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May 19, 2011 - C and swam faster, three days after hatching, than did hatchlings incubated at ... known that incubation temperature often affects hatchling.
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Australian Journal of Zoology, 2011, 59, 18–25

Determining optimal incubation temperature for a head-start program: the effect of incubation temperature on hatchling Burnett River snapping turtles (Elseya albagula) Yvonne A. Eiby A,B and David T. Booth A A B

School of Biological Sciences, The University of Queensland, Brisbane, Qld 4072, Australia. Corresponding author. Email: [email protected]

Abstract. This study monitored natural nest temperatures and examined the effect of incubation temperature on hatchling phenotype of the freshwater turtle Elseya albagula to determine the optimal temperature for a potential head-start program. Eggs were incubated at constant temperatures (26C, 28C and 30C) to determine the influence of temperature on incubation period, hatchling morphology, swimming performance and post-hatching growth rate. Hatchlings incubated at 26C had longer plastrons than hatchlings from 30C and swam faster, three days after hatching, than did hatchlings incubated at either 28C or 30C. Incubation temperature also provided a source of variation in hatchling scute patterns. Clutch of origin influenced hatchling mass and size, growth at 184 days after hatching, and the swimming performance of 3-day and 75-day post-hatch hatchlings. Constant temperatures of 26C and 28C produced the highest hatching success and highest-quality hatchlings and are therefore recommended for incubation of eggs in a head-start program. In the field, unshaded nests experienced greater daily fluctuations in temperature and higher temperatures overall compared with shaded nests, such that unshaded nest temperatures approached the upper thermal limit to development. Additional keyword: hatchling.

Introduction Conservation management of threatened and endangered species often uses interventions to facilitate increased survival and recruitment during the species’ most vulnerable life-history stages. In reptiles the most vulnerable life-history stages are typically the eggs and early hatchlings. Head-start programs aim to increase recruitment from these vulnerable stages by artificially increasing hatching success and recruitment of hatchlings. The most common form of head-start in reptiles is typified by management at sea turtle rookeries, where ‘doomed’ nests are relocated to sites further up the beach in areas where nest predation and poaching is low or into protected hatcheries where heavy nest predation or poaching is a problem (Boulan 1999; Mortimer 1999). Sometimes in countries like Malaysia eggs are incubated artificially in sand-filled styrofoam boxes (Mortimer et al. 1994), but more commonly eggs are left to incubate naturally in artificially dug nests. In both cases hatchlings are released into the sea immediately after hatching. Less common are programs such as has been trialed with tuatara (Sphenodon guntheri) that raise hatchlings in captivity for a period before they are released into the wild (Nelson et al. 2002). The aim of head-start programs is to increase recruitment of hatchlings into the adult population, and in reptiles it is well known that incubation temperature often affects hatchling phenotype (Deeming 2004; Booth 2006), so manipulation of incubation temperature has the potential to increase hatchling  CSIRO 2011

survival through the alteration of the phenotype. Each species of reptile has a temperature range (typically 510C) termed the viable incubation range (Birchard 2004), over which a high proportion of viable hatchlings can be produced when incubated at a constant temperature. Incubation temperature–induced phenotypic variation can affect hatchling morphology and size (Packard et al. 1987; Brooks et al. 1991; Janzen 1995; Shine 1995; Booth 1999), behaviour (O’Steen 1998; Webb et al. 2001), locomotor performance (Janzen 1993; Elphick and Shine 1998; Doody 1999; Brana and Ji 2000; Ashmore and Janzen 2003; Du and Ji 2003; Booth 2004; Burgess et al. 2006), sex determination (reviewed by Deeming 2004), and post-hatching growth rates (Brooks et al. 1991; Bobyn and Brooks 1994; O’Steen 1998; Booth 1999, 2004). The aim of this study was to investigate the optimal constant temperature for incubation of Burnett River snapping turtle (Elseya albagula) eggs and to investigate whether incubation temperature affects hatchling morphology, swimming performance and post-hatching growth rates of this species so that recommendations could be made on the incubation temperature to be used in a possible head-start program. In addition, temperatures experienced by eggs incubated at natural nest sites were investigated. The Burnett River snapping turtle (E. albagula) has recently been described by Thomson et al. (2006), and is the largest freshwater turtle inhabiting the waterways of the Burnett, Mary 10.1071/ZO10080

