Comparative Biochemistry and Physiology Part A 129Ž. 2001 461᎐471

The effects of thermal and hydric environments on hatching success, embryonic use of energy and hatchling traits in a colubrid , carinata

Xiang JiU, Wei-Guo Du

Department of Biology, Hangzhou Normal College, Hangzhou 310036, Zhejiang, PR China

Received 18 October 2000; received in revised form 10 December 2000; accepted 31 December 2000

Abstract

We examined the effects of thermal and hydric environments on hatching success, the embryonic use of energy and hatchling traits in a colubrid snake, Elaphe carinata. The eggs were incubated at four temperatures ranging from 24 to 32ЊC on substrates with water potentials of 0 and y220 kPa using a 4=2 factorial design. Both thermal and hydric environments affected the water exchange between eggs and their surroundings. Eggs incubated in wetter substrates gained mass throughout the course of incubation, whereas eggs in drier substrates gained mass during the first half of incubation and lost mass thereafter. Hatching success was noticeably higher at 26 and 30ЊC than at 24 and 32ЊC, but among treatments, differences in hatching success were not significant. Temperature significantly affected the duration of incubation and most hatchling traits examined. Deformed hatchlings were found in all temperature treatments, with more deformities observed at 32ЊC. Hatchlings from eggs incubated at different temperatures differed in wet body mass, but the differences stemmed mainly from variation in water contents. Embryos at different temperatures completed development at nearly the same expenditure of energy and catabolized nearly the same amount of lipids, but hatchlings from different temperatures differed in the development condition of carcass at hatching. Hatchlings from eggs incubated at 26ЊC were larger in SVL than those from other higher or lower incubation temperatures, characteristically having larger carcasses; hatchlings from 32ЊC eggs were smaller in SVL and had smaller carcasses but larger residual yolks than those from lower incubation temperatures. Hatchlings from eggs incubated at 24ЊC were shorter in tail length but greater in sizeŽ. SVL -specific body wet mass than those from higher incubation temperatures. Within the range from y220 to 0 kPa, the substrate water potential did not affect hatching success, the embryonic use of energy and all hatchling traits examined, and the effects of temperature were independent of the effects of substrate water potential. Therefore, our data add evidence showing that embryonic development in with pliable-shelled eggs is relatively insensitive to variation in hydric environments during incubation. ᮊ 2001 Elsevier Science Inc. All rights reserved.

Keywords: Reptilia; ; ; Elaphe carinata; Egg; Incubation; Hatching success; Calorimetry; Hatchling trait

U Corresponding author. Tel.: q86-571-8082506; fax: q86-571-8804145. E-mail address: [email protected]Ž. X. Ji .

1095-6433r01r$ - see front matter ᮊ 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3Ž. 0 1 00271-9 462 X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471

