J Cmp Physil B (1989) 159 : 183-193 Jurnal f Cmparative Systemic, ~.m,... and nvirn~ Physilgy B ~"' Physilgy (~ Springer-Verlag 1989 ffects f incubatin temperature n grwth and develpment f embrys f Alligatr mississippiensis D.C. Deeming and M.W.J. Fergusn* Department f Cell and Structural Bilgy, University f Manchester, Cupland Building, Cupland Street, Manchester M13 9PL, United Kingdm Accepted February 7,1989 Summary. ggs f Alligatr mississippiensis were incubated at 3 ~ and 33 ~ thrughut incubatin up t hatching. very fur days several eggs were pened and the albumen, ylk and extra-embrynic fluids remved and weighed. The embry was remved and fixed prir t being staged, weighed and measured fr varius mrphmetric criteria. Develpment at 33 ~ was accelerated cmpared with 3 ~ in terms f ylk and albumen utilizatin and embry grwth. Significant lsses in ylk mass did nt ccur until stage 22 at 33 ~ but ccurred at stage 18 at 3 ~ Different patterns in grwth were bserved in embrys at the tw temperatures at similar mrphlgical stages: between stages 18 and 22 embrys at 33 ~ were smaller (in mass and length) cmpared with embrys at 3 ~ despite being mrphlgically similar. The differences in grwth and physilgy between embrys at 3 ~ (females) and 33 ~ (males) were dependent n incubatin temperature but nt sex. ncubatin at 33 ~ accelerated bth grwth and develpment in Alligatr; initially mrphgenesis was accelerated by the higher temperature but later, grwth rate was accelerated. Key wrds: Crcdilians - Mrphmetrics - xtra-embrynic fluids - Grwth sex determinatin ntrductin Temperature has prfund effects upn the physilgy f all animals. n particular, the rate f develpment f reptilian embrys is temperature-dependent; high temperatures (within the viable range) accelerate embrynic develpment cmpared with lwer temperatures. This is usually b- * T whm ffprint requests shuld be sent served in the length f the incubatin perid, because as incubatin temperature increases s hatching ccurs earlier (wert 1979, 1985; Packard and Packard 1988). Reprts f the effects f temperature n embrynic develpment are rarer. mbrynic grwth rates at different temperatures have been reprted in nly a few species f turtles (Pieau and Drizzi 1981 ; Packard et al. 1987), ne species f snake (Vinegar 1973), a lizard (Madersn and Bellairs 1962; Hlder and Bellairs 1962) and three crcdilian species (Webb et al. 1987; Lang et al. in press; Deeming and Fergusn 1988). Sme data n the effects f temperature n the embrynic utilisatin f ylk and albumen are knwn fr Crcdylus jhnstni (Manlis et al. 1987). This lack f data cncerning the effects f temperature n embrynic grwth is surprising, cnsidering the significance f temperature in the bilgy f reptiles. n crcdilians alne, incubatin temperature has been shwn t affect sex determinatin (Fergusn and Janen 1982, 1983; Webb and Smith 1984; Webb et al. 1987; Huttn 1987; Lang et al., in press), embrynic grwth rates (Deeming and Fergusn 1988), pigmentatin patterns f hatchlings (Deeming and Fergusn, in press a; Murray et al., in press), pst-hatching thermregulatin (Lang 1987) and grwth (Janen et al. 1987). This paper presents data n the effects f incubatin at 3 ~ and 33 ~ n the develpment f embrys f Alligatr mississippiensis, using a sample f eggs different t that described in Deeming and Fergusn 1988, in press b). American alligatrs have temperature-dependent sex determinatin and these temperatures prduce 1% female and male hatchlings, respectively (Fergusn and Janen 1982, 1983). Changes in the extra-embrynic fluids, albumen and ylk are recrded thrughut the incubatin perid, tgether with
184 D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment the patterns f embrynic grwth using mass and mrphmetric measurements (Deeming and Fergusn, in press b). Mrphmetric criteria are used t generate equatins t predict age and stage f develpment f embrys incubated at 3 ~ and 33 ~ Materials and methds ggs f Alligatr mississippiensis were cllected in 1988 frm several nests f wild alligatrs at the Rckefeller Wildlife Refuge, Luisiana, USA, n the first r secnd day after laying and were immediately transprted by air t Manchester, UK. On arrival at the labratry (day 3 r 4 depending n clutch) the eggs were weighed and set in incubatrs (Vinden Scientific Ltd) accurate t _+.1 ~ ggs were incubated at either 3 ~ r 33 ~ (temperatures which prduce 1% females and 1% males, respectively) and clutches were randmised between