0004-959X/11/010018

Effect of incubation temperature on E. albagula hatchlings

and Fitzroy River catchments of central eastern Queensland (Limpus et al. 2002). Its ecology is poorly understood and little is known of its nesting behaviour. E. albagula lays its eggs in late autumn or early winter and the eggs hatch in late spring to early summer. This is an unusual breeding strategy for a subtropical freshwater turtle species that typically nests in late spring and early summer (Booth 1998a). Autumn–winter breeding is, however, shared with C. expansa (Booth 2002), a long-necked turtle that also inhabits the Burnett, Mary and Fitzroy River systems (Limpus et al. 2002). Recent studies have shown E. albagula to be herbivorous, feeding primarily on algae, submerged roots and flowers that fall into the water from the riparian vegetation above (Armstrong and Booth 2005). This species is considered highly vulnerable to the impact of habitat modification because of its limited geographic distribution, late sexual maturation, dependence on riparian vegetation for food and nesting sites and susceptibility to nest predation (Limpus et al. 2002; Armstrong and Booth 2005). Materials and methods Natural nest temperature monitoring In July and August of 2003, four temperature data loggers (thermochrom DS1922 L set to 0.06C resolution; Dallas Semiconductor Corporation, Dallas, TX) set to record every 2 h were buried 18 cm below the surface in nests constructed by E. albagula. Two nests were located on a sand island in the Burnett River at Gayndah, Queensland (25370 1100 S, 151380 0400 E), one in the shade of a Eucalyptus tree and one exposed to full sun throughout the day. Two other nests were constructed on the banks of Barambah Creek, a tributary of the Burnett River near Gayndah (25370 4800 S, 151420 3900 E). Both of these nests were shaded, one by a Eucalyptus tree, and the other by a Casuarina tree. Egg collection and incubation Eight female E. albugala turtles were collected at different times within the Burnett River or Barambah Creek near Gayndah between May and July 2003. Of these, one female was found dead and a second was euthanaised because of injuries sustained during a recent flood event. These two turtles were dissected to remove their eggs. The other six females were captured by hand and X-rayed to verify that they were gravid. Injections of 11 mL of synthetic oxytocin (Synocinon, NOVARTIS Pharmaceuticals Australia Pty Ltd, 10 IU mL–1) into a hind leg muscle induced laying of eggs (Ewert and Legler 1978). All eggs were marked with a unique number using a pencil, and placed in moist vermiculite (water potential –150 kPa) in loosely sealed containers. In total, 72 intact eggs from eight clutches were collected. These eggs were transported to the laboratory at The University of Queensland’s St Lucia campus in an insulated container cooled with ice bricks within four days of being harvested from females. Once in the laboratory each egg’s mass (0.001 g), length and width (0.1 mm) were measured. Eggs were then half buried in moist vermiculite (–150 kPa) in loosely sealed plastic containers that allowed for the exchange of water vapour and other gases. During this time the eggs were held at 18C to ensure that their embryos remained in secondary diapause at the late-gastrula stage of development, which often