1. Introduction different species respond differently to variations in hydric environments encountered during in- Thermal and hydric environments affect em- cubation. In addition, investigators using different bryogenesis in reptilesŽ Packard, 1991; Overall, experimental designs and types of eggs might 1994. . Theoretically, embryos can develop suc- reach different conclusions on the same topic. cessfully at temperatures between the upper and Reptiles lay either pliable-shelled eggs or lower limits of incubation temperatures, yielding rigid-shelled eggs, and most squamate reptiles lay viable hatchlingsŽ. Vinegar, 1974 . A high inci- pliable-shelled eggs. Unlike rigid-shelled eggs, pli- dence of abnormalities and failure of embryonic able-shelled eggs gain or lose mass rapidly due to development occurs at extreme temperatures, so the active exchange of water with their surround- the proportion of abnormal hatchlings and hatch- ingsŽ. Vleck and Hoyt, 1991; Vleck, 1991 . Conse- ing success can be used to assess the range of quently, pliable-shelled eggs show greater tem- viable incubation temperatures for any given poral changes in mass during incubation than do speciesŽ Ji and Brana,˜ 1999; Lin and Ji, 1998; Ji et rigid-shelled eggs, providing a greater variation in al., 1999b. . The range of viable incubation tem- hydric conditions inside the eggŽ Vleck, 1991; Ji, peratures may vary among and within species that 1992; Overall, 1994; Ji et al., 1996, 1997a,b; Lin differ in distribution and habitat use, and is gen- and Ji, 1998; Ji and Brana,˜ 1999. . Due to this erally wider in reptiles than in birdsŽ Ferguson, difference, we hypothesized that pliable-shelled 1985; Deeming and Ferguson, 1991; Lin and Ji, eggs would be more sensitive to variations in 1998; Ji et al., 1999b. . Within this range, tempera- hydric conditions than rigid-shelled eggs and ture may subtly affect the embryonic use of en- would, therefore, be more suitable to test for the ergy and thermally plastic traits of hatchlingsŽ Lin subtle influence of incubation hydric environ- and Ji, 1998; Ji and Brana,˜ 1999; Ji et al., 1999b; ments. Brana˜ and Ji, 2000. . Some influence could be Elaphe carinata is a large sized colubrid snake, ecologically and biologically important, and would, and females normally lay a single clutch with therefore, have a long-term effect on an individ- 5᎐17 pliable-shelled eggsŽ.Ž means9.7 Ji et al., ual’s fitnessŽ Webb and Cooper-Preston, 1989; 2000. per breeding season. The snake is widely Burger, 1991, 1998; Van Damme et al., 1992; distributed in southern China, including Taiwan, Allstead and Lang, 1995; Shine, 1995; Shine et al., northward to the provinces of Henan, Shaanxi 1997a,b; Brana˜ and Ji, 2000. . In species with TSD and Gansu; it is also found in northern Vietnam Ž.temperature sex determination , temperature and JapanŽ Ryukyu Island, including the Senkaku also influences the sex ratio of hatchlingsŽ Bull, Group.Ž Zhao and Adler, 1993 . . Previous work on 1980; Deeming and Ferguson, 1988; Janzen and egg incubation of this species focused on the Paukstis, 1991; Mrosovsky and Pieau, 1991. . embryonic use of energy and nutrients and the Studies examining the effects of hydric environ- function of residual yolk. Results showed that E. ments on eggs and the hatchlings of reptiles are carinata embryos use yolk as the source of all limited, and the conclusions reached seem to be organic and most inorganic nutrients, and the contradictory. Some studies have shown pervasive eggshell as the additional source of calcium, and effects of incubation hydric environments on the residual yolk could be used to support the hatching success, duration of incubation and some subsequent growth of newly emerged hatchlings important hatchling traitsŽ e.g. mass and linear during their first days of lifeŽ. Ji et al., 1997a . measurements of hatchlings and size of residual However, the eggs used in previous work were yolk.Ž Lutz and Dunbar-Cooper, 1984; Packard incubated using just one combination of tempera- and Packard, 1984, 1986, 1988; Gutzke and ture and moisture, so the extent to which temper- Packard, 1987; Packard, 1991. . Others, however, ature, moisture and their interaction affect eggs considered that the influence of hydric environ- and hatchlings remains unclear. Thus, we in- ments on egg incubation in reptiles, if any, was cubated E. carinata eggs at four temperatures less importantŽ Tracy, 1980; Hotaling et al., 1985; ranging from 24 to 32ЊC on substrates with water Plummer and Snell, 1988; Ratterman and Acker- potentials of 0 and y220 kPa using a 4=2 fac- man, 1989; Lin and Ji, 1998; Ji and Brana,˜ 1999; torial design. These temperatures and water po- Ji et al., 1999b. . This contradiction is not so tentials were selected because they were mostly surprising, however, and suggests that the eggs of within the range of thermal and hydric environ- X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471 463 ments found in the E. carinata nests that were We moved containers every day among shelves located, although in natural nests, the thermal in the incubators according to a predetermined and hydric conditions normally fluctuateŽ Ji et al., schedule to minimize any influence of thermal unpublished data. . Our aims were to examine the gradients inside the incubator. Incubation tem- effects of incubation temperature, substrate mois- peratures in close proximity to eggs were moni- ture and their interaction on the embryonic use tored twice daily using a digital thermometer. of energy and mass, linear measurements, mor- Eggs were weighed at 5-day intervals. Containers phology and the composition of newly emerged were weighed daily and, if necessary, distilled hatchlings. water was mixed evenly into substrates to com- pensate for small evaporative losses and water absorbed by eggs, thereby maintaining the sub- 2. Materials and methods strate water potential at a constant. When eggs were found to have pipped, we Forty-seven gravid female snakesw snout-vent moved them individually into covered glass con- lengthŽ. SVL : 95.0᎐144.0 cm; post-oviposition tainersŽ. 250 ml , thereby assuring positive identi- body mass: 276.5᎐788.0 gx were collected in mid- fication of emergent young. The duration of in- June 1998 from Lishui and Jiande, Zhejiang, east- cubation, measured as the number of days to ern China. The were subsequently pipping, was recorded for each egg. Upon emer- transported to our laboratory at Hangzhou Nor- gence, mass, SVL and tail length were measured mal College, where they were randomly assigned on each hatchling. Deformed hatchlings were ex- 50=45=35 cm3 Ž. length=width=height wire cluded from further statistical analyses. Hatch- cages of which each contained 1᎐2 individuals. lings Ž.Ns204 were then killed by freezing to We fed snakes with commercial quail ŽTurnix y15ЊC for later study. Upon thawing, we sepa- tanki. eggs and water enriched with vitamins and rated each hatchling into carcass, residual yolk minerals. Cages were placed in an air-conditioned and fat bodies. The three components were oven room, where the temperature varied from 26 to dried to constant mass at 65ЊC, weighed and 30ЊC. Snakes laid eggs between 24 June and 19 preserved frozen for the later determination of July. Cages were checked at least twice every day composition. We assessed the sex of hatchlings by for eggs and more frequently when females were pressing on both sides of the tail base for the seen to lay eggs. At oviposition, the eggs were presence or absence of hemipenes and considered numbered, measured and weighed individually on those with hemipenes as males. a GB303 electronic balanceŽ Mettler᎐Toledo In- We extracted non-polar lipids from dried sam- struments, China. to the nearest 0.001 g. Detailed ples in a Soxhlet apparatus for a minimum of 5.5 data on reproductive female and their eggs will be h using absolute ether as a solvent. The amount reported elsewhere. All post-oviposition females of lipids in a sample was determined by subtract- were released unharmed at the sites where they ing the lipid-free dry mass from the total sample were collected. dry mass. The total lipid in each hatchling was We incubated the eggs systematicallyŽ such that calculated as the sum of the lipids in its carcass, eggs from single clutches were distributed as residual yolk and fat bodies. We determined the equally as possible among treatments. at 24, 26, energy density of the dried samples using a GR- 30 and 32 Ž."0.3 ЊC in plastic containers Ž 250= 3500 adiabatic bomb calorimeterŽ Changsha In- 180=70 mm. that were covered with a perfo- struments, China. . Titrations were performed on rated plastic membrane to retard water loss. The the residue after calorimetry to correct for ni- containers contained known amounts of vermi- trogenous wastes. Further corrections were per- culite and distilled water to produce approxi- formed for fuse wire burning. We determined ash mately y220Ž. 1 g waterr1 g vermiculite and 0 contentŽ. inorganic content in each sample using kPaŽ. 3 g waterr1 g vermiculite water potentials. a muffle furnace at 700ЊC for a minimum of 12 h Substrate water potentials were calibrated with a and weighing the remaining ash. Wescor HR-33T microvoltmeterŽ. dew point mode A preliminary statistical analysis showed that Ž.Lin and Ji, 1998 . One-third of the egg was there were no between-sex differences in all buried in the substrate, with the surface near the hatchling traits examined, so we pooled data for embryo being exposed to air inside the container. both sexes. All data were tested for normality 464 X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471

