Heteromorphic sex chromosomes are absent from all species of Crocodilia, (1) and rare in Testudines with only 7 species possessing them.

Without these heteromorphic sex chromosomes other forms of sex determination are needed. In reptiles, sex determination is controlled by

genetic (GSD) and temperature-dependent (TSD) influences.

These aspects are thought to be fundamentally different by many researchers in the field, yet some argue that they are intrinsically linked (2).

This poster aims to investigate both the genetic and temperature-dependent factors of sex determination, while comparing the systems

involved in the orders of Crocodilia and Testudine.

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In most turtles, the female-producing temperature is on average 31oC, with males typically forming at around 26oC (7). The aromatase expression around these male and female producing temperatures is considerably higher and lower respectively (Figure 2.). In other species, such as fish and amphibians, altering the temperature during gonadal-development drastically changes the expression of aromatase, and subsequently sex-ratios. In the turtles, DNA methylation in the TATA box was found to be significantly lower in the female gonads respective to the males (8).

Of 28 species studied in a 20 year investigation, 15 species displayed cool-males and warm-females (similar to that of the slider turtles (8)). These species are generally found to have larger females than males on average. Another 10 species of turtle were found to have cool and warm females, with intermediate temperatures giving rise to males. The size of the sexes in these species varied between smaller females on average, or both species being dimorphic. The remaining 3 species were shown to relate directly to genetic sex determination, as found with the work of Matsumoto once more (7).

Early literature suggested that crocodilians display 2 patterns of TSD: low incubation temperatures (≤30ᵒC) cause the production of females and higher temperatures (≥33ᵒC) produce only males, or both low and high temperatures produce females and males are produced at intermediate temperatures (3). Later papers revised the first pattern, concluding only one pattern is present (female-male-female) (4). Laboratory tests with Alligator mississippienis eggs found temperatures of ≤31.5 and ≥35ᵒC produced only females and 32.5-33ᵒC produced only males, with intermediate temperatures producing varying sexes (5). Early tests found the thermosensitive period was 20-35 days after egg laying (4), but more recent tests showed this actually occurred at days 30-45 (5). Under field conditions, there was a significant correlation (P<0.001) between expected and actual sex ratios of Alligator mississippienis hatchlings (4). Transaction temperatures vary through species. Crocodylus johnstoni produce only a small amount of males at any given temperature, but Alligator mississippienis produce 100% males at some intermediate temperatures (5). Sex is unchangeable after hatching (6). Incubation temperature affects post hatching growth rates, with hatchlings from intermediate temperatures having a faster rate (3).

Matsumoto, 2013

Currently, no evidence exists for a link between TSD and GSD within crocodilia, but the evidence provided allows for a well-supported speculation that this link exists. As there is an identified thermo-sensitive period in crocodilia, it can be speculated that the aromatase gene itself is primarily switched on during this period. By moving the eggs in between temperatures during this time, it can be further speculated that the aromatase expression is altered and causes the shown impact of the sex-ratios of hatchlings, as it also does in the testudines.

Figure 1. Comparison of base-pair alignment of the aromatase gene across turtles and other species

Sex determination is recognized as being attributed to external environmental temperature and the genetics of the species itself. In turtles, these two elements are directly linked.

Unlike with crocodilia, the results of the testudine investigations strongly suggest that environmental temperature has a direct influence on aromatase transcription itself, linking both the GSD and TSD

elements of this investigation. Crocodilians appear to rely solely on TSD, with no proven link to GSD.

The reason for temperature dependent sex determination is largely unknown. One paper hypothesizes that there is an optimum temperature for male sex differentiation and either side of that temperature a female will form. Variation in quantity of the male sex determining factor (MSD) affects the initial sex, but both high and low temperatures are disruptive to this factor (3). This remains unproven.

Cytochrome P450 aromatase, or aromatase, is another key factor in the initial sex determination, and involves the irreversible catalyzation of androgens into oestrogens. (8) (Figures 2 and 3.)

Figure 2. Molecular representation of the effect of aromatase http://www.functionalps.com/blog/wp-content/uploads/2011/12/Picture-3.png

1 - Ferguson M.W.J, Joanen T. (1982). Temperature of egg incubation determines sex in Alligator mississippiensis. Nature. 296:850-853 2 – Crews D., Bull J. (1994). Temperature-dependent sex determination in reptiles: Proximate mechanisms, ultimate outcomes, and practical applications. Developmental Genetics. 15:297-312 3 - Deeming D.C, Ferguson M.W.J . (1989). The Mechanism of Temperature Dependent Sex Determination: A Hypothesis. Amer. Zool. 29:973-985. 4 - Lang J.W, Rhodes W.E. (1998). Alligator Nest Temperatures and Hatchling Sex Ratios in Coastal South Carolina. Proc. Annu. Conf. SE Assoc. Fish and Wildlife Agencies . 50:521-531. 5 - Lang J.W, Andrews H.V. (1994). Temperature-dependent sex determination in crocodilians. Journal of Experimental Zoology. 270:28-44. 6 - Michael A Ewert & Craig E Nelson. (1991). Sex Determination In Turtles: Diverse Patterns and Some Possible Adaptive Values. Copeia. 1:50-69.

7 - Yuiko Matsumoto et al. (2013). Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles. PLOS One. (1)

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