incubatrs. Bth incubatrs were maintained at a relative humidity as clse as pssible t 1%. Starting n day 8 f incubatin, and n every furth day thereafter (time f vipsitin = start f incubatin), eggs were remved frm the incubatrs. gg mass (g), length and width (in millimetres measured using Vernier calipers t the nearest.1 mm) were measured and the eggs were placed n ice prir t pening. During the first three weeks f incubatin the embry was remved first and placed in 1% frmal saline (a slutin f.133m1"1-1 HCHO,.37m1.1-1 Na2HPO4 and.25 tl.l-1 NaH2PO4). The sub-embrynic fluid was drained frm the egg and was weighed. Ylk sac, albumen and eggshell mass were then recrded. After day 24 f incubatin, allantic fluid and amnitic fluid were remved prir t remval f the embry and ther cmpnents. On day 6 f incubatin at 33 ~ and day 68 at 3 ~ embrys had withdrawn their ylk sac int the abdminal cavity (stage 28; Fergusn 1985). These embrys were weighed intact prir t remval f the ylk sac by dissectin and the embry was reweighed befre fixatin. The egg cmpnents were kept frzen fr future analysis. Ninety eggs were measured fr each temperature with a minimum sample f 4 n each day f incubatin. After fixatin the embrys were staged using the tables f develpment presented by Fergusn (1985). The fllwing mrphmetric parameters were measured (illustrated and described mre fully in Deeming and Fergusn, in press b): embry mass, ttal embry length, tail length, length f the trunk (calculated), nape-t-rump length, distance between the limbs, length f the fre and hind limbs, length, height and width f the head, eye length and length and width, at the nstrils and at the midpint, f the snut. Data were analysed using Minitab (Ryan et al. 1985) and S.A.S. (Allen Ray 1982) statistical packages n an Amdahl 589-3 mainframe cmputer. The develpment rate cefficient (Webb et al. 1987) was calculated fr embrys at 33 ~ (DRC33) as fllws. d embrys incubated at 33 ~ were allcated a 'mrphlgical age' (in days) derived frm the time that each stage ccurs in embrys at 3 ~ 'Mrphlgical age' was pltted against real age (days) and this relatinship was analysed using linear regressin (the line was frced thrugh zer). DRC33 is equal t the slpe f the regressin line (Webb et al. 1987). Linear regressin estimates fr mrphmetric measurements against time and stage were calculated n untransfrmed, square rt transfrmed and lge transfrmed data depending n the crrelatin cefficient f the regressin estimate. The equatins with the highest R 2 (i.e. the equatin which explained the maximum prprtin f the vari- Table 1. Mean dimensins f all Alligatr eggs used in the present study Temperature 3 ~ 33 ~ Mean S Mean S nitial egg mass (g) 72.66 6.14 72.37 5.95 gg length (mm) 71.43 2.81 71.41 3.18 gg breadth (ram) 41.55 1.18 41.34 1.15 gg vlume (can 3) 64.68 5.17 63.99 5.34 Weight lss lg" day- 1).87.34.89.36 ance in the data) were used (Deeming and Fergusn, in press b). Data frm the tw incubatin temperatures were cmpared using GLM (S.A.S.) analysis f variance (using type sums f squares). Principle cmpnents analysis (S.A.S.) was used t derive predictive equatins fr age and stage f develpment using a cmbinatin f 1 mrphmetric measurements cndensed int ne, PRN (Deeming and Fergusn, in press b). Results gg dimensins and cntents Mean values fr initial egg mass, egg dimensins and vlume are shwn in Table 1. The eggs lst water thrughut incubatin despite the high humidity within the incubatr, but the mean rates f water lss were similar at bth temperatures (Table 1). nitial egg mass culd nt be strictly cntrlled between samples and s, in rder t reduce the effects f egg mass n the egg cntents, data were standardised t a mean egg mass f 72.19 g (the mean value fr initial mass f 6 alligatr eggs transprted t Manchester in 1988). Mean eggshell mass was nt significantly different at the tw temperatures (9.66-t-.91 g at 3 ~ and 9.61 _+ 1.5 g at 33 ~ The patterns f change in the extra-embrynic cmpnents f the eggs are shwn in Fig. 1. Utilisatin f albumen by the embry during incubatin (Fig. a) was cntinuus at 33 ~ but at 3 ~ arund 6 g f albumen persisted during the middle third f the incubatin perid. Analysis f variance revealed that althugh temperature had n significant effect n albumen mass at each stage f develpment there was a significant difference in the patterns f utilisatin at the tw temperatures. At 3 ~ the albumen cntent f the egg remained at a mean f 6 g frm stage 17 t 23 cmpared with a mean f 1 g between stages 12 and 2 at 33 ~ (Fig. 1 a). Similarly changes in ylk sac mass with time were significant (Fig. 1 b). Analysis f variance shwed that ylk
