted t ny given inution temperture using the formul: THB = temperturespeifi hert rte totl minutes of developmentl time (= from oviposition to hthing) of eggs inuted t tht temperture. Seondly, the totl numer of effetive hert ets (TEHB) ws lulted y sutrting hert rte t developmentl zero from the formul, i.e. TEHB = (hert rte t tht temperture hert rte t the developmentl zero) totl minutes of developmentl time. The hert rte t developmentl zero ws estimted from the eqution desriing the reltionship etween hert rte nd temperture within eh speies. This seond prmeter thus fouses only on hert ets likely to ontriute to emryogenesis, i.e. those ove the sl hert et rte t whih emryos survive ut do not develop. Sttistil nlysis We used the softwre pkge of STATISTICS 6.0 to nlyse dt. Normlity of distriutions nd homogeneity of vrines were tested

3 1304 W.-G. Du nd others using the Kolmogorov Smirnov test nd Brtlett s test, respetively. One-wy nlysis of vrines (ANOVAs) were onduted to test for the influene of inution temperture on the totl numer of hert ets nd the totl numer of effetive hert ets, nd Tukey s post-ho multiple omprisons were used to distinguish mong men vlues of hert ets t eh inution temperture. RESULTS Men egg msses for B. duperreyi, T. septentrionlis nd P. sinensis were 0.447±0.017g (±s.e.m.), 0.271±0.009g (±s.e.m.) nd 4.738±0.166 g (±s.e.m.), respetively. In ll three speies, higher inution tempertures resulted in fster rtes of emryoni hert et (Fig.1A), shorter totl inution periods (Fig.1B) nd higher developmentl rtes (Fig. 1C). Bsed on these liner reltionships etween developmentl rte nd inution temperture, we estimted developmentl zero tempertures (minimum for emryogenesis) s 14.2 C for oth lizrds (B. duperreyi nd T. septentrionlis) nd 16.0 C for the turtle (P. sinensis) (Fig. 1C). In ll three speies, sttistil nlysis showed tht the totl numer of hert ets over the ourse of inution differed signifintly mong inution tempertures (P. sinensis: F 4,170 =32.75, P<0.0001; T. septentrionlis: F 4,65 =53.10, P<0.0001; B. duperreyi: F 5,150 =29.56, P<0.0001). Emryos tht were inuted t low tempertures ompleted more hert ets prior to hthing thn did onspeifi nimls inuted t higher tempertures. The totl numer of hert ets over the ourse of inution inresed onsiderly for emryos developing t tempertures elow 26 C in B. duperreyi nd T. septentrionlis nd t tempertures elow 28 C in P. sinensis (Fig.2). By ontrst, the totl numer of effetive hert ets over the ourse of inution ws similr ross inution tempertures in ll speies (P. sinensis: F 4,170 =2.14, P=0.08; T. septentrionlis: F 4,65 =1.68, P=0.16; B. duperreyi: F 5,150 =1.01, P=0.42) (see Fig.2). The totl numer of hert ets required to omplete emryoni development t stndrd inution temperture (30 C) differed signifintly mong the three speies (F 2,72 =344.78, P<0.001), with P. sinensis requiring more hert ets over the ourse of inution ( vs 4.8 for B. duperreyi nd for T. septentrionlis). A similr interspeifi differene ws evident for the totl numer of effetive hert ets (F 2,72 =252.58, P<0.001), whih verged 5.5 for P. sinensis, for B. duperreyi nd for T. septentrionlis. DISCUSSION Although the emryos of the three speies were proly t different developmentl stges t the time we monitored their hert et rtes, ontogeneti hnges in hert rtes in these speies re so minor tht they would hve little or no impt on our lultions (Rdder nd Shine, 2006) (W.