Reduced Egg Shell Permeability Affects Embryonic Development And

Integrative Zoology 2018; 13: 2–45 doi: 10.1111/1749-4877.12269

1 ORIGINAL ARTICLE 1 2 2 3 3 4 4 5 5 6 6 7 Reduced egg shell permeability affects embryonic development 7 8 8 9 and hatchling traits in Lycodon rufozonatum and Pelodiscus 9 10 10 11 sinensis 11 12 12 13 13 14 Wenqi TANG,1,2 Bo ZHAO,3 Ye CHEN3 and Weiguo DU1 14 15 15 1 16 Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 16 2 3 17 University of Chinese Academy of Sciences, Beijing, China and Hangzhou Key Laboratory for Animal Evolution and Adaptation, 17 18 College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, China 18 19 19 20 20 21 21 22 Abstract 22 23 The response of embryos to unpredictable hypoxia is critical for successful embryonic development, yet there 23 24 remain significant gaps in our understanding of such responses in reptiles with different types of egg shell. We 24 25 experimentally generated external regional hypoxia by sealing either the upper half or bottom half of the surface 25 26 area of eggs in 2 species of reptiles (snake [Lycodon rufozonatum] with parchment egg shell and Chinese soft- 26 27 shelled turtle [Pelodiscus sinensis] with rigid egg shell), then monitored the growth pattern of the opaque white 27 28 patch in turtle eggs (a membrane that attaches the embryo to the egg shell and plays an important role in gas ex- 28 29 change), the embryonic heart rate, the developmental rate and the hatchling traits in turtle and snake eggs in re- 29 30 sponse to external regional hypoxia. The snake embryos from the hypoxia treatments facultatively increased 30 31 their heart rate during incubation, and turtle embryos from the upper-half hypoxia treatment enhanced their 31 32 growth of the opaque white patch. Furthermore, the incubation period and hatching success of embryos were 32 33 not affected by the hypoxia treatment in these 2 species. External regional hypoxia significantly affected embry- 33 34 onic yolk utilization and offspring size in the snake and turtle. Compared to sham controls, embryos from the 34 35 upper-half hypoxia treatment used less energy from yolk and, therefore, developed into smaller hatchlings, but 35 36 embryos from the bottom-half hypoxia treatment did not. 36 37 37 38 Key words: egg shell, embryonic metabolism, hatchling, heart rate, hypoxia, reptile 38 39 39 40 40 41 41 42 INTRODUCTION 42 43 43 44 Gas exchange is essential for successful embryonic 44

45 development. Oxygen (O2) diffuses from air to the em- 45 46 bryo and is needed in aerobic metabolism, whereas wa- 46 Correspondence: Weiguo Du, Key Laboratory of Animal 47 ter vapor and carbon dioxide diffuse in the opposite di- 47 48 Ecology and Conservation Biology, Institute of Zoology, rection (Deeming & Thompson 1991; Mortola 2009). 48 Chinese Academy of Sciences, Beijing 100101, People’s 49 Gaseous O2 diffuses through the egg shell and under- 49 50 Republic of China neath shell membranes that consists of 2 layers (a thick 50 51 Email: [email protected] fibrous membrane and a thin diffusion-limiting mem- 51

1 brane) (Kern & Ferguson 1997). Under the shell mem- egg when eggs are partially immersed in liquid water 1 2 branes is the chorio-allantoic membrane (CAM) (Ribat- (Booth 1998; Corona & Warburton 2000). In response 2 3 ti 2016), which is highly vascularized to provide a large to external regional hypoxia, reptile embryos are able to 3 4 surface for gas exchange (Birchard & Reiber 1993; Co- shunt blood away from non-exchanging areas (via hy- 4

5 rona & Warburton 2000). After gas exchange, O2 is poxic vasoconstriction), increase cardiac output and 5

6 transported from the gas exchange surface to the tissue blood O2-carrying capacity by polycythemia (increase 6 7 via the blood vessel and cardiovascular system (Nechae- in erythrocyte numbers), and allosterically modify he- 7 8 va et al. 2007). Viviparous embryos maintain a relative- moglobin (e.g. Corona & Warburton 2000; Cordero et 8 9 ly stable gas exchange between the mother and them- al. 2017). Nevertheless, some fundamental questions re- 9 10 selves through complex extra-embryonic membranes or main unanswered with respect to external regional hy- 10 11 placenta, whereas oviparous embryos are exposed to the poxia. First, does the response of reptile embryos to 11 12 external environment and may face unpredictable avail- external regional hypoxia differ among species with dif- 12

13 ability of O2 and water (Deeming & Thompson 1991; ferent types of egg shell? We expected that the sensitiv- 13 14 Mess & Ferner 2010; Liang et al. 2015). Identifying ity of embryos to external regional hypoxia would in- 14 15 how embryos tolerate such unpredictability is crucial for crease from parchment-shelled non-avian reptile eggs 15 16 the understanding of embryonic responses to environ- to rigid-shelled non-avian reptile eggs, because the per- 16