D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 185 16 -& v12 g _g 8-5 4 5 ~ - g x ~1 ~ ~12 ]6 J k \ i t. L a ~ ~ ' 2 ~ 4' '''b;'~'''2'~' 6 8 16 i 1 4 &' 8' 8 16 24 32 28 24 ~k,',- V~-',,~ \.-& 4 "8 3 2 A~ '/ m ~ 2 >'16 ~ ~2 \ \ "8 d i ~ 4' 1 6 8',~. ' 16 ' 2~ ' 4 b ' 2' 41 ' &' \ 8'6 8 ' ' " 6 ' 24 ' ' rr 12 -&~ '\k Fig. 1 a-e. The influence f incubatin temperature n the mass f the extra-embrynic cmpnents (standardized t an initial egg mass f 72.19 g) during incubatin and at different stages thrugh develpment. Values are means f at least fur samples. Open symbls = 3 ~ clsed symbls = 33 ~ a albumen, b ylk sac, e sub-embrynic fluid, d amnitic fluid and e allantic fluid r ' 2;' L' d' ~; i i J d 1 16 ' 214 ' sac mass, pltted against stage f develpment, was significantly affected by temperature (P <.5) and the patterns f ylk utilisatin were significantly different at the tw temperatures. At 3 ~ the size f the ylk sac diminished after stage 17 but at 33 ~ this did nt ccur until stage 21 (Fig. b). Mass f sub-embrynic fluid in the eggs was significantly affected by temperature n bth the different days f incubatin and the different stages f develpment (Fig. 1 c). At 33 ~ there was a rapid lss in the fluid after a shrt peak in mass but at 3 ~ the maximum amunt f sub-embrynic fluid was present fr a lnger perid f time. Changes in the amunt f amnitic fluid with time were different at the tw temperatures but they ccurred at equivalent stages f develpment (Fig. 1 d). Detectable amunts f amnitic fluid were present n day 2 f incubatin (stage 17 at 33 ~ and stage 15 at 3 ~ Frmatin f allan-
186 D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment Table 2. Mean values (_ SD) fr stage f develpment (Fergusn 1985) thrugh incubatin fr embrys f Alligatr mississippiensis at 3 ~ and 33 ~ Day 3 ~ 33 ~ 8 8.. 7.6.5 12 1.. 11.2.8 16 14.. 15.. 2 15.. 17.. 24 17.. 19.2.4 28 18.. 21.8.4 32 19.. 22.. 36 21.. 23.. 4 21.. 23.. 44 23.. 24.. 48 23.. 24.. 52 24.. 25.. 55 24.. 26.2 1.1 6 25.. 27.9.3 64 25.. 28.. 68 26.6.9 -- - 75 28.. -- - tic fluid ccurred earlier in incubatin and at an earlier stage f develpment in eggs at 33 ~ cmpared with eggs at 3 ~ (Fig. l e). Substantial amunts f allantic fluid were present at the end f incubatin but this zed ut and was lst during hatching. mbry grwth Develpment f Alligatr embrys, as assessed by stage, was accelerated by incubatin at 33 ~ cmpared with 3 ~ (Table 2). DRC33 was 1.29+.19. 28 (equivalent t hatchling) was attained by day 6 at 33 ~ and by day 68 at 3 ~ Hatching ccured by 64 days at 33 ~ but at 3 ~ embrys were terminated at 75 days f incubatin when they were clse t hatching. mbrys grew expnentially (as measured by mass after fixatin) up t hatching at bth temperatures but the rate f grwth was slwer at 3 ~ than at 33 ~ (Fig. 2). Fr a lng perid f incubatin (days 24 t 55) embry mass at 3 ~ was 5% f that at 33 ~ and incubatin at the lwer temperature retarded grwth by 8 days. Temperature did nt significantly affect grwth at equivalent stages althugh analysis f variance shwed that the patterns f grwth at the tw temperatures were different (Fig. 2). Between stages 18 and 22 embrys at 33 ~ were smaller than at 3 ~ but shwed rapid grwth between stages 22 and 25. At 3 ~ grwth was mre sustained with maximal grwth ccurring during stages 23 t 27. This pattern was repeated fr mst f the mrphmetric parameters measured. mbrynic c 3 +_.x 4 4 / 2O "6 1 x i i i i i i i t 1 2 4O 6O 8O j ' ' 8 1" 6 ' 24 J ' Fig. 2. Grwth f the alligatr embry, measured as mass after fixatin, thrugh time at tw incubatin temperatures. mbry mass at different incubatin stages is shwn. Open symbls = 3 ~ clsed symbls = 33 ~ S "B JC: Q3 g 3 2 1 7 / /?? trt "e'9 -'z' 4 6''d; 1; 2'4 Fig. 3. The influence f temperature n grwth f the alligatr embry, measured as ttal length, pltted against time and different stages f develpment. Open symbls = 3 ~ clsed symbls = 33 ~ grwth thrugh time, as measured by linear dimensins, was enhanced by increased incubatin temperature but the patterns f grwth at equivalent mrphlgical stages were different at the tw temperatures. Ttal length f the embry increased thrugh time at a faster rate at 33 ~ cmpared with embrys at 3 ~ but grwth was nt cntinuus (Fig. 3). There was a distinct plateau in embry!
