The Charnov-Bull model of differential fitness is often used to explain the evolution and maintenance of temperature-dependent sex determination (TSD). Most tests of the model focus on morphological proxies of fitness, such as size traits, whereas early life physiological traits that are closely related to lifetime fitness might provide a framework for generalising the Charnov-Bull model across taxa. One such trait is the strength of early life immune response, which is strongly linked to early life survival and fitness. Here, we manipulate temperature, variance in temperature, and sex to test the Charnov-Bull model using a physiological trait, immune system strength, in the snapping turtle (Chelydra serpentina L. 1758). We find no evidence of sex-specific differences in bactericidal capacity of hatchling blood, and no evidence that mean temperature influences bactericidal capacity. However, we find that fluctuating incubation temperature (i.e., a more naturalized incubation regime) is associated with a greater bactericidal capacity compared to constant temperature incubation. We also find that egg mass, a proxy for maternal provisioning, is positively associated with bactericidal capacity. Our findings suggest that the evolution of temperature-dependent sex determination in reptiles is unrelated to our measure early-life innate immunity. Our study also underlines how immune response is condition-dependent in early life, and questions the biological relevance of constant temperature incubation in experimental studies on ectotherm development.

Snapping turtle eggs were collected soon after laying and were incubated in one of four temperature regimes (24C +/- 0C, 24C +/- 4C, 28C +/- 0C, 28C +/- 4C). Eggs in each temperature treatment were subjected to either an aromatase inhibitor or a control treatment creating eight total experimental groups. 

On hatching, blood was extracted from hatchlings and this was subjected to a bacterial killing assay with E.coli. To determine the proportion of bacteria killed by hatchling plasma we measured absorbance with a spectrophotometer. Each hatchling BKA was run in duplicate. 

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