17 mental changes, because both O2 and water availabili- meability to gas decreases from parchment-shelled eggs 17 18 ty can significantly affect embryonic development and to rigid-shelled eggs. Second, do embryos respond dif- 18 19 hatchling phenotypes in oviparous vertebrates (Nechae- ferently to hypoxia occurring in different regions of egg 19 20 va 2011; Zhao et al. 2013; Sun et al. 2014; Bodenstein- shell? Given that reptile embryos generally attach to the 20 21 er et al. 2015). uppermost section of the egg (Ferguson 1982; Thomp- 21 22 The egg shells of amniotes provide protection for son 1985), it is expected that the embryo would respond 22 23 embryos against predation and hazardous environ- differently if external regional hypoxia occurs at the up- 23 24 ments, such as dehydration and microbial infection, but per half or bottom half of egg shells. 24 25 may also constrain gas exchange between embryos and To test these 2 hypotheses, we experimentally gener- 25 26 air (Deeming & Ferguson 1991; Chang & Chen 2016). ated external regional hypoxia by sealing either the up- 26 27 Gas permeability of parchment-shelled non-avian rep- per half or bottom half of the egg surface of snakes and 27 28 28 tile eggs is roughly 20 times higher than that of hard- turtles, thus rendering it impermeable to O2 and prevent- 29 shelled non-avian reptile eggs (Ackerman 1991; Deem- ing water exchange (Corona & Warburton 2000; Du et 29 30 ing & Thompson 1991). Therefore, embryos wrapped al. 2010b). We then monitored the water exchange of 30 31 in hard-shelled reptile eggs suffer more dramatic ill ef- eggs, the growth pattern of the opaque white patch in 31 32 fects from hypoxia (Booth 2000; Nechaeva 2011; Liang turtle eggs, the embryonic heart rate, the developmen- 32 33 et al. 2015). The opaque white patch is originated from tal rate and hatchling traits (morphology and locomo- 33 34 the position where the embryo attaches; the loss of wa- tor performance) to understand how embryos respond to 34 35 ter makes the egg shell opaque (Ewert 1985; Thompson external regional hypoxia. 35 36 1985; Deeming & Ferguson 1991). The opaque patch 36 37 37 increases the conductance of the egg shell and mem- MATERIALS AND METHODS 38 brane and plays an important role in gas exchange in 38 39 rigid-shelled eggs because the opaque patch is more per- Study species and egg collection 39 40 40 meable to O2 and carbon dioxide (CO2), and, thus, facil- 41 We used 2 species of reptiles to study the influence 41 itates diffusive O2 and CO2 exchange (Thompson 1985). 42 of external regional hypoxia on embryonic develop- 42 Obtaining sufficient O for development is a big chal- 43 2 ment and offspring traits. The red-banded snake (Lyco- 43 lenge for embryos inside eggs. Access to O from inside 44 2 don rufozonatum Cantor, 1842) is a medium-sized (to 44 the nest chamber depends on maternal nest-site selec- 45 1600-mm snout–vent length [SVL]) nocturnal colubrid 45 tion, climate events such as excess rainfall and flood- 46 snake widespread in Asia (Zhao & Adler 1993), and can 46 ing, and the metabolism of microbes and sibling eggs 47 be found in agricultural areas and forests. Females pro- 47 that may modify the composition of gases within the 48 duce approximately 8 parchment-shelled eggs per clutch 48 nest chamber (Seymour et al. 1986; Joanen & McNease 49 in spring and summer (Zhang & Ji 2002). The Chinese 49 1989; Booth 1998; Ackerman & Lott 2004). In addi- 50 soft-shelled turtle [Pelodiscus sinensis Wiegman, 1835], 50 tion, external regional hypoxia may occur within an 51 originally inhabiting rivers, lakes and ponds, is now 51

