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Previous in situ sex determination studies conducted on hawksbill turtles (Eretmochelys imbricata) (Wibbels 2003) have indicated male bias from one clutch laid in Florida (Dalrymple et al. 1985); a smaller percentage of female hatchlings (no % given) in Antigua, (Mrosovsky et al. 1992) than previously reported for loggerhead turtle (Caretta caretta) hatchlings in Florida (Mrosovsky and Provancha 1989); male biased (67.7%) on the southern (cooler = 30.3 °C) coast and female biased (80.6%) on the western (warmer = 32.4 °C) coast of Barbados (Horrocks & Scott 1991); female bias (estimated >90%) for several consecutive nesting seasons in Bahia, Brazil (Godfrey et al. 1999) and female bias during the peak nesting period at Milman Island, Australia (Loop et al. 1995).

Herein we report on in situ sand incubation temperatures during the summer nesting months at Milman Island, the in situ sex ratios in E. imbricata clutches laid in December 2004, just prior to the peak nesting season at Milman Island (Dobbs et al. 1999), and an ex situ incubator based study to estimate the pivotal temperature for the population. All hatchlings used for sex ratio determination were euthanized with an injection of 1.0 ml of Nembutal; the kidney and adhering gonads were dissected out and preserved in 10% formalin. Gonads were prepared for histological examination following procedures in Humason (1962). Sex was determined by microscopic examination of gonadal tissue following established criteria (Miller & Limpus 1981, 2003, Limpus et al. 1985). In this study, percent females produced was based on the number of hatchlings produced, not the number of eggs incubated because embryos died at different times (and hence differing expressions of gonadal development) during the incubation period.

in situ studies. Incubation temperatures were recorded in four E. imbricata clutches laid on 4 December 1994 on Milman Island, Australia. These eggs were relocated within one hour of oviposition to two unshaded sand (clutches A & B) and two shaded sand (under Premna serratifolia, clutches D & E) sites on the north-western end of the island; these were the same sites used in a 1991 study (Loop et al. 1995). Two control nests (without eggs) were constructed, one in each environment type: clutch C in unshaded sand, clutch F in shaded sand (Loop et al. 1995). Clutches containing eggs were placed approximately 1.0 m apart from one another, forming a triangle with the ‘control nest’ in each environment. Four temperature probes were placed in each clutch at average nest depths to the top and bottom of clutches (based on Loop et al. 1995) of 25 cm (top), 45 cm (bottom) and two at 35 cm (one at the side of the egg chamber, and one in the middle of the eggs); three probes were placed in each ‘control nest’ at the same depths. Temperatures were recorded once a day in late afternoon. Daily temperature fluctuations were recorded at two-hour intervals beginning at 1020 h on 9 December 1994, 1610 h on 25 December 1994 and 1618 h on 7 January 1995 (Loop 1996).

Sand temperatures were monitored using a Cole Parmer Type T Thermocouple Thermometer Model 08500-40 (± 0.4% accuracy of reading, ±1 digit). Air temperature in each environment type (shaded, unshaded) was recorded with a probe placed 40-50 cm above the clutches. At the northern end of Milman Island, air temperature was recorded using a mercury minimum/maximum thermometer hung in the shade of a tree and rainfall was measured using a plastic rain gauge placed 20 cm above the ground away from overhanging vegetation.

Figure 1. Incubation temperatures (°C) of unshaded clutches between 4 December 1994 and 25 January 1995. Panel A: Control clutch and rainfall (mm, right axis); Panel B: Clutch A; Panel C: Clutch B. Depths: 25 cm (diamond), 35 cm in the middle of eggs (square), 35 cm at side of egg chamber (triangle), 45 cm (circle).

Temperatures did not differ significantly with depth among the unshaded clutches A and B or among shaded clutches D and E (Table 1) during the term of incubation. Temperatures recorded in the unshaded control were not significantly different from the unshaded clutches with eggs (Fig. 1, Table 1). However, significant differences occurred between the shaded control and clutches D and E at 35 cm depth at the egg chamber side (Fig. 2, Table 1).

Figure 2. Incubation temperatures (°C) of shaded clutches between 4 December 1994 and 25 January 1995. Panell A: Control clutch and rainfall (mm, right axis); Panel B: Clutch D; Panel C: Clutch E. Symbol definitions same as Fig 1.

Rainstorms were followed by decreases in incubation temperatures 1-2 days later (Figs. 1, 2). Within a 24-h period, temperature fluctuated most (up to 2.9 °C ) at 25 cm depth in unshaded sand and fluctuated least (varying 0.5 °C) in shaded clutches at 35 cm depth in middle of the clutch and 45 cm depth. Night time incubation temperatures were higher than daytime temperatures.