-G.D. nd R.S., unpulished dt). Our mesurements of hert et rtes in lizrd nd turtle emryos revel rod similrity to the results of previous studies on irds in terms of the solute numer of hert ets required to omplete emryogenesis [5 to in the two lizrd speies, in the turtle speies (Fig.2) nd pproximtely 6 to in irds (Ar nd Tzw, 1999)]. The totl numer of hert ets my, however, spn wider rnge in some other verterte lineges, suh s mmmls [e.g. humn emryos tke more thn hert ets to omplete emryogenesis wheres mouse emryos tke out hert ets: lulted from dt in Meier et l. (Meier et l., 1983)]. Lizrds nd turtles lso resemle other tx in showing rod onstny of totl hert et numers within emryos of single speies (Fig. 2). Tht reltive onstny pplies despite Hert rte (ets min 1 ) Inution durtion (dys) Developmentl rte 160 Pelodisus sinensis HR=0.134T T , R 2 =0.997 Tkydromus septentrionlis 140 HR=0.045T T , R 2 =0.999 Bssin duperreyi 120 HR=0.093T T 6.811, R 2 = A B C DR=0.058T 0.917, R 2 =0.980 DR=0.054T 0.773, R 2 =0.993 DR=0.067T 0.947, R 2 = Inution temperture ( C) Fig. 1. Effets of men inution temperture on (A) hert et rtes (ets min 1 ), (B) totl durtion of inution nd (C) developmentl rtes of the turtle Pelodisus sinensis nd the lizrds Tkydromus septentrionlis nd Bssin duperreyi. (A) Hert et rtes were mesured y non-invsive methods nd re expressed s mens ± s.e.m. (B) Men inution durtions were olleted from pulished reports on B. duperreyi (Shine nd Hrlow, 1996), T. septentrionlis (Du, 2003) nd P. sinensis (Du nd Ji, 2003; Ji et l., 2003). (C) Developmentl rte t eh temperture ws lulted y dividing inution durtion y the shortest inution durtion reorded in the lortory nd tking the inverse of this vlue. Dt on T. septentrionlis nd P. sinensis were tken from Du (Du, 2003) nd Du nd Ji (Du nd Ji, 2003), respetively. Dt on B. duperreyi were tken from Shine nd Hrlow (Shine nd Hrlow, 1996). HR, hert rte; T, temperture; DR, developmentl rte. twofold vrition in totl inution durtion in the non-vin reptiles we studied, vrition driven y inution temperture (Fig.1B). The numers quoted ove tully underestimte the degree of similrity etween irds, turtles nd lizrds in totl numer of hert ets during inution. Both turtles nd irds ly eggs with reltively undeveloped emryos, so tht the period from oviposition to hthing enompsses ll of emryogenesis (Booth nd

4 Totl numer of hert ets in reptiles 1305 Hert ets Pelodisus sinensis Tkydromus septentrionlis Bssin duperreyi Totl hert ets Totl effetive hert ets Inution temperture ( C) Thompson, 1991; Andrews, 2004). By ontrst, most squmtes (inluding the two lizrd speies tht we studied) dely oviposition until the emryo is prtwy (typilly round 25%) through the totl period of emryoni development (Shine, 1983; Andrews, 2004); nd, thus, the numer of hert ets estimted for lizrds over the period from oviposition to hthing is sustntilly less thn the totl numer over the entire ourse of emryogenesis. It would e of gret interest to study lizrds tht ly their eggs t muh erlier stges of emryoni development, s in some hmeleons (Andrews et l., 2008) nd to determine whether the inlusion of this missing frtion (pre-lying) rings the sum hert et ount for lizrds up to the sme level s in irds nd turtles. Despite these similrities, the totl numer of hert ets for emryoni development lerly is not fixed numer nd is ffeted oth y developmentl ftors (suh s the degree of Fig. 2. Therml dependene of the totl numer of hert ets prior to hthing nd the totl numer of effetive hert ets (i.e. exluding those tht our t tempertures too low for emryoni growth nd differentition) in the non-vin reptiles Pelodisus sinensis, Tkydromus septentrionlis nd Bssin duperreyi. Totl hert ets over the ourse of inution inresed t lower tempertures wheres the totl numer of effetive hert ets did not differ signifintly mong tempertures. Dt re expressed s mens ± s.e.m. Mens with different letters ove their error rs re sttistilly different (Tukey s post-ho test)., offspring development t lying nd t hthing) nd y eologil ftors (suh s nest temperture). For exmple, the offspring of ltriil irds hth fter fewer hert ets thn do the offspring of preoil speies (Ar nd Tzw, 1999; Tzw et l., 2001). Similrly, egg size strongly ffets hert et rtes in irds (Ar nd Tzw, 1999) nd developmentl time in mny orgnisms (Gillooly et l., 2002). In non-vin reptiles, therml onditions in the nest will proly e the most importnt proximte ftor ffeting oth hert rtes nd the durtion of inution. Lortory experiments generlly hve reported little to no effet of hydri