1 widely raised in turtle farms for food in China. This tur- and “bottom-half hypoxia” treatment was 50% covered 1 2 tle lays clutches of small rigid-shelled eggs from May to with non-toxic and oxygen-impermeable beeswax on 2 3 August in eastern China, clutch size ranges from 20 to the upper half and bottom half of the eggs, respectively. 3 4 40 eggs (Zhang et al. 1998). We defined the “upper” section of eggs as the origin po- 4 5 We collected 68 clutches of 621 freshly laid tur- sition where the embryo attaches, which was usually at 5 6 tle eggs, and 124 freshly laid snake eggs with unknown the uppermost section of the egg. The upper half or bot- 6 7 maternal identity from a private farm in Hangzhou City tom half of eggs were quickly (1 s) dipped into the liq- 7 8 of Zhejiang Province, China. The eggs were measured uid beeswax at a temperature of 65 °C, and then dipped 8 9 to 0.1 mm and weighed to ±1 mg using an electronic into 25 °C water for 30 s to cool down. The thickness of 9 10 balance (Mettler Toledo AB 135-S) (average egg mass beeswax coat was approximately 1 mm. The sham con- 10 11 = 4.87 g for the turtle, 6.63 g for the snake) and indi- trols of the upper-half or bottom-half sealed eggs were 11 12 vidually numbered with pencil on the egg shell for later wrapped with a piece of thin plastic film, and dipped in 12 13 identification. beeswax. The plastic film and beeswax coat were then 13 14 14 We measured hatching success and incubation period removed immediately after cooling. In this way, the 15 15 and embryonic heart rate and hatchling phenotypes (in- control eggs experienced heat stress (65 °C) as did their 16 16 cluding body mass, body size, residual yolk mass, car- siblings from the hypoxia treatments, but not the hypox- 17 17 cass mass and locomotion) for turtles and snakes. In ad- ic condition. 18 18 dition, we measured water content of the opaque and The reptile eggs were then placed on cystosepiment 19 19 translucent shells in turtle eggs to verify whether or not with egg-size holes suspended 10 mm above a free wa- 20 20 the opaque patch is caused by water loss. We also moni- ter surface within plastic boxes (220 × 100 × 80 mm) 21 21 tored the growth of the opaque shell of turtle eggs to de- to allow exchange of O2 and water vapor between the 22 22 termine the role of opaque shell in gas exchange. eggs and their surroundings (Du et al. 2010b). The rel- 23 ative humidity within boxes averaged 89.2% ± 1.9% 23 24 Water content of opaque and translucent shells (n = 5), which was monitored with HoBo (Onset, U12- 24 25 in turtle eggs 011, USA). Each box contained 15 eggs and was cov- 25 26 ered with plastic wrap (sealed with a rubber band). The 26 27 For the turtle eggs, we collected 16 eggs from dif- boxes were then placed in an incubator set at a constant 27 28 ferent clutches to determine the water content of the temperature of 28 °C, which is the optimal egg incuba- 28 29 opaque and translucent shells. The eggs were placed on tion temperature in these species (Zhang & Ji 2002; Du 29 30 cystosepiment with egg-size holes suspended 10 mm & Ji 2003). We weighed the eggs once per week to esti- 30 31 above the free water surface within plastic boxes (220 × mate water exchange during incubation. 31 32 100 × 80 mm). The box was then placed in an incubator 32 33 set at a constant temperature of 28 °C. Once the white Growth of the opaque white patch in turtle eggs 33 patch (opaque shell) covered half of the shell (7 days af- 34 A total of 125 turtle eggs from 15 clutches were used 34 ter incubation), we dissected the eggs along the bound- 35 in the white patch growth experiment. During the exper- 35 ary of the white patch to divide the whole egg shell into 36 iment, 6 eggs died, and were, thus, excluded from the 36 2 parts: opaque and translucent shells. The egg contents 37 analysis. The eggs were weighed and their diameter was 37 and the albumen sticking to the shell were all removed, 38 measured using a digital caliper (CD-6″ CSX, Mitutoyo, 38 and the opaque and translucent shells were weighed. 39 Tokyo, Japan). The white patch was round-shaped and 39 These shells were then oven-dried at 65°C for 48 h and 40 the diameter of the edge was measured to 0.1 mm ev- 40 reweighed. 41 ery day initially and every 3 days thereafter. We calcu- 41 42 Egg incubation lated the percentage of the opaque white patch relative 42 43 to egg surface as the spherical cap area of the opaque 43 44 Turtle eggs from the same clutches were assigned shell (2πRH)/sphere area (4πR2), where H (spherical cap 44 45 to different treatments of “upper-half hypoxia,” “bot- height) = R − (R2 − d2/4)1/2, R = egg length/2, d = diame- 45 46 tom-half hypoxia” and sham controls using a split- ter of the edge of the white patch. 46 47 clutch design. Snake eggs were randomly assigned to 47 48 the treatments and sham controls instead of the split- Heart rates of embryos 48 clutch design to minimize the potential clutch effect be- 49 We used 240 turtle eggs from 23 clutches and 124 49 cause we did not know the maternal identity. Prior to in- 50 snake eggs to determine embryonic heart rates. Of these 50 51 cubation, the egg shell from the “upper-half hypoxia” 51

