Experimental assessment of the effects of moisture on loggerhead sea turtle hatchling sex ratios

Many reptiles have temperature-dependent sex determination (TSD). Sex determination in marine turtles is described by a cool-male, warm-female pattern. Nest sand temperature strongly influences sea turtle embryo development and sex differentiation. Yet, variation in hatchling sex ratios is explained only partially by nest temperature and can be predicted only at the warmest and coolest temperatures. Hence, other factors during development influence sex determination. Rainfall is a common environmental variable that may impact development and sex determination. We experimentally evaluated bias in sex ratio production associated with nest moisture. Conditions tested in surrogate nests were sand moisture in combination with (i) very restricted evaporation, (ii) moderate evaporation (allowing evaporative cooling), and (iii) evaporative cooling plus cooling from rain-temperature water. We collected eggs from 32 unique loggerhead (Caretta caretta L.) turtle clutches, distributed them among the three different conditions, and incubated the eggs until they hatched. All hatchlings were raised for several months and sex was verified laparoscopically to establish sex ratios for each treatment. The nests were expected to produce 50:50 sex ratios or a moderate female bias (∼70%) based on incubation temperatures. All experimental treatments produced high male bias (87-96%). These results support the hypothesis that moisture impacts sex ratios through evaporation and rainfall-based cooling. High male bias was observed in nests with and without restricted evaporative cooling and no direct cooling due to watering as well as those nests hydrated via cool (rainwater temperature) water. High moisture conditions may produce males through thermal or other mechanisms, highlighting the importance of examining other nest environmental factors on sex determination.