onditions on inution durtion (e.g. Fltt et l., 2001; Ji nd Du, 2001; Booth, 2002) or hert rtes (Du nd Shine, 2008) wheres temperture strongly ffets oth of these vriles (Birhrd nd Deeming, 2004; Rdder nd Shine, 2006) (see Fig.1A nd Fig. 1B). The therml dependenies of emryogenesis nd of hert rtes effetively nel eh other out over rnge of wrm onditions, i.e. development elertes with temperture s the sme rte s hert et, thus mintining n pproximte onstny in totl hert ets prior to hthing (Fig. 2). Tht equivlene disppers t lower tempertures, however, suh tht ool-inuted emryos omplete more hert ets prior to hthing thn do their wrm-inuted silings (Fig. 2). Although therml limtion might ffet the rte of emryoni hert ets [s found in the snpping turtle, Chelydr serpentin (Birhrd nd Reier, 1996)], most studies on this topi hve onluded tht the growth effiienies nd energetis of reptilin emryos show little evidene of therml limtion (Whitehed et l., 1992; Angillett et l., 2006). We ttriute the inrese in umultive numer of hert ets t lower tempertures to thermlly-driven shift in the mgnitude of mintenne osts reltive to the rdiovsulr effort devoted to produtive emryogenesis. At the developmentl zero temperture, hert et rtes deline ut do not ese. Under these onditions, ll of the emryo s metoli work is direted to mintenne. Below the developmentl zero temperture, the emryo ontinues to respire (nd thus its hert ontinues to et, leit slowly) ut it does not grow or differentite. Thus, for exmple, n emryo exposed to flututing therml regime tht rises ove developmentl zero for only few hours per dy will neessrily umulte lrge numer of mintenne hert ets tht hieve little or nothing towrds furthering its ontogeneti development. By the end of inution, suh n emryo will thus hve ompleted mny more hert ets, in solute terms, thn wrm-inuted siling. This interprettion is supported y the onstny of umultive numer of effetive hert ets ross wide rnge of inution onditions, in ontrst to the therml dependene of totl numer of hert ets (Fig. 2). These results support nd extend ides regrding onstny in the numer of degree-dys needed to omplete the developmentl proess (Trudgill et l., 2005). The onept of the totl effetive hert et provides funtionl explntion for the reltionship etween inution temperture nd developmentl time. Why don t ird eggs show similr therml dependene in totl numers of hert ets? They my well do so ut ny therml effet is msked y the ft tht: (1) mternl rooding redues the vrine in therml regimes during inution, oth mong eggs within luth nd mong luthes; nd (2) temperture my hve little effet on hert et rte within the ird s thermoneutrl zone (Ar nd Tzw, 1999; Tzw, 2005). Non-vin reptiles experiene different onditions. Mny emryos of oviprous reptiles develop in shllow nests tht disply wide therml flututions on diel nd/or sesonl yle (Shine et l., 1997; Akermn nd Lott, 2004; Shine, 2004; Du nd Feng, 2008). Aordingly, emryos often

5 1306 W.-G. Du nd others experiene tempertures elow the developmentl zero for onsiderle periods of time. For exmple, developmentl zero is 14.2 C in T. septentrionlis nd B. duperreyi ut tempertures in nturl nests hve een reorded to fll to 11.6 C for T. septentrionlis (Du nd Ji, 2006) nd 9 C for B. duperreyi (Shine nd Hrlow, 1996). Beuse they experiene more vrile nd less preditle therml environment (unlike mternlly-rooded ird eggs), squmte eggs thus must e le to djust their totl rdiovsulr effort flexily to onditions in the nest. We thnk Melnie Elphik nd Rory Telemeo for olleting B. duperreyi eggs from the field, nd Hu Ye for ssistne in the lortory. Keith Simpson of Avin Bioteh helped us to refine the use of the infrred monitoring system. Ethis pprovl ws given y the University of Sydney Animl Ethis Committee. This work ws supported y grnts from the Nturl Siene Foundtion of Chin ( ) nd the University of Sydney to W.-G.D., nd y the Austrlin Reserh Counil to R.S. REFERENCES Akermn, R. 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