1 incubated eggs, 19 turtle eggs and 18 snake eggs died tee at the Institute of Zoology, Chinese Academy of Sci- 1 2 during the experiments and were, thus, excluded in the ences. 2 3 analysis. We measured heart rates of embryos on day 3 Data analysis 4 18 (approximately 33% of incubation, at developmen- 4 5 tal stage of 16 and 6 for turtles and snakes based on the We tested the normality of distributions and the ho- 5 6 classification schemes of Tokita and Kuratani [2001] mogeneity of all variances in the data using the Kolm- 6 7 and Boughnera et al. [2007], respectively) and 36 (ap- ogorov–Smirnov test and Bartlett’s test prior to analysis. 7 8 proximately 66% of incubation, at developmental stage We conducted Student’s t-test for dependent samples 8 9 of 21 and 9 for turtles and snakes, respectively) after to compare the water content between the opaque and 9 10 coating the egg surfaces with beeswax to examine the translucent shells. Repeated-measures analyses of vari- 10 11 chronic effect of external regional hypoxia. The heart ance (ANOVAs) with clutch origin as a random factor 11 12 rates of embryos were measured by placing the eggs in were used to detect the influence of external regional hy- 12 13 random order on an infrared heart rate monitor (“Bud- poxia on growth of the white patch and water exchange. 13 14 dy” system, Avian Biotech, Cornwall, England) that was The c2 test was used to examine the influence of exter- 14 15 set up in the 28 °C incubator where the snake and tur- nal regional hypoxia on hatching success. ANOVAs or 15 16 tle eggs were incubated (see Du et al. 2009 for details of mixed-model ANOVAs with clutch origin as a random 16 17 methods for measuring heart rate). The heart rate moni- factor were used to examine the influence of external re- 17 18 tor determines the degree of infrared light absorption by gional hypoxia (and developmental stage) on heart rates 18 19 embryonic blood as an indirect measure of the heart rate and incubation periods in snakes or turtles, respectively. 19 20 (Lierz et al. 2006). We used ANCOVAs or mixed-model ANCOVAs with 20 21 21 Hatchling phenotypes initial egg mass (or hatchling size for swimming per- 22 formance) as a covariate to test the effect of external re- 22 23 A total of 120 turtle eggs from 15 clutches and 124 gional hypoxia on body size, components and swim- 23 24 snake eggs were incubated at different hypoxic condi- ming performance of hatchlings in snakes or turtles, 24 25 tions to determine the effect of external regional hy- respectively. Tukey’s multiple comparisons were used 25 26 poxia on hatchling phenotypes. A total of 26 turtle eggs as post hoc tests to determine the differences among the 26 27 and 18 snake eggs died during experiments and were hatchling traits from different treatments. Significance 27 28 excluded from the analysis. Upon emergence, hatch- was assumed if P < 0.05. 28 29 lings were collected and weighed (± 0.001 g), and car- 29 30 apace size of turtles and SVL of snakes were measured RESULTS 30 31 to 0.1 mm. After the measurements, a sample of snakes 31 32 (n =106) and turtles (n = 68) were killed with an injec- Water content of the shell and water exchange 32 33 tion of sodium pentobarbital to determine composition. 33 34 The hatchlings were separated into carcass and residu- The translucent shell contained more water than did 34 35 al yolks, and weighed. The remaining snakes and tur- the opaque shell in turtle (P. sinensis) eggs (9.01% ± 35 36 tles were individually kept in cages placed in a tem- 0.24% vs 6.02% ± 0.12%, n = 16) (t = 14.85, df = 15, P 36 37 perature-controlled room at 28 ± 1 °C and with a 12-h < 0.0001). 37 38 light/12-h dark cycle. Swimming capacity is important During incubation, snake (L. rufozonatum) eggs 38 39 for fitness-related behavior like foraging and predator from the bottom-half hypoxia treatment gained less wa- 39 40 avoidance and has been used as a fitness index in both ter than did sham controls (F3,116 = 46.04, P < 0.0001) 40 41 snakes and turtles (Winne & Hopkins 2006; Du et al. (Fig. 1a). P. sinensis eggs from the upper-half hypox- 41 42 2009b). The next day after hatching, we determined the ia and bottom-half hypoxia treatments lost less wa- 42 43 swimming capacity of hatchlings in this room by chas- ter than did sham controls (F3,108 = 46.04, P < 0.001). P. 43 44 ing each hatchling along a 1.2-m racetrack filled with 40 sinensis eggs lost more water later in incubation com- 44 45 mm of water at 28 °C. The swimming performance of pared to earlier in incubation (F15,540 = 22.44, P < 0.0001) 45 46 hatchlings was recorded using a Panasonic video cam- (Fig. 1b). 46 47 47 era, and the videotapes were later examined for sprint Growth of the opaque white patch in turtle 48 speed in the fastest 250-mm interval (Du et al. 2010a). 48 49 All experiments in this study were performed with (Pelodiscus sinensis) eggs 49 50 50 approval (IOZ14001) from the Animal Ethics Commit- The opaque white patch grew rapidly in the first 5 51 51

1 days and covered the upper half of egg shells (F5,590 = patch did not differ among the treatments during this 1

2 1929.4, P < 0.0001), but the growth rate of the white period (F3,118 = 0.18, P = 0.91) (Fig. 2). Growth of 2 3 the white patch was then arrested until day 23 (50%) 3 4 through incubation, and then started to grow again 4 5 (Fig. 2). At the later stage of white patch growth, growth 5 6 of the white patch was invisible in the “bottom-half hy- 6 7 poxia” treatment due to the beeswax cover. The growth 7 8 of the opaque white patch at this stage differed signifi- 8 9 cantly among treatments, with a larger percentage of 9 10 opaque white patch in eggs from the “upper-half hy- 10

11 poxia” treatment than in sham controls (F2,88 = 28.65, 11 12 P < 0.0001) (Fig. 2). 12 13 13 14 Embryonic heart rate 14 15 Embryonic heart rates were affected significantly by ex- 15

16 ternal regional hypoxia in L. rufozonatum (F3,102 = 14.85, 16

17 P < 0.0001) and P. sinensis (F3,106 = 3.99, P = 0.01). Embry- 17 18 os from the upper-half hypoxia treatment had higher heart 18 19 rates than other treatments in L. rufozonatum, but not in 19 20 P. sinensis (Fig. 3). In addition, embryonic heart rates 20