Ž.Kolmogorov᎐Smirnov test and homogeneity of Eggs incubated in wetter substrates gained mass variancesŽ. Bartlett test , and Ln transformed when throughout incubation whereas eggs incubated in necessary to satisfy the conditions for using para- drier substrates gained mass during the first half metric tests. We used linear regression analysis, of incubation and lost mass thereafterŽ. Fig. 1a᎐d. one-way and two-way analyses of variance All mass gains were assumed to be a result of the Ž.ANOVA , one-way and two-way analysis of co- absorption of water. The final mass of eggs was varianceŽ. ANCOVA , when the assumptions of positively correlated with the initial mass of eggs parametric analyses were met. Non-parametric in all treatmentsŽ. all P-0.0001 . Therefore, the analyses were used when these assumptions were initial mass of eggs was used as the covariate and " violated. Values are presented as mean 1 S.E., a two-way ANCOVAŽ with temperature and ␣s and the significance level is set at 0.05. water potential as the factors. was performed. s - Incubation temperature ŽF3,192 10.17, P s 0.0001.Ž and substrate water potential F1,192 3. Results 137.74, P-0.0001. affected the final egg mass Ž.Fig. 1a᎐d . Eggs incubated at lower temperatures 3.1. Changes in egg mass gained more mass than did eggs at higher temper- atures but at the same water potentialŽ one-way In all treatments, the finalŽ. pre-hatching mass ANCOVA, both P-0.0001. ; eggs incubated in of eggs was greater than the initial mass of eggs. wetter substrates gained more mass than did eggs