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remains unbroken if warm incubation commences immediately following laying in freshwater turtles that are autumn/winter nesters (Booth 2002). Containers were weighed weekly and lost water was replaced as necessary to maintain the water potential of the vermiculite. Fortnightly for the first month, then weekly thereafter, the mass of each egg was recorded. After 47–107 days of cool incubation, the eggs were redistributed among eight containers and warm incubation commenced. Two containers, with 12 eggs each, were placed in each of the three incubators (26C, 28C and 30C), such that each incubator had equal numbers of eggs from each clutch, thereby removing the overall effect of clutch from the experimental design. At hatching From 60 days after warm incubation commenced, the eggs were checked several times a day to record accurately the day and time of pipping (breaking of shell) and hatching (full emergence) for each hatchling. Once fully emerged, each hatchling was rinsed with water to remove adhering vermiculite and placed in a loosely sealed plastic jar containing wet paper toweling and labelled with the hatchling’s egg number and day of hatching. The jars were then returned to their original incubator for 48 h to allow the hatchling to absorb residual yolk fully and for the carapace and plastron to assume its normal post-hatch shape. Eggs that failed to hatch were dissected and the embryo, if present, was weighed to determine the amount of development that had occurred. Forty-eight hours after hatching, each hatchling’s mass and head width, carapace length, carapace width, carapace depth, plastron length and plastron width were measured using a caliper. Hatchling scute counts were compared with the standard scute pattern for E. albagula (Limpus et al. 2002) and deviations from the norm recorded. Each hatchling was scute-marked with its corresponding egg number using a scalpel to ensure identification of individuals. Hatchlings were held in a water-filled tank in the laboratory from 48 h after hatching to 3 days of age. Swimming performance Hatchlings performed their first swimming trial 24 h after entering the water, three days after hatching. Due to differences in hatching time, the second swimming trial was conducted en masse 75 days after the mean hatch day; hatchlings were between 58 and 89 days old. Possible effects of body temperature differences on performance were removed by keeping the hatchlings in 28C water for 30 min before the trials. The swimming pool was a Perspex tray (800 mm  800 mm) with concentric circles marked 20 mm apart on the bottom, containing 40 mm of 28C water. A hatchling was placed in the centre and the time taken to swim, in a straight line without pausing, over 6 concentric lines was recorded using a stopwatch. Thus, each trial covered a distance of 120 mm and each individual performed the trial 15 times consecutively. These trials measure burst speed, which is considered the most appropriate swimming test for freshwater turtles because they rely primarily on anaerobic pathways to fuel swimming activity (Miller et al. 1987).

Australian Journal of Zoology

Results Natural nest temperatures All field nests were depredated within the first week of being constructed; however, the data loggers were buried in the nest without eggs so that indicative nest temperatures could be obtained. All four nests had a seasonal increase in temperatures through winter and spring that peaked in summer (Fig. 1). The three shaded nests experienced lower temperatures than the nest in full sun. During all seasons, temperatures within the shaded nests fluctuated by less than 3C on a daily basis, but during summer the nest in full sun showed daily fluctuations of up to 10C (Fig. 2).

Nest 1 (shaded) Nest 2 (shaded) Nest 3 (shaded) Nest 4 (unshaded)

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Nest temperature (°C)

30

25

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15

4 -F eb

-0

4 01

-J an 01

ec -0 -D 01

-N

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3

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3

10

01

Statistical analysis Analysis of Variance (ANOVA) was used to detect differences in initial egg mass, length and width between incubation temperatures and clutches and differences in incubation period between temperatures (temperature was a fixed factor and clutch was a random factor). A Chi-square test was used to detect differences in hatching success between temperatures. Differences in pre-pip egg mass, hatchling mass, head width, carapace length, width and depth and plastron length and width between temperatures were analysed using Analysis of Covariance (ANCOVA), where initial egg mass was the covariate and temperature was a fixed factor and clutch was a random factor. If treatment differences were indicated, a Tukey–Kramer Test for unequal sample sizes was used to test for significant differences between particular treatments. Swimming performance was analysed using one-way ANOVAs (temperature was a fixed factor) to detect differences resulting from incubation temperature, and two-way repeatedmeasures ANOVA, where incubation temperature and the age of hatchlings were fixed factors. To test whether the relative swimming performance was repeatable through time, the ranks of hatchling speed from the initial swimming trial three days after hatching were plotted against the ranks determined at the later swimming trial (between Days 58 and 89). The 30C incubation treatment produced just two hatchlings that survived more than two weeks, so statistical analysis of post-hatching growth was not undertaken for this treatment. Statistica ver. 5.1 was used to analyse data and SigmaPlot 8.02 to create graphics. Results are reported as means, leastsquare adjusted means and s.e.m.