21 decreased as embryos developed in P. sinensis (F1,106 = 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 40 Fig. 1. Water exchange in the snake (Lycodon rufozonatum) 41 41 and turtle (Pelodiscus sinensis) eggs incubated in different hy- 42 42 poxic environments. Repeated-measures ANOVAs indicat- Fig. 2. Growth of the opaque white patch in the turtle (Pelodis- 43 43 ed that water exchange differed among different hypoxic treat- cus sinensis) eggs in the different regional hypoxia treatments. 44 44 ments in both snake (P < 0.0001) and turtle (P < 0.001) eggs. Repeated-measures ANOVAs indicated that external region- 45 Data are expressed as least square mean ± standard error, ad- al hypoxia significantly affected growth of the opaque white 45 46 justed for initial egg mass (standardized to 6.65 g for the snake, patch (P < 0.0001). Data are expressed as mean ± SE. Eggs 46 47 and 4.46 g for the turtle) by analysis of covariance with initial egg were sealed over the upper or bottom half of their surface area 47 48 mass as the covariate. The sample sizes for the treatments of up- to induce hypoxia, whereas sham control eggs were unsealed. 48 49 per-half hypoxia, bottom-half hypoxia, upper-half control and The sample sizes for the treatments of upper-half hypoxia, bot- 49 50 bottom-half control were 30, 30, 31 and 29 for the snake, and tom-half hypoxia, upper-half control and bottom-half control 50 51 19, 23, 31 and 39 for the turtle, respectively. were 21, 28, 31 and 39, respectively. 51

1 In both the snake and turtle, external regional hypox- 1 2 ia neither affected hatching success nor incubation pe- 2 3 riod (Table 1). External regional hypoxia significantly 3

4 affected hatchling body mass (L. rufozonatum: F3,101 = 4

5 5.21, P < 0.003; P. sinensis: F3,35 = 7.92, P < 0.001) and 5

6 body size (L. rufozonatum SVL: F3,101 = 8.17, P < 0.001; 6

7 P. sinensis carapace length: F3,35 = 4.57, P < 0.01). In 7 8 both species, hatchlings from eggs with the upper half 8 9 of their surfaces sealed were smaller or lighter than their 9 10 siblings from the sham control. The body size and mass 10 11 did not differ significantly between hatchlings from the 11 12 bottom-half hypoxia and sham control treatments in 12 13 snakes. However, the body mass was smaller for hatch- 13 14 lings from the bottom-half hypoxia than sham control 14 15 treatments in turtles (Fig. 4). 15 16 The body composition of hatchlings was also affect- 16 17 ed by external regional hypoxia. Hatchlings from the L. 17 18 rufozonatum eggs that had their upper half sealed with 18 19 19 beeswax had a smaller carcass (F3,101 = 10.08, P < 0.001), 20 but no significant difference was found in residual yolk 20 21 21 mass (F3,101 = 0.96, P = 0.41) (Fig. 5a). Hatchlings from 22 the P. sinensis eggs that had their upper half sealed with 22 23 23 beeswax had a smaller carcass (F3,31 = 3.18, P < 0.05), 24 24 but larger residual yolk mass (F3,31 = 5.63, P < 0.01) 25 than their siblings from bottom-half hypoxia and sham 25 26 control treatments (Fig. 5b). 26 27 27 The swimming speed of hatchlings did not differ be- 28 28 tween control and hypoxia-treatment animals both in 29 29 L. rufozonatum (upper-half hypoxia: 0.326 ± 0.019 m/ 30 Fig. 3. Mean heart rates of snake (Lycodon rufozonatum) and 30 s, sham control of the upper-half control: 0.408 ± 0.018 31 turtle (Pelodiscus sinensis) embryos from eggs in different re- 31 m/s, bottom-half hypoxia: 0.368 ± 0.019 m/s, sham con- 32 gional hypoxia treatments. Eggs were sealed over the upper 32 trol of the bottom-half control: 0.396 ± 0.019 m/s; F 33 or bottom half of their surface area to induce hypoxia, where- 3,100 33 =1.06, P = 0.37) and P. sinensis (upper-half hypoxia: 34 as sham control eggs were unsealed. We measured heart rates 34 0.249 ± 0.015 m/s, sham control of the upper-half con- 35 18 and 36 days after this manipulation at 28°C. Mixed-model 35 ANOVAs indicated that external regional hypoxia significant- trol: 0.268 ± 0.014 m/s, bottom-half hypoxia: 0.259 ± 36 36 ly affected embryonic heart rates in the snake (P < 0.0001) and 0.014 m/s, sham control of the bottom-half control: 0.264 37 37 turtle (P = 0.01). Different letters above the box plots indicated ± 0.015 m/s; F = 0.94, P = 0.43). 38 3,33 38 statistical difference among treatments (Tukey’s post-hoc test). 39 39 The sample sizes for the treatments of upper-half hypoxia, bot- DISCUSSION 40 tom-half hypoxia, upper-half control and bottom-half control 40 41 were 26, 26, 29 and 25 for both 18 and 36 days in the snake, As we expected, embryos responded differently to 41 42 and were 29, 30, 30 and 30 for 18 days, and 29, 18, 27 and 28 external regional hypoxia as well as to hypoxia occur- 42 43 for 36 days in the turtle. ring in different regions of egg shell in oviparous rep- 43 44 tiles with different types of egg shell. Snake and turtle 44 45 embryos survived the external regional (half-egg shell) 45 46 hypoxia with some physiological adjustments. In addi- 46 47 47 147.1, P < 0.0001), but not in L. rufozonatum (F1,102 = tion, reptile embryos are more sensitive to hypoxia oc- 48 0.75, P = 0.38). curring in the upper half of the egg shell than the bottom 48 49 half of the egg shell. 49 50 Hatching success, incubation period and 50 The gas permeability of egg shell may account for 51 hatchling traits 51