D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 187 #- 15 4 1 J/ /./ q~ l "S 2 Cn C cl "s 5 a 1 / / / 4` 8 '; i 2 16 12 n~ Cl _c 8 ~6 a ' 2 '' 2'd'' 8 ; / g.j 5 / / "r- b ' '' ' 24` ' 'd'a'' ;' Fig. 5a, b. Grwth f the head f the alligatr embry as affected by temperature and pltted against time and stage f develpment. Open symbls = 3 ~ clsed symbls = 33 ~ a length f head and b height f the head 5 4` = 3 2 2O s b 2; i J l i L, ~ i 4, 6' 8' 8 16 ' 2'4` 21 // ""e"~ /,r / 4`l 6 ; ; ; 16 214` 1 Fig. 4a-c. Grwth f varius parts f the bdy f the alligatr embry as affected by incubatin temperature pltted against time and different stages f develpment. Open symbls= 3 ~ clsed symbls=33 ~ a length f the tail, b length f the trunk and e length f the frelimb length which was achieved well befre hatching. mbry length at equivalent stages shwed a similar pattern t mass; at 33 ~ embrys at stages 19 t 22 were smaller than embrys at 3 ~ but grew faster during later stages f develpment (Fig. 3). These patterns f grwth were als present in the tail (Fig. 4a), the nape t rump length, distance between the limbs, trunk length (Fig. 4b), and the limbs (length f frelimb is shwn in Fig. 4c). Grwth f the head, as measured by length (Fig. 5 a) shwed similar patterns t ttal embry length but changes in the height and width f the head were different. At bth temperatures, increases in the height f the head shwed a distinct plateau arund 12 mm fr 15 days (stages 17 t 22), befre grwth reached anther plateau prir t hatching (Fig. 5 b). The changes in head width (nt illustrated) were similar with a plateau at 11.5 mm, but fr nly 8 days (stages 19 t 23). ye length increased t a plateau prir t hatching but shwed a different pattern frm stage t stage f develpment (Fig. 6). ye length shwed a plateau at similar stages f develpment t head height and width but during this perid the eye was larger in embrys at 3 ~ cmpared with 33 ~ The snut was measurable frm stage 17 f incubatin (2 days at 33 ~ and 24 days at 3 ~ and increased in length up t hatching (Table 3). The width f the snut shwed a plateau during a simi-
188 D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 1 "~ 8 v 6 ~S _c 4 2 J ' 2' /4' 1 6 ' 8' ; ' ~} ' ] ~ 24l ' Fig. 6. The effects f temperature n grwth f the eye f alligatr embrys during incubatin and at different stages f develpment. Open symbls = 3 ~ clsed symbls = 33 ~ 6 HM= 2.62 *.635 M, R2= 77.8 % "~ 56 ; 9 c~.c 52 "S AC "3 = a8 / cj :~ 44 4 9 ' ' "] ;6 ' 8 ' 6 64 68 2 8 4 M-nitt mss f egg (g) Fig. 7. The relatinship between mass f alligatr hatchlings and the initial mass f the egg. Open symbls = 3 ~ clsed symbls = 33 ~ c..c.9 "6 ~ "S _S.8 i# go.7 z % 9.5.1 ' ' '.2 ' '3 ' Mss f ylk saclmass f hatchling Fig. 8. The relatinship between ylk-free embry mass and residual ylk sac mass expressed as a rati f hatchling mass. Open symbls = 3 ~ clsed symbls = 33 ~ 9 Table 3. Length and width at the mid-pint, (mean in mm_+ SD) f the snut thrugh incubatin fr embrys f Alligatr mississippiensis at 3 ~ and 33 ~ Day Length Width at mid-snut 3 ~ 33 ~ 3 ~ 33 ~ 2 - -- 2.34.15 -- - 2.82.34 24 2.7.41 4.64.38 3.24.6 4.16.26 28 4.3.12 5.96.6 3.76.6 4.8.15 32 4.98.21 7.1.29 4.16.32 4.68.58 36 6.1.17 8.96.27 4..16 6.34.31 4 7.3.31 9.58.15 4.84.23 7.6.51 44 7.96.49 11.16.5 5.9.35 8.86.71 48 9.42.39 11.32 1.3 6.78.44 9.36 1.7 52 1.86.3 13.72.6 8.56.36 11.7.73 55 11.2.3 14.42.42 8.64.27 12.6.24 6 11.98.39 15.13.53 9.9.59 13.8.63 64 13,24.64 15.63.99 11.5.69 13.34.85 68 14.4.51 -- - 12.44.65 - - 75 14.3.4 -- - 12.5.49 - -- lar perid f incubatin as head height and width (width at the mid-pint f the snut is shwn in Table 3) befre recmmencing grwth. Grwth f the snut fllwed the patterns exhibited by ther dimensins f the embry. The effects f egg mass n embry size Data fr all embrys at stages 23 t 28 f develpment were examined fr any indicatin f egg size affecting embry mass r embry mrphmetrics. mbrys at earlier stages were cnsidered t be t small t be affected by egg size. Regressin estimates fr fixed embry mass against initial egg mass did nt shw any effect until the final stage f develpment (stage 28) but the crrelatin cefficient was nly 22.3%. There was n significant relatinship between egg length and embry length; at stage 28 the crrelatin cefficient was 7.4%. Hatchling mass (all embrys at stage 28) was related t initial egg mass (Fig. 7) and n average cnstituted 66.76 _ 3.55% f the initial mass f the egg. There was a clear relatinship between embry mass and ylk sac mass; larger embrys had smaller ylk sacs (Fig. 8). Fixatin als reduced embry mass (Fig. 9). Use f mrphmetrics t predict embrynic develpment Principle cmpnent analysis applied t mrphmetric data against days f incubatin shwed that
