DNA-methylation to improve conservation of TSD species

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Global warming threatens over 400 species that have temperature-dependent sex determination (TSD), including all sea turtles. In Cabo Verde - home of the world's largest aggregation of loggerhead turtles (Caretta caretta) - a predicted increase of 3C will result in an extreme sex ratio bias, with >99% female neonates by 2100. If these predictions are correct, this population, and species, will go extinct soon. To mitigate that possibility, we need a better molecular understanding of sex determination that can be applied to new management strategies.Since they first appeared, ~120 million years ago, sea turtles have been exposed to dramatic climatic changes. It is therefore likely they have evolved still undescribed molecular mechanisms that buffer against extreme sex ratio bias driven by temperature variation. Yet, these mechanisms may not keep up with the pace of current global warming, as we already see populations with >95% adult females3. But in contrast, if they do, our models that predict the future are wrong. Hence we may need rethinking mitigation strategies such as hatcheries that maximise the survival of neonates but produce unknown sex ratios, and may even amplify the problem.In sea turtles, sex determination is a plastic trait with only a narrow variation in the pivotal temperature making an entire clutch being composed of either males or females. From a mechanistic perspective, epigenetic processes have regularly been proposed to control phenotypic plasticity that can accelerate adaptive responses to natural selection. Although several epigenetic mechanisms can facilitate phenotypic plasticity, DNA methylation - the addition of a methyl group to cytosine nucleotides - is one of the best characterized to date. DNA methylation is particularly interesting because, we can differentiate environmentally-induced marks and possibly inherited ones. While, understanding if such mechanisms exist is of global conservation importance, before that we need to innovate methods that enable sexing neonates without killing them as is currently the case. Here, we will take advantage of a large field experiment conducted as part of a NERC project led by the applicant (NE/V001469/1). Specifically, we collected blood samples of nesting loggerhead turtles, and moved their egg clutches into an experimental hatchery, where they were exposed to natural incubation conditions, but were protected from predation. Each egg clutch was split into two groups to be exposed to male incubating temperature (deeper depth) or female incubating temperature (shallow depth), mimicking future climate warming. Upon hatching, we collected blood samples and conducted fitness tests to determine the effect of temperatures on dispersal of neonates. Given the successful completion of this experiment, we offer to sequence neonates and mother blood samples for whole-genome bisulfite sequencing (WGBS) to determine genome-wide DNA methylation marks. Backed up by our newly annotated high quality reference genome, we will identify the genes and gene networks involved in sex determination at birth and compare the gold standard WGBS with portable sequencing solution such as MinION. Crucially, because this project focuses on offering conservation-based solutions, we do not investigate the early development stages, since they would require sacrificing individuals and are therefore unlikely to shed light onto conservation strategies needed to respond to the challenge of climate warming.Overall, this project will fill major gaps i) in conservation by determining the potential persistence of sea turtles in the face of global warming and offering practical diagnostic tools to non-lethally determine a clutch sex ratio at birth; ii) in evolutionary biology, because it will provide a new understanding of the mechanisms associated with temperature sex determination. While this project focuses on sea turtles, its significance extends to >400 vertebrate species with TSD.