40

-J ul

Post-hatching growth and water temperature After their first swimming trial all hatchlings were housed in an outside tank 2 m in diameter and 800 mm in height; mean water depth was 150 mm and mean water volume was 47 L. The tank received direct sunlight in the morning but was shaded in the afternoon and hatchlings had unrestricted access to food and basking opportunities. Every 28 days from the date the first turtle hatched, morphological measurements were taken from each turtle as at hatching.

Y. A. Eiby and D. T. Booth

01

20

Date Fig. 1. Mean daily nest temperatures for four natural nests of Elseya albagula along the Burnett River from July 2003 to January 2004. Note that the unshaded nest has higher daily mean temperature fluctuations.

(recorded at the time of laying) varied between clutches, but not temperature treatments (Table 2). During cool incubation mean egg mass remained unchanged, but increased by an average 3.9 g for viable eggs through warm incubation. The mass of viable eggs incubated at 26C and 30C increased as incubation proceeded, reached a peak about two weeks before hatching, then decreased until pipping. Viable eggs incubated at 28C increased in mass for the first six weeks of incubation, then stabilised before increasing during the final three weeks of incubation. Pre-pip egg mass (the mass of an egg preceding pipping) was significantly affected by incubation temperature (Table 2) such that at pipping eggs incubated at 30C were lighter than eggs incubated at 28C (P < 0.01) or 26C (P < 0.01), but there were no differences in eggs incubated at 26C and 28C. The warm incubation period lasted between 67 and 99 days (Table 1) and was affected by incubation temperature and clutch (Table 2). Incubation periods for the two clutches that produced hatchlings at all three incubation temperatures averaged 93, 80 and 72 days at 26, 28 and 30C respectively. The two clutches that were dissected from dead females failed to produce any hatchlings (Table 1), so analysis of hatching success for each treatment was limited to six clutches. Overall, 97% of eggs were fertile, as indicated by the appearance of a white patch on the upper surface on the egg (Thompson 1985) and most embryonic mortality happened early in development (Table 3). Incubation at 30C resulted in significantly lower hatching success (c2, P < 0.001) than at 26C and 28C, but there was no significant difference in hatching success between 26C and 28C (c2, P = 0.528), and averaged 71%.

Eggs, incubation period and hatching success

Morphology, swimming performance and post-hatching growth

Egg mass ranged from 27.0 to 39.1 g and clutch size ranged from 10 to 16 eggs (Table 1). Initial egg mass, length and width

Hatchling mass was correlated with initial egg mass (R2 = 0.318, P = 0.002, y = 7.335 + 0.25x, n = 27). For hatchlings that

Effect of incubation temperature on E. albagula hatchlings

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(a) Nest 1 (shaded) Nest 2 (shaded) Nest 3 (shaded) Nest 4 (unshaded)

Nest temperature (°C)

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10 40

(b) Nest temperature (°C)

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Artificial incubation (c)

Nest temperature (°C)