D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 189 5 3 x L~ / //O // 9 // 9 9 Table 4. Develpment rate cefficients at 33 ~ (DRC33) and the difference in days in incubatin perid at 3 ~ and 33 ~ fr fur species f crcdilian (DRC3 = 1.) Species DRC33 Reference ~4 ~5 l Lt3 LL 3 /// // 9,,~ %-: 3.26+ 1.2 W M,RZ= 95.8 % 28 3'2 36 4' 44 4'8 52 6'6 WM- Mass fwet embry (g) Fig. 9. The effects f fixatin upn the mass f stage 28 embrys. Open symbls = 3 ~ clsed symbls = 33 ~ 24 2 16 Z ~12 C~ 8 4 a / iii /11 ii --3~ 33c b f t i i i i 2 2 6 8 ; A Dc~ys Fig. 1. The effect f incubatin temperature n the relatinships between PRN and develpmental time (plt a), and stage f develpment (plt b) temperature had a significant effect upn the relatinship between time and PRN (Fig. 1a): At 3 ~ PRN =.31. + 5.87; R 2 = 96.6%, At 33 ~ PRN =.284. + 4.97; R 2 = 98.5%. By cntrast, values fr PRN versus stage f develpment generated fr bth temperatures were similar t each ther (Fig. 1b): At 3 ~ PRN =.419. +.793 ; R 2 = 97.1%, At 33 ~ PRN =.451. +.216; R 2 = 98.4%. Alligatr mississippiensis 1.29" 8 Janen et al. 1987 Crcdylus palustris 1.256 1 l Lang et al., in press Credylusprsus 1.28 15 Webb et al. 1987 Credylusjhnstni 1.329 22 Webb et al. 1987 a Present study Discussin ffects f incubatin temperature n grwth and mrphgenesis Grwth f Alligatr embrys thrugh time was significantly affected by incubatin temperature, cnfirming previus reprts that increasing incubatin temperature accelerates the grwth rate f reptilian embrys (Vinegar 1973; Deeming and Fergusn 1988). Mrphlgical stages are ften used t assess rate f develpment, and increasing incubatin temperature accelerates develpment (Pieau and Drizzi 1981; Webb et al. 1987; Lang et al., in press). ncubatin temperature als had significant effects n mrphgenesis f Alligatr embrys which was accelerated at 33 ~ with each stage f develpment ccurring earlier during incubatin than at 3 ~ The develpment rate cefficient at 33 ~ (DRC33; Webb et al. 1987) recrded fr embrys f A. mississippiensis was lwer than has been recrded fr ther crcdilian species (Table 4). There is a clear crrelatin between DRC33 and the differences in the length f the incubatin perid at 3 ~ and 33 ~ Temperature has less effect upn the rate f embrynic develpment in A. mississippiensis than in Crcdylus species (Table 4). ncubatin at 33 ~ appeared t increase the rate f mrphgenesis at the expense f increase in the size f embrys; at 3 ~ grwth and mrphgenesis shwed a clser assciatin. At particular stages f develpment differences in the size f embrys were bserved. During stages 19 t 22 embrys at 3 ~ were heavier than thse embrys at 33 ~ but after stage 22 grwth rates f embrys at 33 ~ were increased rapidly up t stage 25. This pattern was reflected in bth mass and many f the linear dimensins recrded. At stage 2 the eyes f embrys at 3 ~ were larger than in embrys at 33 ~ despite the similarity in verall mrphlgy. Mrphgenesis at the expense f grwth has been bserved in the embrynic gnad f A. mississippiensis (Deeming and Fergusn
19 D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 1988). At 33 ~ there is rapid differentiatin f the medulla f the embrynic gnad and by 34 days f incubatin there are distinct signs f testicular rganisatin; at 3 ~ cellular rganisatin f the gnad t frm an vary is nt apparent until after day 47 (Deeming and Fergusn 1988). The pattern f pigmentatin in hatchlings f A. mississippiensis is als affected by incubatin temperature (Deeming and Fergusn, in press a). The pigmentatin pattern becmes apparent at stage 22 when melanin is depsited in the skin (Fergusn 1985). At 33 ~ pigmentatin is bserved n day 36 f incubatin cmpared with day 44 at 3 ~ (Murray et al., in press). ncubatin temperature significantly affected extra-embrynic cmpnents and therefre, by implicatin, embrynic physilgy. Previusly nly ne reprt has detailed the changes in the extraembrynic fluids f a reptile egg (Crcdylusjhnstni, Manlis et al. 1987) but the effects f temperature were nt extensively studied. xaminatin f the changes in the extra-embrynic fluids thrugh time and at different stages f develpment in A. mississippiensis has revealed interesting effects f temperature. The prcesses f fluid frmatin and utilisatin are mre rapid at 33 ~ The amunt f ylk did nt begin t decline until stage 22 f develpment in eggs at 33 ~ cmpared with stage 18 at 3 ~ This reflects the reduced mass f embrys at 33 ~ between these stages. Patterns f albumen utilizatin thrugh develpmental time vary accrding t incubatin temperature (Manlis et al. 1987), but in additin there were differences at different stages f develpment. The transfer f water frm the albumen t the sub-embrynic fluid (wert 1979; Manlis et al. 1987) cntinued up t stage 17 at 3 ~ but had stpped by stage 12 at 33 ~ The rapid decline in albumen mass ccurred after stage 2 at 33 ~ and after stage 23 at 3 ~ The amunts f subembrynic fluid increased and declined rapidly at 33 ~ but maximum levels were prlnged at 3 ~ n C. jhnstni the maximum amunt f sub-embrynic fluid ccurred earlier at 33 ~ cmpared with 3 ~ but ccurred at similar stages f develpment (Manlis et al. 1987). n bth A. mississippiensis and C. jhnstni, at 3 ~ and 33 ~ lss f sub-embrynic fluid ccurred at stage 24 (Manlis et al. 1987). Frmatin f allan- tic fluid was accelerated at 33 ~ ccurring bth at an earlier time and an earlier stage, mirrring the decline in sub-embrynic fluid. Later in develpment the amunt f allantic fluid in eggs at the tw temperatures was similar. n summary, incubatin at 33 ~ accelerates bth differentiatin and grwth. The rates f utilisatin f albumen and ylk and frmatin f the extra-embrynic fluids are als accelerated. These bservatins reflect different physilgical prcesses f the embry at different incubatin temperatures: develpment f Alligatr embrys at 33 ~ is nt simply scaled up frm develpment at 3 ~ the patterns are different. These results highlight the imprtance f incubatin temperature in the develpment f reptilian embrys. Althugh staging embrys may be useful fr standardizing develpment (Miller 1985; Fergusn 1985, 1987) the effects f temperature must be appreciated in any experimental design. A previus examinatin f embrynic grwth patterns revealed that A. mississippiensis grew differently t Crcdylus prsus and C. jhnstni (Deeming and Fergusn, in press b). Cmpared with tw species f Crcdylus, Alligatr embrys develped very quickly sustaining a rapid rate f grwth thrughut incubatin and emerging as lng but light hatchlings (Deeming and Fergusn, in press b). n Crcdylus hatchlings were lng but heavier, having spent mre time in the egg (Deeming and Fergusn, in press b). The lw DRC33 recrded in this study cnfirms that develpment is less temperature-dependent in Alligatr than in ther crcdilians. This may be imprtant in the bilgy f A. mississippiensis which ccurs at the nrthernmst limit fr crcdilians (Grmbridge 1987). t hibernates during the cl winter mnths and hatchlings have nly a few weeks in rder t feed befre hibernatin. Selectin has acted upn alligatr ppulatins t prduce embrys which develp rapidly (Deeming and Fergusn, in press b). nterestingly, the rate f embrynic develpment in different ppulatins f turtles als shws much less dependence n incubatin temperature depending n latitude (wert 1985; Legler 1985). Hatching, egg size and hatchling mass mbry mass frm stages 23 t 28 did nt shw any significant relatinship t either egg mass r egg length, cntrary t reprts fr ther species f crcdilian (Webb etal. 1983a, 1983b, 1987). By cntrast, the mass f hatchling alligatrs (embry plus abdminal ylk sac) was related t initial egg mass. n birds, egg size begins t affect embry size after day 16 f incubatin and the effect is very clear at hatching (Burtn and Tullett 1985). The results f the present study may reflect the small variatin in sizes between samples and further investigatin is required t dcument fully the
D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 191 effects f initial egg mass n the patterns f embrynic grwth in crcdilians. The rati f hatchling mass t initial egg mass in Alligatr was similar t that reprted fr preccial birds, several lizards and turtles but lwer than that reprted fr anther species f crcdilian, ther turtles and squamates (wert 1979). Ylkfree hatchling mass was inversely related t abdminal ylk mass and at 3 ~ relatively mre ylk had been cnverted int tissue cmpared with hatchlings frm eggs at 33 ~ This cnfirms a previus reprt which shwed that the energetic requirements f the crcdilian embry are greater at lwer incubatin temperatures: mre ylk is utilised during the prlnged incubatin perid and embry mass is greater at hatching (Manlis et al. 1987). The amunt f abdminal ylk is related t incubatin length in ther reptiles. n sme turtles, as the nest substrate becme prgressively drier the incubatin perid is shrter and hatchlings cntain mre abdminal ylk (Mrris et al. 1983). During hatching f Alligatr embrys allantic fluid was lst frm the egg, cnfirming reprts f fluid lss frm hatching eggs f the gharial (Gavialis gangeticus) and sme squamate reptiles (Bustard 1966; Badham 1971; Subb Ra 1987; Deeming, in press). At hatching, the amunt f allantic fluid present in crcdilian eggs (Manlis et al. 1987), and prbably thse f mst ther reptiles, is relatively large but its significance has been largely ignred (Deeming, in press). n crcdilian embrys urea is the main nitrgenus excretry prduct: it is stred in the allantic fluid (Manlis et al. 1987). By cntrast, avian embrys cnvert urea int uric acid (Rmanff 1967) which is left as slid depsits in the eggshell after hatching; the water in allantic fluid is absrbed prir t hatching. Crcdilians appear then t leave a slutin f their nitrgenus waste in the eggshell at hatching (Deeming, in press). The amunt f