Fig. 1. Temporal changes in mass of viable Elaphe carinata eggs incubated in different thermal and hydric environments. Data are expressed as mean and positive and negative bars of standard error. X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471 465 in drier substrates, but at the same temperature 3.3. Size and morphology of hatchlings Ž.one-way ANCOVA, all P-0.0001 .

3.2. Duration of incubation, hatching success and Incubation temperature affected SVL, tail sex ratio of hatchlings length, wet body mass, carcass dry mass and resid- ual yolk dry mass of hatchlings, but it did not Initial egg mass did not affect the duration of affect total hatchling dry mass and fat body dry Ž. Њ incubation in all treatmentsŽ. all P)0.05 . There- mass Table 2 . Overall, eggs incubated at 26 C Ž. fore, the covariate was eliminated from consider- produced the largest SVL hatchlings, character- ation and a two-way ANOVAŽ with temperature istically having the largest carcasses, and those Њ and water potential as the factors. was performed incubated at 32 C produced the smallest hatch- on the duration of incubation. Incubation tem- lings, characteristically having the smallest car- s cassesŽ. Table 2 . Differences in hatchling wet perature affected duration of incubation ŽF3,196 5034.80, P-0.0001. and substrate water poten- mass stemmed mainly from variation in water s s contents, as there were no differences in the total tial did not Ž.F1,196 0.07, P 0.798 . The interac- tion of temperature and water potential did not dry mass among hatchlings from different incuba- s s tion temperaturesŽ. Table 2 . Hatchlings from eggs affect duration of incubation ŽF3,196 0.38, P 0.769. . Incubation length decreased as tempera- incubated at 24ЊC had shorter tails than did those ture increased, but not in a linear way: the aver- from higher incubation temperaturesŽ. Table 2 . age days of incubation decreased 15.9 days from More yolk remained unused at hatching in eggs 24 to 26ЊC, 13.7 days from 26 to 30ЊC and 1.8 days incubated at 32ЊC, and residual yolks in hatch- from 30 to 32ЊCŽ. Table 1 . lings from the three lower incubation tempera- Hatching success was noticeably higher at 26 tures were nearly the sameŽ. Table 2 . and 30ЊC than at 24 and 32ЊC, but among treat- Compared with incubation temperature, sub- ment differences in hatching success were not strate water potential was not an important source significant Ž.Ž.Gs3.20, d.f.s7, P)0.75 Table 1 . of variation for all hatchling traits reported in A total of 19 hatchlings exhibited trunk andror Table 2. The effects of incubation temperature on tail malformations. Deformed hatchlings were the examined hatchling traits were independent found in seven of the eight treatments; the fre- of the effects of substrate water potentialŽ Table quency of deformity was independent of treat- 2.. ments Ž.Ž.Gs7.54, d.f.s7, P)0.25 Table 1 . The Analysis of covariance of linear dimension and incubation thermal and hydric environments did mass of hatchlings where hatchling SVL was the not affect the sex ratio of hatchlings ŽGs1.97, covariate showed influence of incubation temper- d.f.s7, P)0.95. , but the overall sex ratio was ature on morphology of hatchlings. Hatchlings female-biasedŽ femalesrmales s 138r66; G s from eggs incubated at 24ЊC had shorter SVL- s - s - 25.97, d.f. 1, P 0.01. . specific tail length Ž.F3,202 24.50, P 0.0001 but

Table 1 The effects of thermal and hydric environments on duration of incubation, hatching success, and sex ratio and abnormality of hatchlings in a colubrid snake, Elaphe carinataa

Temperature Moisture Incubated Duration of Hatching Sex ratio Abnormality Ž.ЊC eggs incubation success Ž.Ž.&&r(( % Ž.days Ž. %

