printed by Chemical Defense in Hatchling Wood Turtles, Glyptemys insculpta: Function of the Mysterious Rathke’s Gland Robert A. Mitchell, Mary Kate Donais, and B. J. Wicklow Department of Biology, Saint Anselm College, Manchester, NH, USA Turtles rely on long-lived females to continue a modest annual reproductive output spread over many years to increase the chance that some eggs will hatch and that some of those hatchlings will survive to adults. Eggs and hatchlings are extremely vulnerable to predators (Figure 1). We discovered that hatchling wood turtles, Glyptemys insculpta, emit a strong, unpleasant odor when the skin of the turtle is lightly scraped. This substance is released from the axillary region just below the carapace and above the leg. Previous studies show the presence of a gland, Rathke’s gland, with ducts leading to openings in the shell. In 2009, Plummer and Trauth described the structure of Rathke’s gland in softshelled turtles. They conclude, however “the gland’s function remains largely unknown and offers a challenging opportunity for behavioral and chemical ecologists”. We hypothesize that Rathke’s gland is the source of the substance emitted from hatchling wood turtles and that the substance functions as a chemical defense. We dissected a previously dead adult male and a pre-hatched individual collected that died at a late stage of development in We used a polar solid phase microextration fiber to collect the volatile component of the substance then used an Agilent GCD Plus gas chromatograph for analysis. We designed a preference experiment with rats to test the defensive function of the substance. This is the first anatomical and functional description of Rathke’s gland in wood turtles. T The spectrogram of the defensive chemical showed a very distinct peak that was absent in the controls. The preliminary preference experiment support the hypothesis of a defensive function of the Rathke’s gland in the hatchling turtles. LOGO Figure 1. Wood turtle just emerging from the nest (left) and a hatchling turtle that survived a predator attack. Figure 2. Preference experiments show a significant difference in the choice of food items with and without the defensive substance (^X 0.025< p<0.05). Figure 3. Rathke’s gland in wood turtles. (A) Anatomical location of the gland orifice in an adult shell. (B) Pre-hatchling used to locate the gland. (C) Location of the gland orifice in the pre-hatchling (red arrow), unidentified gland (gl), above striated muscle (stm), cranial direction (white arrow). (D) Exposed Rathke’s gland (rgl). (E) Exposed duct leading from Rathke’s gland to orifice (rgd) Figure 4. Spectrograms of the control, hatchling 807 without induced defense (upper left); released defensive substance from hatchling 807 (upper right); released defensive substance from hatchling 605 (lower left); spectrum of peak in induce hatchling 807 (lower right). Introduction Methods Results Conclusions rgl rgd stm 1 mm rgl 1 mm gl AB C D E