allantic fluid that wuld have zed frm the egg wuld prbably have been greater if it had nt lst water during incubatin. The extent f water lst frm eggs in the present study was prbably abnrmal (Fergusn 1985), but the effects f excessive lss f water (> 15% f initial egg mass) n embrynic physilgy have nt been examined in crcdilians (Manlis et al. 1987). The use f mrphmetrics t predict embrynic age and stage Recent studies f embrynic develpment in crcdilians have ften relied upn field preservatin f intact eggs (Webb and Manlis 1987). Many studies have described staging criteria and grwth f embrys fixed in frmal saline (Magnussn and Taylr 198; Webb et al. 1983a, 1983b; Fergusn and Janen 1982, 1983; Fergusn 1985, 1987; Deeming and Fergusn 1988, in press b) but sme studies describe fresh embry mass (Manlis et al. 1987). Fixatin causes a significant lss f water frm tissue leading t shrinkage f the embry. Future studies f embrynic develpment must take shrinkage assciated with fixatin int accunt, particularly in cmparative studies. ncubatin temperature significantly affects the develpment rate f Alligatr embrys. Predictive equatins fr embrynic age and stage, generated fr embrys at 3 ~ and 33 ~ are useful in labratry studies but equatins generated frm pled data frm a different sample f eggs incubated at a variety f temperatures (Deeming and Fergusn, in press b) remain mre useful fr field studies. The incubatin temperature f a nest is nt accurately reflected by a single temperature recrding n any particular day; reptile nests increase in temperature thrughut incubatin, but temperature may als reflect the climate (Packard and Packard 1988). quatins using a mean incubatin temperature are mre useful in allcating an age t embrys in nests. quatins t predict stage f develpment f embrys are less affected by incubatin temperature and are f particular value in assigning a stage t a particular embry, particularly if the investigatr has a pr knwledge f embrylgy. mbry grwth and sex determinatin The tw temperatures in this study were chsen because they prduce hatchlings f nly ne sex; in A. mississippiensis eggs at 3 ~ prduce 1% females, eggs at 33 ~ 1% males (Fergusn and Janen 1982, 1983). This study has demnstrated perids during which embrys incubated at 3 ~ and 33 ~ shw differences in size and physilgy: interestingly these crrespnd t imprtant times fr sex determinatin (Deeming and Fergusn 1988). Differential grwth f particular types f embrynic tissue has been suggested t be imprtant in the mechanism f temperature dependent sex determinatin. n mammals, male embrys grw faster than females frm the earliest stages (Cattanach et al. 1988) and differences in the rate f grwth f the embrynic gnad have been suggested t be imprtant in the sex determining mechanism (Mittwch 1985). n additin, differential grwth f the adrenal-kidney cmplex relative
i92 D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment t the gnad at different incubatin temperatures has been suggested t be part f the mechanism f temperature-dependent sex determinatin in C. jhnstni (Webb and Smith 1984). Whether the differences in embrynic grwth between 3 ~ C and 33 ~ are a cause r an effect f sex determinatin remains unclear (Deeming and Fergusn 1988, in press a). Acknwledgements. We thank Ted Janen, Larry McNease and Dave Richard f the Rckefeller Wildlife Refuge, Luisiana Department f Wildlife and Fisheries, USA fr their invaluable assistance ver several years with the cllectin and transprtatin f eggs f A. mississippiensis. We thank Dr..K. Kyprianu and Dr. C.T. Rberts fr advice with the statistical analysis, and Marin Pultn, Chris rwin and Alan Rgers fr invaluable assistance with the preparatin f Alligatr eggs. DCD is funded by the University f Manchester Research Supprt Fund. References Allen Ray A (1982) SAS Users Guide: Statistics, 1982 ditin. SAS nstitute, Cary, Nrth Carlina Badham JA (1971) Albumen frmatin in eggs f the agamid Amphiblurus barbatus barbatus. Cpeia: 543-545 Burtn FG, Tullett SG (1985) The effects f egg weight and shell prsity n the grwth and water balance f the chicken embry. Cmp Bichem Physil 81A:377-385 Bustard HR (1966) Ntes n the eggs, incubatin and yung f the bearded dragn, Amphiblrus barbatus barbatus (Cuvier). Br J Herpetl 3 : 252-259 Cattanach BM, Rasberry C, Beechey CV (1988) Sex reversal and retardatin f embrynic develpment. Prceedings f the 6th nternatinal Wrkshp n Mlecular Genetics f the Muse, Abstract N 3 Deeming DC (in press) The residues in the eggs f squamate reptiles. Herp J Deeming DC, Fergusn MWJ (1988) nvirnmental regulatin f sex determinatin in reptiles. Phil Trans R Sc Lnd B 322:19-39 Deeming DC, Fergusn MWJ (in press a) The mechanism f temperature dependent sex determinatin in crcdilians: a hypthesis. Am Zl Deeming DC, Fergusn MWJ (in press b) Mrphmetric analysis f embrynic grwth in Alligatr mississippiensis, Crcdylusjhnstni and Credylus prsus. J Zl Lnd wert MA (1985) mbrylgy f turtles. n: Gans C, Billet F, Madersn PFA (eds) Bilgy f the reptilia, vl 14: Develpment A. Wiley, New Yrk, pp 75-267 wert MA (1979) The embry and its egg: develpment and natural histry. n: Harless M, Mrlck H (eds) Turtles perspectives and research. Wiley-nterscience, New Yrk, pp 333-413 Fergusn MWJ (1985) The reprductive bilgy and embrylgy f crcdilians. n: Gans C, Billet F, Madersn PFA (eds) Bilgy f the reptilia, vl 14: Develpment A. Wiley, New Yrk, pp 329-491 Fergusn MWJ (1987) Pst-laying stages f embrynic develpment fr crcdilians. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 427-444 Fergusn MWJ, Janen T (1982) Temperature f egg incuba- tin determines sex in Alligatr mississippiensis. Nature (Lnd) 296: 85-853 Fergusn MWJ, Janen T (1983) Temperature dependent sex determinatin in Alligatr mississippiensis. J Zl (Lnd) 2:143-177 Grmbridge B (1987) The distributin and status f wrld crcdilians. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 9-21 Hlder L, Bellairs Ad'A (1962) The use f reptiles in experimental embrylgy. Br J Herpet 3 : 54-61 Huttn JM (1987) ncubatin temperatures, sex ratis and sex determinatin in a ppulatin f Nile crcdiles (Crcdylus niltieus). J Zl (Lnd) 211:143-155 Janen T, McNease L, Fergusn MWJ (1987) The effects f egg incubatin temperature n pst-hatching grwth f american alligatrs. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pry, Sydney, pp 533-537 Lang JW (1987) Crcdilian thermal behaviur. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 31-317 Lang JW, Andrews H, Whitaker R (in press) Sex determinatin and sex ratis in Credylus palustris. Am Zl Legler JM (1985) Australian chelid turtles: reprductive patterns in wide-ranging taxa. n: Grigg G, Shine R, hmann H (eds) Bilgy f Australasian Prgs and reptiles. Surrey Beatty Pty, Sydney, pp 117-123 Madersn PFA, Bellairs Ad'A (1962) Culture methds as an aid t experiment n reptile embrys. Nature (Lnd) 195 : 41-42 Magnussn W, Taylr JA (198) A descriptin f develpmental stages in Crcdylus prsus, fr use in aging eggs in the field. Aust Wildl Res 7:479-485 Manlis SC, Webb GJW, Dempsey K (1987) Crcdile egg chemistry. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 445-472 Miller JD (1985) Criteria fr staging reptilian embrys. n: Grigg G, Shine R, hmann H (eds) Bilgy f Australasian frgs and reptiles. Surrey Beatty Pry, Sydney, pp 35-31 Mittwch U (1985) Males, females and hermaphrdites. Ann Hum Genet 5:13-121 Mrris KA, Packard GC, Bardman TJ, Paukstis GL, Packard MJ (1983) ffect f the hydric envirnment n grwth f embrynic snapping turtles (Chelydra serpentina). Herpetlgica 39:272-285 Murray JD, Deeming DC, Fergusn MWJ (in press) Size dependent pattern frmatin in embrys f Alligatr mississippiensis: time f initiatin f pattern generatin mechanism. Prc R Sc B Packard GC, Packard MJ (1988) The physilgical eclgy f reptilian eggs and embrys. n: Gans C, Huey RB (eds) Bilgy f the reptilia, vl 16 : clgy B. Alan R. Liss, New Yrk, pp 523-65 Packard M J, Packard GC, Miller K, Bardman TJ (1987) nfluence f misture, temperature and substrate n snapping turtle eggs and embrys. clgy 68:983 993 Pieau C, Drizzi M (1981) Determinatin f temperature sensitive stages fr sexual differentiatin f the gnads in embrys f the turtle mys rbieularis. J Mrphl 17: 373-382 Rmanff AL (1967) Bichemistry f the avian embry. Wiley- nterscience, New Yrk Ryan BF, Jiner BL, Ryan TA (1985) Minitab handbk, 2nd edn. Duxbury Press, Bstn Subb Ra MV (1987) ncubating eggs f the Gharial (Gavialis
D.C. Deeming and M.W.J. Fergusn: Temperature effects n alligatr develpment 193 gangeticus) fr cnservatin purpses. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 53-55 Vinegar A (1973) The effects f temperature n the grwth and develpment f embrys f the indian pythn, Pythn rnlurus (Reptilia, Serpentes : Bidae). Cpeia : 171-173 Webb GJW, Manlis SC ( 987) Methds fr retrieving crcdilian embrys. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pty, Sydney, pp 423-426 Webb GJW, Smith AMA (1984) Sex rati and survivrship in a ppulatin f the Australian freshwater crcdile Crcdylus jhnstni. n: Fergusn MWJ (ed) The structure, develpment and evlutin f reptiles. Academic Press, Lndn, pp 319-355 Webb GJW, Buckwrth R, Manlis SC (1983a) Crcdylus jhnstni in the McKinlay River, N.T. V. Nesting bilgy. Aust Wildl Res 1:67 637 Webb GJW, Buckwrth R, Sack GC, Manlis SC (1983b) An interim methd fr estimating the age f Crcdylusprsus embrys. Aust Wildl Res 1:563-57 Webb GJW, Beal AM, Manlis SC, Dempsey K (1987) The effects f incubatin temperature n sex determinatin and embrynic develpment rate in Crcdylus jhnstni and C. prsus. n: Webb GJW, Manlis SC, Whitehead PJ (eds) Wildlife management: crcdiles and alligatrs. Surrey Beatty Pry, Sydney, pp 57 531