24 D 35 74.3"0.4Ž. 71.6᎐77.3 68.6 Ž. 24r35 12r12 5.7 Ž. 2r35 W 37 73.8"0.3Ž. 70.3᎐77.9 75.7 Ž. 28r37 18r10 8.1 Ž. 3r37 26 D 24 58.0"0.3Ž. 55.4᎐62.0 87.5 Ž. 21r24 13r8 4.2 Ž. 1r24 W 34 58.1"0.3Ž. 54.3᎐62.8 82.4 Ž. 28r34 20r8 5.9 Ž. 2r34 30 D 25 44.4"0.2Ž. 42.8᎐46.6 92.0 Ž. 23r25 18r500 Ž.r25 W 38 44.4"0.2Ž. 42.0᎐46.8 89.5 Ž. 34r38 25r9 10.5 Ž. 4r38 32 D 35 42.5"0.2Ž. 40.7᎐44.4 65.7 Ž. 23r35 16r7 11.4 Ž. 4r35 W 34 42.6"0.2Ž. 40.6᎐44.3 67.6 Ž. 23r34 16r7 8.8 Ž. 3r34

a Data on duration of incubation are expressed as mean"S.E.Ž. range . D: drier substrate Žy220 kPa . ; W: wetter substrate Ž. 0 kPa . 466 X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471

Table 2 Linear dimension, mass and components of Elaphe carinata hatchlings from eggs incubated in a different thermal and hydric environmentsa

Hatchling Temperature Ž.ЊC Effects traits Moisture 24 26 30 32 Moisture Temperature Interaction

FF1,195 3,195 F 3,195

Initial egg D 28.1"1.2 28.3"1.1 26.8"1.0 28.0"1.3 massŽ. g W 28.6"1.0 28.0"0.9 29.9"1.6 28.0"1.1 U UUU U Snout-vent D 370.4"5.0 378.5"6.0 360.4"5.0 344.4"5.2 0.09 ns 13.43 1.98 ns lengthŽ. mm W 361.9"5.7 374.4"4.6 373.8"5.2 355.2"5.1 24baabc , 26 , 30 , 32 U UUU U Tail length D 79.8"1.7 87.2"2.3 85.5"1.7 83.1"1.7 0.06 ns 14.99 0.56 ns Ž.mm W 77.6"1.8 85.9"1.6 88.5"1.3 84.4"1.5 24baaa , 26 , 30 , 32 UUU U Wet body D 20.9"0.9 20.9"0.9 19.4"0.8 19.8"0.8 2.91 ns 3.87 1.22 ns massŽ. g W 21.3"0.7 21.1"0.7 21.3"0.8 20.5"0.8 24ab , 26 a , 30 b , 32 b UU U Dry body D 5.34"0.25 5.50"0.31 5.15"0.27 5.24"0.27 0.05 ns 0.57 ns 2.07 ns massŽ. g W 5.56"0.24 5.37"0.22 5.50"0.26 5.42"0.28 U UUU U Carcass D 3.29"0.14 3.29"0.14 2.90"0.11 2.74"0.23 3.83 ns 26.99 1.58 ns Ž.g W 3.03"0.12 3.25"0.12 3.18"0.12 2.69"0.11 24babc , 26 , 30 , 32 U UUU UU Residual D 1.24"0.09 1.34"0.12 1.49"0.15 1.74"0.13 2.95 ns 12.83 5.19 yolkŽ. g W 1.73"0.12 1.28"0.09 1.51"0.12 1.95"0.17 24bbba , 26 , 30 , 32 UU U Fat bodies D 0.81"0.05 0.86"0.07 0.77"0.05 0.76"0.05 0.36 ns 2.08 ns 0.37 ns Ž.g W 0.80"004 0.84"0.05 0.83"0.06 0.77"0.05

a Data are expressed as mean"S.E. F values correspond to single effects and factor interactions in two-way ANOVAsŽ with initial egg mass as the covariate.Ž.Ž. . D: drier substrate y220 kPa ; W: wetter substrate 0 kPa . Symbols immediately after F values represent significant levels. UUUUUU ns P)0.05; P-0.01; P-0.001. Means with different superscripts differ significantlyŽ. Tukey’s test, ␣s0.05, a)b)c.

greater SVL-specific body massŽ dry mass, F3,202 important source of variation for energy, lipid and s - s - 4.65, P 0.004; wet mass, F3,202 4.99, P ash contents of hatchlingsŽ. Table 3 . The effects 0.003. than did those from higher incubation tem- of incubation temperature on these hatchling peratures. traits were also independent of the effects of substrate water potentialŽ. Table 3 .