1 Table 1 Statistical comparisons of hatching success and incubation period among hypoxic treatments in different species of reptiles 1 2 2 Upper-half Bottom-half Sham control of the Sham control Statistical significance 3 hypoxia hypoxia upper-half hypoxia of the bottom- 3 4 half hypoxia 4 5 Lycodon rufozonatum 5 6 Hatching success 83.8% (26/31) 83.8% (26/31) 93.5% (29/31) 80.6% (25/31) c2 = 2.34, df = 3, P = 0.505 6 7 7 Incubation period 50.9 ± 0.1 51.5 ± 0.1 50.7 ± 0.1 51.1 ± 0.1 F3,103 = 1.0, P = 0.38 8 Pelodiscus sinensis 8 9 Hatching success 82.6% (19/23) 95.8% (23/24) 95.6% (22/23) 83.3% (20/24) x2 = 4.04, df = 3,P = 0.258 9 10 10 Incubation period 51.7 ± 0.1 51.9 ± 0.1 51.7 ± 0.1 51.8 ± 0.1 F3,36 = 0.4, P = 0.73 11 df, degrees of freedom. 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 Fig. 4. Effects of external regional hypoxia on body mass and body size of hatchling snakes (Lycodon rufozonatum) and turtles 46 47 (Pelodiscus sinensis). Hatchlings from eggs sealed over the bottom half of their surface were smaller than their counterparts from 47 48 eggs sealed over the upper half of their surface and control eggs. Mixed-model ANCOVAs indicated that external regional hypoxia 48 49 significantly affected body size and mass in both species (all P < 0.01). Different letters above the box plots indicated statistical dif- 49 50 ference among treatments (Tukey’s post-hoc test). The sample sizes for the treatments of upper-half hypoxia, bottom-half hypoxia, 50 51 upper-half control and bottom-half control were 26, 26, 29 and 25 for the snake, and 19, 21, 20 and 20 for the turtle, respectively. 51

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 Fig. 5. The body component of hatchling snakes (Lycodon rufozonatum) and turtles (Pelodiscus sinensis) from eggs in different re- 30 gional hypoxia treatments. Hatchlings from eggs sealed over the bottom half of their surface had a smaller carcass and residual yolk 31 31 than their counterparts from eggs sealed over the upper half of their surface and control eggs. Basically, mixed-model ANCOVAs 32 32 indicated that external regional hypoxia affected the carcass and residual yolk of hatchlings in both species (P < 0.05). Different let- 33 33 ters above the box plots indicated statistical difference among treatments (Tukey’s post-hoc test). The sample size for the treatments 34 34 of upper-half hypoxia, bottom-half hypoxia, upper-half control and bottom-half control were 26, 26, 29 and 25, respectively, in the 35 35 snake; the sample size for all treatments is 17 in the turtle. 36 36 37 37 38 38 39 39 40 the among-species difference in hypoxic responses. the second half of incubation when the embryos grow 40 41 Most squamates (lizards and snakes) produce parch- rapidly and O2 consumption and carbon dioxide produc- 41 42 ment-shelled eggs with high gas permeability (Deem- tion increase exponentially (Vleck & Hoyt 1991; An- 42 43 ing & Thompson 1991; Packard & DeMarco 1991), and drews 2004). The opaque shell of turtles and crocodiles, 43 44 the fibrous layer of parchment-shelled snake eggs de- including our species of the Chinese soft-shelled turtle, 44 45 creases during incubation to accommodate the increas- P. sinensis, contained less water than that of the translu- 45 46 ing embryonic respiration (Stahlschmidt et al. 2010). cent shell. Importantly, the removal of liquid water that 46 47 In contrast, many turtles and all crocodiles produce rig- is in the porous spaces of the egg shell greatly increas- 47 48 id-shelled eggs with low gas permeability (Thompson es the oxygen and carbon dioxide gas permeability of 48 49 1985; Deeming & Ferguson 1991). To compensate the the egg shell, and, thus, facilitates diffusive exchange of 49 50 low gas permeability, turtle and crocodile eggs have the oxygen and carbon dioxide (Lomholt 1976; Thompson 50 51 opaque shell that can cover the whole egg surface during 1985). Moreover, the growth of the opaque white patch 51

1 was enhanced during the second half of incubation in P. poxia stress imposed on embryos. Therefore, quantify- 1

2 sinensis embryos; such facultative responses to external ing the supply of O2 to embryos would be critical for de- 2