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PM

0 :0 12

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10 :0

observed for 67%, 31% and 0% of all hatchings incubated at 26C, 28C and 30C respectively (Table 4). The swimming performance of hatchlings was affected by incubation temperature at 3 days after hatching, but not 58–89 days after hatching (Table 2). Hatchling mass was not correlated with swimming speed at 3 days (R2 = 0.111, P = 0.088, n = 27) or 58–89 days (R2 = 0.019, P = 0.092, n = 27) after hatching. There was no correlation between individual swimming performance at 3 days and 58–89 days (Fig. 3). Mean swimming speed was higher for hatchlings at 58–89 days than at 3 days (Table 2). A two-way repeated-measures ANOVA detected an effect of hatchling age (P = 0.004) and an interaction between temperature and hatchling age (P = 0.020) on swimming performance. Clutch of origin influenced swimming performance at 3 days and 58–89 days (Table 2). Post-hatching growth, measured as a function of time since incubation commenced or time since hatching, was unaffected by incubation temperature (Fig. 4). Because there was no significant effect of temperature on post-hatching growth, data for hatchlings from 26C and 28C were combined to investigate the influence of clutch on post-hatching growth and hatchlings from different clutches were found to grow at different rates, so that by 185 days after hatching, turtle mass and dimensions differed between clutches (P = 0.004 for all measured traits) (Fig. 5). Discussion

10 40

12

21

Time Fig. 2. A sample of temperatures recorded from Elseya albagula nests along the Burnett River, every 30 min, over a three-day period during (a) winter, (b) spring and (c) summer. Note the increased variance and maximum temperatures experienced in the unshaded nest compared with the shaded nests.

survived at least two weeka after hatching, plastron length at hatching, but no other morphological measurement, was affected by the incubation temperature (Table 2). Additionally, clutch of origin affected carapace length and plastron width (Table 2) in these hatchings. The standard scute pattern for E. albagula was

Incubation of eggs at 2628C maximised hatching success, therefore this range represents the optimum temperature range for the artificial constant temperature incubation of E. albagula eggs and is comparable with other freshwater turtle species (Christens and Bider 1987; Eckert and Eckert 1990; Pena et al. 1996). Typically, for any reptilian species, hatching success is highest within a temperature range of 510C (Birchard 2004) so it would be of interest to incubate E. albagula eggs at temperatures below 26C to determine the lower threshold temperature for successful embryonic development. Interestingly, the low hatching success seen at 30C in this species was also observed in the broad-shelled river turtle (Chelodina expansa). Both species breed in the cooler months, undergo secondary diapause and experience comparable nest temperatures (Booth 1998b, 1999). The influence of incubation temperature on the scute pattern of E. albagula hatchlings was a novel finding as no other studies have examined this effect, but abnormal scute patterns can be induced in freshwater turtles through dehydration of embryos (Lynn and Ullrich 1950). In the wild, adults from the same population were found to have a relatively high incidence of individuals with abnormal scute counts (Limpus et al. 2002) so it appears to be an attribute with considerable plasticity in this species. Differences in hatchling swimming performance had disappeared after 75 days (Table 2), implying that any survival advantage these faster hatchlings may have had over their slower counterparts is short-lived. The lack of repeatability of swimming performance between the two trials (Fig. 3) implies that the speed of each hatchling, relative to the speed of other

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Australian Journal of Zoology

Y. A. Eiby and D. T. Booth

Table 1. Clutch and egg parameters for Elseya albagula collected from the Burnett River in May–June 2003 Hatch success does not include the 2 clutches (1 and 4) that failed to produce hatchlings or infertile eggs. Values for egg mass and size are mean  s.e.m. n.a. = information not available Clutch # Female mass (kg) 1 2 3 4 5 6 7 8

6.5 5.7 6.5 5.2 5.2 n.a. n.a. 5.2

Mean

5.7

Egg harvest method

Clutch size

Dissected Induced Induced Dissected Induced Induced Induced Induced

16 10 14 10 14 12 12 12 12.5

Mass (g)

Egg Length (mm)

Width (mm)

Cold period (days)

29.5 ± 0.4 36.7 ± 0.6 34.0 ± 0.6 30.0 ± 0.4 28.2 ± 0.3 36.5 ± 0.6 31.3 ± 0.4 34.5 ± 0.4

54.1 ± 0.3 51.9 ± 0.6 55.0 ± 0.7 51.5 ± 0.4 49.9 ± 0.3 54.5 ± 0.7 52.9 ± 0.5 52.6 ± 0.2