3.4. Embryonic use of energy and material

4. Discussion Hatchlings from different incubation tempera- tures did not differ in energy and lipid contents 4.1. Water flux and influence of substrate water Ž.Table 3 . This indicated that, within the tempera- potential ture range from 24 to 32ЊC, embryos of E. cari- nata completed development at nearly the same expenditure of energy and catabolized nearly the In this study, a net mass gainŽ and, hence, a net same amount of lipids. Incubation temperature water gain. occurred in eggs in all treatments significantly affected embryonic use of ashŽ in- Ž.Fig. 1 . However, eggs incubated at different organic materials. , as indicated by the fact that temperatures had different patterns of water ex- hatchlings from different incubation tempera- change even when substrate water potentials were tures differed in ash contentsŽ. Table 3 . Hatch- identical, and eggs incubated in different water lings from eggs incubated at 26ЊC apparently con- potentials had different patterns of water ex- tained more ash than did those from eggs in- change even when temperatures were identical. cubated at 32ЊCŽ. Table 3 , indicating that more Eggs incubated at lower temperatures absorbed inorganic materials were transferred from the egg more water than eggs at higher temperatures, into the hatchling when eggs were incubated at largely due to the longer developmental time and 26ЊC. smaller transpirational losses of water from the Again, substrate water potential was not an egg at lower temperatures. This result is consis- X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471 467

Table 3 The effects of incubation thermal and hydric environments on energy contents, non-polar lipid mass and ash mass of Elaphe carinata hatchlingsa

Temperature Moisture N Energy Non-polar lipids Ash mass Ž.ЊCkJgg Ž. Ž. Ž.

24 D 24 130.7"7.0 1.34"0.08 0.56"0.02 Ž.Ž.81.2᎐197.0 0.68᎐2.07 Ž. 0.38᎐0.72 W 28 133.1"6.0 1.46"0.07 0.58"0.02 Ž.Ž.76.7᎐197.9 0.83᎐2.18 Ž. 0.37᎐0.87 26 D 21 139.1"9.9 1.36"0.10 0.59"0.03 Ž.Ž.77.8᎐258.2 0.67᎐2.22 Ž. 0.41᎐0.85 W 28 127.6"5.7 1.34"0.07 0.59"0.02 Ž.Ž.70.9᎐184.6 0.55᎐2.02 Ž. 0.41᎐0.83 30 D 23 122.8"7.4 1.28"0.08 0.55"0.02 Ž.Ž.79.4᎐223.6 0.58᎐2.02 Ž. 0.41᎐0.78 W 34 132.1"7.5 1.36"0.09 0.59"0.02 Ž.Ž.75.9᎐262.2 0.70᎐2.71 Ž. 0.39᎐0.91 32 D 23 123.5"7.1 1.32"0.09 0.56"0.02 Ž.Ž.73.7᎐194.4 0.74᎐2.25 Ž. 0.41᎐0.82 W 23 128.6"7.0 1.40"0.09 0.56"0.02 Ž.Ž.82.6᎐195.5 0.64᎐2.06 Ž. 0.39᎐0.83