3 regional hypoxia may facilitate O2 diffusion and trans- termining the actual hypoxic condition experienced by 3 4 port. embryos in such kinds of studies, although we did not 4 5 The among-species difference in hypoxic respons- measure O2 due to methodological difficulty. 5 6 es is also associated with among-species difference in External regional hypoxia significantly affected em- 6 7 physiological responses. Snake embryos with parch- bryonic yolk utilization and offspring size but not swim- 7 8 ment egg shells increased heart rate and therefore car- ming performance in the snake and turtle. Compared 8 9 diac output in response to external regional hypoxia. to those controls, embryos from the upper-half hypoxia 9 10 This phenomenon of tachycardia in response to hypox- treatment utilized less yolk to hatchling carcass during 10 11 ia has also been reported in other reptile species like the incubation (Fig. 5) and, therefore, developed into small- 11 12 scincid lizard [Bassiana duperreyi Gray, 1838] (Du et er hatchlings, but embryos from the bottom-half hypox- 12 13 al. 2010b) and the snapping turtle (Chelydra serpentina ia treatment did not. Lower water availability may re- 13 14 Linnaeus, 1758) (Eme et al. 2013). In contrast, hypox- tard utilization of yolk inside eggs, resulting in a large 14 15 ia reduces the heart rate of American alligators [Alliga- residual yolk at hatching in some turtles (Packard 1999) 15 16 tor mississippiensis (Daudin, 1802)] (Crossley & Alti- and snakes (Aubret et al. 2003; Brown & Shine 2006). 16 17 miras 2005a) and some species of birds (VanGolde et However, the reduced yolk utilization by embryos in 17 18 al. 1997). In addition, other mechanisms such as an en- this study is unlikely attributed to lower availability of 18 19 larged heart (Snyder et al. 1982; Kam 1993; Crossley water for embryos because eggs with the bottom half 19 20 & Altimiras 2005b), region-specific changes in vascu- of their egg shell sealed lost less water than sham con- 20 21 lar density on the chorioallantoic membrane, increas- trols (Fig. 1). Instead, it is a response to hypoxia, be- 21 22 es in hematocrit and an acceleration and redistribution cause hypoxia reduces energy metabolism of embryos 22 23 and yields small hatchlings with large residual yolk sac 23 of blood flow have been demonstrated to increase O2 24 transport rates under hypoxic conditions (Kam 1993; (Owerkowicz et al. 2009). The difference in embryonic 24 25 Warburton et al. 1995; VanGolde et al. 1997; Coro- yolk utilization between the upper-half and bottom-half 25 26 na & Warburton 2000). Further studies on these aspects hypoxia indicated that O2 deficiency in early develop- 26 27 would be of great interest and important to our under- ment may impose a significant effect on embryonic de- 27 28 standing of the physiological responses of amniote em- velopment, although O2 consumption increases dramat- 28 29 bryos to hypoxia. ically only in later developmental stage when embryos 29 30 grow fast (Vleck & Hoyt 1991; Andrews 2004). Despite 30 We did not detect negative consequences of external 31 the difference in yolk utilization by embryos between 31 regional hypoxia on the developmental rate and hatch- 32 the upper-half and bottom-half hypoxia treatments, re- 32 ing success of reptile embryos. These results are consis- 33 gional hypoxia treatments produced smaller hatchlings 33 tent with some studies on the snapping turtle and alliga- 34 compared to those of sham controls. Hypoxia inducing 34 tor (Eme et al. 2011; Eme et al. 2014), but inconsistent 35 smaller hatchlings has also been reported in other reptile 35 with other studies in turtles, crocodiles and birds show- 36 species (Eme et al. 2011; Lungman & Piña 2013). In- 36 ing decreased embryonic growth and survival induced 37 terestingly, the effect of regional hypoxia treatments on 37 by hypoxia (Snyder et al. 1982; Seymour et al. 1986; 38 hatchling components showed between-species differ- 38 Kam 1993; Owerkowicz et al. 2009; Nechaeva 2011). 39 ences. Carcass mass and residual yolk differed signifi- 39 This discrepancy may reflect methodological differenc- 40 cantly between the upper-half and bottom-half hypox- 40 es in experimentally-induced hypoxia among studies. 41 ia treatments in turtles, but not in snakes. This is likely 41 Unlike previous studies applying low-concentration O 42 2 related to the difference in egg shell structures between 42 to eggs (e.g. Kam 1993; Owerkowicz et al. 2009), we 43 the 2 species. In turtles, the opaque patch occurs in the 43 used external regional hypoxia with half of the egg sur- 44 upper half of the egg shell and facilitates gas permeabil- 44 face left for gas exchange, suggesting that reptile em- 45 ity. The upper-half hypoxia might lead to a severe hy- 45 bryos may be able to survive losing half of the respirato- 46 poxia condition that would induce larger residual yolk 46 ry egg surface with the help of physiological adjustment 47 than did the bottom-half hypoxia, although the fast- 47 like compensatory responses of cardiovascular functions 48 er growth of the opaque patch in the upper-half hypoxia 48 (Kam 1993; Miller et al. 2002; Du et al. 2010b). In ad- 49 treatment could partly compensate for gas exchange (Fig. 49 dition, gaseous O could circulate from uncovered egg 50 2 2). However, this phenomenon does not apply to snake 50 regions to covered regions, which may reduce the hy- 51 eggs with parchment shells. 51