The middle third of incubation, when sex is determined (Georges et al. 1994), was from 22 December 1994 through 7 January 1995. During this time, rain fell on five days, but ≤1.0 mm each day. Average air temperature above unshaded and shaded clutches during this time was 35.1 °C (range 24.9-42.9 °C) and 33.1 °C (range 28.2- 41.7 °C), respectively. Average incubation temperature at 35cm nest depth in the middle of the monitored clutches was 34.6 °C (±0.76, range 33.5-36.0 °C) and 34.7 °C (±0.72, range 33.6-35.7 °C) for the unshaded clutches and 30.7 °C (±0.43, range 29.8-31.6 °C) and 32.4 °C (±0.96, range 31.0-34.3 °C) for the shaded clutches.

Table 1. Incubation temperature (oC) from 4 December 1994 to 25 January 1995. * = no probes placed here; ** = probe malfunctioned. Column ANOVAs are by depth across clutches; Row ANOVAs are within clutches

The in situ sex ratio was determined from hatchlings collected from clutch D (n=50) and three unshaded (n=25/clutch) and two shaded (n=25/clutch) clutches that were marked for incubation success at the beginning of the monitoring period. Nests were marked with surveyor’s tape immediately after oviposition at the beginning of the field season. These nests were checked daily for evidence of emergence beginning 45 days after oviposition (Loop 1996). All 175 hatchlings collected were female.

ex situ incubation and pivotal temperature. Freshly oviposited eggs were collected from four hawksbill turtle clutches laid at Milman Island on 31 January 1998 and transported under refrigeration to the Cape Pallarenda office of the Queensland Environmental Protection Agency (Miller & Limpus 1983, Harry & Limpus 1989). Eggs were divided into groups of 10 per clutch; each group was buried in a 2.5 litre plastic container that held 300g vermiculite mixed with ≈20g of distilled water (approx. -350 kPa). Containers were placed into constant temperature incubators (28.0, 29.5, 31.0, 32.5 °C); temperatures were monitored using TidBit data-loggers (Onset Computer Corp) placed in the centre of the incubator. The position of the containers on the shelf was shifted clockwise each week. Eggs were checked for condition weekly; distilled water was added, if necessary, to maintain hydric conditions and dead or mouldy eggs were removed. Pivotal temperature results were analysed using logistic regression constrained to reach 100% sex ratio at extremes of incubation temperature ranges within R (R Development Core Team 2007).

Figure 3. Hatching success (A) and sex ratios (B) from constant temperature incubation of hawksbilll eggs from four females at Milman Island, Great Barrier Reef. Samples of ten eggs per clutch were incubated at four constant temperatures. Expected lethal limit for constant temperature incubation is 34 °C (Miller 1985)

Total temperature variation within the incubators fluctuated within ± 1.0 °C of the preset value during incubation. Egg mortality varied among clutches and temperature treatments (Figure 3A). High incubation success was obtained at 28 °C and 29.5 °C constant temperature incubation. However, incubation success decreased with the higher constant incubation temperatures. These data are consistent with a lethal threshold of incubation temperature at approximately 34 °C, as has been recorded for the other Cheloniid turtles breeding in eastern Australia (Miller, 1985).

E. imbricata eggs from Milman Island display an abrupt transition from temperatures producing 100% male hatchlings to those producing 100% female hatchlings (Figure 3B). Only male hatchlings emerged from eggs incubated at 28 °C and only females emerged from eggs incubated at 32.5 °C; the two intermediate temperatures produced varying sex ratios of hatchlings (Figure 3B). The pivotal temperature calculated for these clutches was 29.2 °C. This study did not test for pivotal temperature variation among individual clutches.

Differences in sand type and shading of turtle nests affect the thermal environment of the embryos, enough to alter sex ratios between nesting beaches. For example, loggerhead turtles nesting at different sites with different types of sand in the southern Great Barrier Reef exhibit variation in the sex ratios of hatchlings produced (Limpus et al. 1983). Hawksbill turtles have been reported to nest on predominantly vegetated beaches (Witzell 1983). Most nests on Milman Island are laid under vegetation (Dobbs et al. 1999, Miller et al. 2008); however, low density nesting occurs on two adjacent, unvegetated islands, Sinclair and Un-named 11-034, and numerous sparsely vegetated islands in the northern Great Barrier Reef (GBR) and Torres Strait (Limpus 1980, Miller et al. 1995, Miller et al. 2008). Air temperatures and rainfall are generally uniform throughout this area (Bureau of Meteorology 1988); weather conditions recorded at Milman Island can be extended to cover the nearby islands and, in general, other islands of the northern GBR and Torres Strait. If this assumption is true, and given the distribution and density of hawksbill turtle nesting in the region (Miller et al. 1995, Miller et al. 2008) islands should produce more female than male hatchling hawksbill turtles during the peak summer nesting months, considering that clutches incubated under shading vegetation during the rainy season produce less than 70% males (Loop et al. 1995).