UU UUab a ab b Effects Temperature F3,195 1.16 ns 0.66 ns 3.36Ž. 24 , 26 , 30 , 32 UU U Moisture F1,195 0.49 ns 0.56 ns 0.35 ns UU U Interaction F3,195 1.50 ns 2.08 ns 1.21 ns a Data are expressed as mean"S.E.Ž. range . F ratios correspond to single effects in two-way ANOVAs Ž with temperature and moisture as the factors.Ž.Ž. using the initial egg mass as the covariate. D: drier substrate y220 kPa ; W: wetter substrate 0 kPa . Symbols immediately after F values represent significant levels. UUU ns, P)0.05; P-0.05. Means corresponding to temperatures with different superscripts differ significantlyŽ Tukey’s test, ␣s0.05, a)b.. tent with that reported for some other reptiles, position of hatchlings are all unaffected within e.g. Pituophis melanoleucus Ž.Žbull snake Gutzke the range of water potential considered in this and Packard, 1987.Ž , Podarcis muralis Mediter- study. Therefore, we conclude that eggs of E. ranean wall lizard.Ž Ji and Brana,˜ 1999 . and Taky- carinata are insensitive to variation in incubation dromus septentrionalis Ž.Žnorthern grass lizard Lin hydric conditions within the range of water poten- and Ji, 1998. . The decline in mass late in incuba- tial we used Ž.y220 to 0 kPa . The hydric insensi- tion when eggs were incubated in drier substrates tivity of egg incubation is considered to be un- is most likely due to a greater rate of transpiratio- usual amongst reptiles, but has been recorded in nal losses of water from the egg. Clearly, the some reptiles with pliable-shelled eggsŽ Muth, external thermal and hydric environments en- 1980; Tracy, 1980; Packard and Packard, 1987; countered by developing eggs significantly affect Plummer and Snell, 1988; Thompson, 1990. . Be- the hydric conditions inside the egg. This fact cause the hydric dependence of egg incubation leads to the following question: do the hydric does exist in many reptilesŽ Packard et al., 1980, conditions a hatchling experienced during em- 1982, 1981, 1983; Morris et al., 1983; Gutzke and bryogenesis subtly affect its development condi- Packard, 1985, 1987; Packard and Packard, 1986; tion at hatching? If so, then any resulting differ- Packard, 1991. , we tend to support the conclusion ences in hydric conditions among eggs of E. cari- that the eggs of different reptilian species may nata in different thermal and hydric environments respond differentially to variance of hydric envi- would, to some extent, affect hatchling pheno- ronmentsŽ. Lin and Ji, 1998; Ji and Brana,˜ 1999 . types. Our data, however, indicate that such ef- fects are unlikely to occur in E. carinata, because 4.2. Influence of incubation temperature hatching success, incubation length, the embry- onic use of energy, and sex, size, mass and com- Thermal variance affects many aspects of egg 468 X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471 incubation in E. carinata, including the aforemen- ergy and catabolize nearly the same amount of tioned water exchanges between eggs and their lipids. However, newly emerged young from dif- surroundings. Our results show that the incuba- ferent temperatures differ in the development tion temperature affects hatching success, incuba- condition of carcass, with hatchling from 32ЊC tion length, the embryonic use of energy and having smaller carcasses but larger residual yolks linear dimension, morphology and composition of than those from lower temperaturesŽ. Table 2 . hatchlings. The result that more yolks remain unused at Significant differences in linear dimension and hatching when eggs are incubated at high temper- wet body mass were found among hatchlings from atures has been reported for all studied reptiles eggs incubated at different temperatures, with and is, therefore, not surprisingŽ Beuchat, 1988; hatchlings from eggs incubated at 26ЊC being Phillips et al., 1990; Van Damme et al., 1992; largerŽ. SVL and heavier than their siblings in- Phillips and Packard, 1994; Ji and Brana,˜ 1999. . cubated at lower or higher temperaturesŽ Table In E. carinata, the unused yolk at hatchingŽ i.e. 2. . However, the differences in wet body mass of residual yolks. is far from trivial and can be used hatchlings result mainly from variation in the for maintenance and carcass growth during the water content, because there are no significant first 3 post-hatching weeks when the hatchlings differences in dry mass among hatchlings from are fasted at temperatures ranging from 26 to different temperatures. Our finding that eggs in- 38ЊCŽ. Ji et al., 1997a . Thus, hatchlings from 32ЊC cubated at medium temperatures such as 26ЊC can be expected to show a subsequent growth of produce larger hatchlings is consistent with re- carcass following the depletion of residual yolk. ports for other reptilesŽ Gutzke et al., 1987; The post-hatching carcass growth may substan- Packard et al., 1989; Burger, 1990; Van Damme tially increase the SVL of hatchlingsŽ Ji et al., et al., 1992. . The larger hatchling size has an 1997a, 1999a,b,c; Ji and Sun, 2000. , thereby mak- association with the greater carcass dry mass Žs ing it possible for hatchlings from 32ЊC to grow to total hatchling dry massyfat body dry massy the size of newly emerged young from low incuba- residual yolk dry mass.