31.1 ± 0.2 34.2 ± 0.2 32.3 ± 0.1 31.7 ± 0.1 31.0 ± 0.1 34.1 ± 0.3 31.2 ± 0.2 33.5 ± 0.1

32.5 ± 3.4 52.8 ± 0.2 32.3 ± 0.1

Hatch success (%) 26C 28C 30C

Mean warm incubation period (days) 26C 28C 30C

104 107 98 98 99 58 58 47

0 33 67 0 67 67 100 100

0 50 50 0 50 100 100 100

0 0 0 0 0 0 33 33

– 80 78.5 – 87.5 97 92.7 94.5

– 67 67.5 – 68 80 79 81.3

– – – – – – 71 73

84

67

76

12

89

76

72

Table 2. Egg and hatchling parameters for Elseya albagula eggs incubated at different constant temperatures For initial egg parameters, n = 24. For pre-pip egg mass, n = 13 (268C), 13 (288C) and 8 (308C). For hatchling parameters, n = 12 (268C), 13 (288C) and 2 (308C); includes all hatchlings that survived >2 weeks. Hatching success does not include Clutches 1 and 4 that failed completely. The incubation period refers to the period that the eggs were incubated in warm conditions. Values are mean  s.e.m, except in cases indicated by an asterisk, in which the values are mean (adjusted by initial egg mass using ANCOVA)  s.e.m. An asterisk also indicates cases where P values were calculated from 268C and 288C only due to limited numbers of hatchlings at 308C Incubation temperature 28C 30C

26C Initial egg mass (g) Initial egg length (mm) Initial egg width (mm) Pre-pip egg mass (g)* Hatching success Incubation period (d) Hatchling mass (g)* Head width (mm)* Carapace length (mm)* Carapace width (mm)* Carapace depth (mm)* Plastron length (mm)* Plastron width (mm)* Swim speed at 3 days (cm s–1) Swim speed at 75 days (cm s–1)

33.4 ± 0.8 52.6 ± 0.5 32.8 ± 0.4 38.6 ± 1.1 67% 89.3 ± 2.1 16.0 ± 0.4 13.0 ± 0.1 49.8 ± 0.5 49.0 ± 0.7 19.3 ± 0.2 38.1 ± 0.3 21.6 ± 0.2 6.6 ± 0.5 6.8 ± 0.3

33.7 ± 0.8 52.8 ± 0.5 32.8 ± 0.3 37.6 ± 1.0 76% 76.2 ± 1.7 15.5 ± 0.4 12.7 ± 0.3 48.3 ± 0.5 47.5 ± 0.5 18.4 ± 0.6 37.7 ± 0.3 21.2 ± 0.3 4.9 ± 0.3 6.9 ± 0.3

Table 3. Mortality during development of Elseya albagula incubated under three constant temperature regimes Excludes Clutches 1 and 4 in which all eggs failed to hatch. Infertile = no white patch developed; all other eggs developed a white patch Developmental stage at which death occurred

26C

Incubation temperature 28C 30C

All

Infertile No visible embryo Embryo 1 g Died at hatching Died at 2 weeks

2 1 7 1 1 0 12

2 3 6 0 0 0 13

1 2 11 2 3 3 2

5 6 24 3 4 3 27

Total

24

24

24

71

33.6 ± 0.8 53.5 ± 0.7 32.6 ± 0.3 34.8 ± 1.0 12% 72.0 ± 0.1 15.8 ± 0.6 13.1 ± 0.1 48.6 ± 3.1 47.8 ± 0.1 17.7 ± 0.3 35.2 ± 3.0 21.7 ± 1.0 5.4 ± 0.2 7.6 ± 0.3

P-values Temperature effect

All temperatures 33.6 ± 0.5 53.0 ± 0.3 32.7 ± 0.2 37.9 ± 0.7 52%

0.958 0.428 0.887 0.065