Within-season changes in weather and temperature can also produce variations in sex ratio across seasons, such as been reported for C. caretta in the southeastern United States for the loggerhead turtle (Mrosovsky et al. 1984a) and for C. mydas and Dermochelys coriacea in Suriname (Mrosovsky et al. 1984b; Godfrey et al. 1996). The temporal and spatial differences in temperature at Milman Island are reflected in the sex ratios of the resulting hatchlings. When nest temperatures span across the pivotal temperature reported here, as occurred for clutches laid during the peak nesting period of January 1991, unshaded clutches produced 92 and 100% female hawksbill turtle hatchlings, whilst shaded clutches produced 44% and 64% female hatchlings on Milman Island (Loop et al. 1995). However, when nest temperatures exceed the pivotal temperatures across a broad spectrum of available habitats, as occurred during the hot early part of the breeding season in December 1994 (Figures 1 & 2), all hatchlings from shaded and unshaded nests were female. In the 1994/95 season, there was a general trend of increasing temperature within both shaded and unshaded nests as the nesting season progressed (Figs 1 & 2). Shaded nests were cooler than unshaded nests (Table 1) and temperatures recorded in deeper sand were cooler and fluctuated less than temperatures recorded closer to the surface (Miller et al. 2008).

Understanding the temporal and spatial variability of sand temperature at nest depth during mid incubation is crucial for understanding the sex ratio of hatchlings entering the hawksbill turtle population of north-eastern Australia, in part because they nest year-round with a peak in the summer months (Loop 1996). The present in situ study occurred approximately 6 weeks earlier, before peak nesting, than that reported from 1991 (Loop et al. 1995). Data reported herein and unpublished temperature data recorded from Milman Island (Loop 1996) indicate sex ratios probably change over the course of a summer nesting season. Although the summer sex ratio appears biased towards females irrespective of when the incubation occurs (e.g. December, January or February), the sex ratio produced during the remainder of the year is hypothesised to be closer to 50:50 male: female, even potentially being more biased towards males during the winter months, when air temperatures are lower (Bureau of Meteorology 1988). Further support for this idea comes from the estimated pivotal temperature (29.2 °C); as temperatures decrease at the depth of the eggs, the proportion of male hatchlings should increase. Clutches laid in later portions of the peak nesting season when monsoonal rains are occurring or during cooler winter months are likely to experience cooler temperatures than during the peak period. Seasonal variation in temperature and the number of nesting turtles may synergistically work to ensure that male hatchlings are produced at least in low numbers during part of the year.

Local weather conditions (rainfall, cloudiness) during the incubation period dramatically affected the beach temperatures. Heavy rain decreased the temperature experienced by eggs by several degrees in a few days (Figs 1 & 2). Such conditions for a prolonged period of time, especially during the middle third of incubation, could alter sex ratios of hatchlings. However, when considering the cumulative input of males and females into the total population across multiple breeding seasons, very hot periods with nest temperatures that approach or exceed the upper lethal incubation temperature will make a reduced female contribution to the population because of reduced hatchling production. In this present study, two monitored unshaded clutches incubated during the excessively hot December 1994 – January 1995 period that was biased to female hatchling production. Similar outcomes would apply for other cheloniid turtles when incubation temperatures approach and exceed 34 °C (Matsuzawa et al. 2002, Miller, 1985).

Given the variation in sex ratios that may be produced at a nesting beach (among habitats, among beach aspects, among different periods of the nesting season, among years), and among beaches in response to spatially and temporally variable nest temperatures, a better understanding of the incubation environment (sensu Ackerman 1997) and its effects on the sex of marine turtle hatchlings will help to identify the contributions made to the regional population by numerous wide-spread hawksbill turtle sub-populations nesting in the northern GBR and Torres Strait. Further, the definition and documentation of variation in beach conditions experienced by incubating eggs can contribute to the development of heuristic models to support development of strategies for the conservation and management of sea turtle nesting beaches (Mrosovsky & Yntema 1982) on a regional basis. Better understanding of the inter- and intra-seasonal variation of beach conditions will provide the context in which critical experiments can be conducted. In the future, the thermal impacts of climate change may also alter sex ratios (Limpus 1993, Loughland & Miller 2006) and having a detailed understanding of beach conditions will allow predictions based on robust models and contribute to the development of management plans that emphasise regional protection of nesting beaches.

Acknowledgments: Partial funding provided by Queensland Turtle Research program of the Queensland Environmental Protection Agency, Japan Bekko Association, Research Foundation of Texas A&M University, and International Women’s Fishing Association. Thanks to the many volunteers who assist with this study as part of the Queensland Turtle Research program. The authors would also like the reviewers for their constructive comments and to Andrea Whiting for assistance with the figures.

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