Ž Ji et al., 1997a, 1999a,b,c; tion temperatures. Because a part of the energy Ji and Brana,˜ 1999; Ji and Sun, 2000. . Therefore, in the residual yolk is used for maintenanceŽ Ji et the larger size of hatchlings from eggs incubated al., 1997a. and hatchlings from 32ЊC do not differ at 26ЊC is primarily attributed to their larger from those from lower temperatures in energy carcasses, and the smaller size of hatchlings from contents, embryos of E. carinata developing at 32ЊC is primarily attributed to their smaller car- 32ЊC actually have a lower efficiency of convert- cassesŽ. Table 2 . However, size at hatching is not ing energy reserves to tissue. On the contrary, a good indicator for judging the exact size to embryos of E. carinata developing at 26ЊC com- which newly emerged young will grow prior to plete development at nearly the same expenditure feeding, because they may use resources in the of energy as do those at other lower or higher residual yolk to increase size during their first temperatures, but produce larger hatchlings with post-hatching daysŽ Packard, 1991; Ji et al., 1997a, larger carcassesŽ. Table 2 , showing a higher effi- 1999a,b,c; Ji and Sun, 2000. . For example, smaller ciency of converting energy reserves to tissue. hatchlings of E. carinata may increase sizeŽ. SVL Our data show the influence of incubation tem- following the depletion of residual yolk, thereby perature on the morphology of hatchlings, with compensating for their smaller size at hatchingŽ Ji hatchlings from eggs incubated at 24ЊC having et al., 1997a. . The reverse relationship between shorter tails and greater sizeŽ. SVL -specific wet carcass mass and residual mass has also been body mass than do their siblings from higher reported for some reptilesŽ Packard, 1991; Ji et temperatures. However, the explanation for the al., 1997a, 1999a,b,c; Ji and Brana,˜ 1999; Ji and cause and ecological consequence of the thermal Sun, 2000. . dependence of these morphological traits remains Our data show that hatchlings of E. carinata unknown at this time. from different incubation temperatures do not differ in energy and lipid contentsŽ. Table 3 . This 4.3. The suitable temperatures for incubating eggs of indicates that, over the temperature range from E. carinata 24 to 32ЊC, embryos of E. carinata complete de- velopment at nearly the same expenditure of en- Embryos of E. carinata at 24ЊC take almost 2.5 X. Ji, W. Du rComparati¨e Biochemistry and Physiology Part A 129() 2001 461᎐471 469 months to complete developmentŽ. Table 1 , so increasing embryonic mortality. Because the ef- the majority of newborns from this temperature fects of incubation temperature on post-hatching are expected to appear between late September performance and behavior of hatchlings were not and early October. Because hatchlings of E. cari- examined in this study, and because size alone is nata need at least 3 weeks to absorb the yolk sac not a good indicator of a hatchling’s ‘quality’, we and do not feed during this periodŽ Ji et al., do not know what constant temperatures are more 1997a. , their growth period prior to the onset of suitable for incubating the eggs of E. carinata. the first winterŽ. late November through feeding However, taking the energy expenditure and the is approximately 1 month or less. In squamates, rate of embryogenesis into account, we consider incubation length increasingly increases as tem- that the relatively better constant incubation tem- perature decreasesŽ e.g. Van Damme et al., 1992; peratures for E. carinata are within the tempera- Overall, 1994; Lin and Ji, 1998; Ji et al., 1999c; Ji ture range between 26 and 30ЊC. and Brana,˜˜ 1999; Brana and Ji, 2000. . This pat- tern is also found in E. carinata: incubation length decreases by 15 days when the temperature in- Acknowledgements creases from 24 to 26ЊC, and by 14 days when the temperature increases from 26 to 30ЊCŽ. Table 1 . Financial support for the research was obtained Taking the pattern into account, we expect that from the Zhejiang Provincial Natural Science hatchlings of E. carinata from temperatures lower Foundation, the Zhejiang Provincial Department than 24ЊC would have to take the risk of losing of Education, Hangzhou Normal College and the growth period prior to first winter. In addi- local governments of Zhejiang Province and tion, the prolonged duration of incubation at Hangzhou City. We are very grateful to Y. Dai, lower temperatures increases the exposure of eggs J.-Q. Du, X.-Z. Hu, X.-F. Xu and C.-H. Zhang for to the effects of adverse bioticŽ increased mi- their assistance throughout the research. crobial contamination.Ž and abiotic factors ex- treme thermal and hydric conditions. in the in- References cubation environment of the eggs, which may substantially reduce hatching success. Allstead, J., Lang, J.W., 1995. Incubation temperature Because an increase in incubation temperature affects body size and energy reserves of hatchling from 30 to 32ЊC only shortens the incubation American alligators Ž.Alligator mississippiensis . Phys- length by approximately 1.9 days in E. carinata, iol. Zool. 68, 76᎐97. the ecological advantage of the shortened incuba- Beuchat, C.A., 1988. Temperature effects during gesta- tion length can be negligible. 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