1 In future studies, it would be of great interest to de- Bodensteiner BL, Mitchell TS, Strickland JT, Janzen 1 2 termine the ecological consequence of external region- FJ (2015). Hydric conditions during incubation in- 2 3 al hypoxia on hatchling size and fitness, because small fluence phenotypes of neonatal reptiles in the field. 3 4 hatchlings may grow more slowly and have less op- Functional Ecology 29, 710–7. 4 5 portunity to survive to adulthood compared with their Booth DT (1998). Nest temperature and respiratory gas- 5 6 large siblings in many (although not all) reptiles (Janzen es during natural incubation in the broad-shelled riv- 6 7 1993; Sinervo 1993; Shine 2005; Jacobs & Sherrard er turtle, Chelodina expansa (Testudinata : Chelidae). 7 8 2010). In addition, the response of organisms to hypox- Australian Journal of Zoology 46, 183–91. 8 9 ia depends on thermal conditions (Lighton 2007; Liang Booth DT (2000). The effect of hypoxia on oxygen con- 9 10 et al. 2015). Accordingly, the thermal stress experienced sumption of embryonic estuarine crocodiles (Croco- 10 11 by embryos during the beewax treatment might affect dylus porosus). Journal of Herpetology 34, 478–81. 11 12 embryonic responses to hypoxia condition, although 12 Boughnera JC, Buchtováa M, Fua K, Diewertb V, Hall- 13 a proper sham control was used in our study. A better 13 grímssonc B, Richmana JM (2007). Embryonic de- 14 solution to this issue is to add an unmanipulated control 14 velopment of Python sebae – I: Staging criteria and 15 or to use other materials (e.g. non-toxic tissue adhesive) 15 macroscopic skeletal morphogenesis of the head and 16 that do not induce a thermal stress to coat egg shell (Du 16 limbs. Zoology 110, 212–30. 17 et al. 2010b). 17 18 Brown GP, Shine R (2006). Effects of nest temperature 18 and moisture on phenotypic traits of hatchling snakes 19 ACKNOWLEDGMENTS 19 20 (Tropidonophis mairii, Colubridae) from tropical 20 21 We thank BJ Sun, B Sun and L Wang for their assis- Australia. Biological Journal of the Linnean Society 21 22 tance in the laboratory. This work was supported by a 89, 159–68. 22 23 grant from the National Natural Science Foundation of Chang Y, Chen PY (2016). Hierarchical structure and 23 24 China (31525006). mechanical properties of snake (Naja atra) and turtle 24 25 (Ocadia sinensis) eggshells. Acta Biomaterialia 31, 25 26 REFERENCES 33–49. 26 27 Cordero GA, Karnatz ML, Svendsen JC, Gangloff EJ 27 28 Ackerman RA (1991). Physical factors affecting the wa- (2017). Effects of low-oxygen conditions on embryo 28 29 ter exchange of buried reptile eggs. In: Deeming DC, growth in the painted turtle, Chrysemys picta. Inte- 29 30 Ferguson MWJ, eds. Egg Incubation: Its Effects on grative Zoology 12, 148–56. 30 31 Embryonic Development in Birds and Reptiles. Cam- Corona TB, Warburton SJ (2000). Regional hypoxia 31 32 bridge University Press, Cambridge, UK, pp. 193– elicits regional changes in chorioallantoic membrane 32 33 211. vascular density in alligator but not chicken embry- 33 34 Ackerman RA, Lott DB (2004). Thermal, hydric and re- os. Comparative Biochemistry and Physiology A 125, 34 35 spiratory climate of nests. In: Deeming DC, ed. Rep- 57–61. 35 36 tilian Incubation: Environment, Evolution and Be- Crossley DA, 2nd, Altimiras J (2005a). Cardiovascu- 36 37 haviour. Nottingham University Press, Nottingham, lar development in embryos of the American alliga- 37 38 pp. 15–43. tor Alligator mississippiensis: Effects of chronic and 38 39 Andrews R (2004). Patterns of embryonic development. acute hypoxia. Journal of Experimental Biology 208, 39 40 In: Deeming DC, ed. Reptilian Incubation: Environ- 31–9. 40 41 ment, Evolution and Behaviour. Nottingham Univer- Crossley DA, Altimiras J (2005b). Cardiovascular de- 41 42 sity Press, Nottingham, pp. 75–102. velopment in embryos of the American alligator Alli- 42 43 Aubret F, Bonnet X, Shine R, Maumelat S (2003). gator mississippiensis: Effects of chronic and acute 43 44 Clutch size manipulation, hatching success and off- hypoxia. Journal of Experimental Biology 208, 31–9. 44 45 45 spring phenotype in the ball python (Python regius). Deeming DC, Ferguson MWJ (1991). Egg Incubation: 46 46 Biological Journal of the Linnean Society 78, 263– Its Effect on Embryonic Development in Birds and 47 47 72. Reptiles. Cambridge University Press, Cambridge. 48 48 Birchard GF, Reiber CL (1993). A comparison of avian 49 Deeming DC, Thompson MB (1991). Gas exchange 49 and reptilian Chorioallantoic vascular density. Jour- 50 across reptilian eggshells. In: Deeming DC, Ferguson 50 nal of Experimental Biology 178, 245–49. 51 MWJ, eds. Egg Incubation: Its Effects on Embryonic 51

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