Anurans employ a wide variety of anti‐predator mechanisms to defend themselves. In casque‐headed hylids, defence is thought to be a complex combination of several anti‐predator mechanisms. However, the defence traits of only a few species are known; some hypotheses have yet to be addressed, whereas others, already tested in some species, need to be tested in additional taxa. The anti‐predator mechanism of the casque‐headed frog, Argenteohyla siemersi, is described here. It is a complex mechanism consisting of (1) behavioural and ecological traits, including secretive and semi‐phragmotic habits and posture; (2) morphological features, including cryptic and aposematic colourations, a skull covered with bony dermal spines and protuberances that are associated with two types of granular venom glands; and (3) physiological and chemical traits, such as a highly lethal skin secretion. Our results are compared with those of previous studies of defence mechanisms in casque‐headed frogs in an effort to understand the mechanisms and evaluate their potential phylogenetic signal in this group of anurans.

Background: The WHO states that antivenom is the only safe and effective treatment to neutralize snake venom. Snakebite antivenom typically involves horse hyperimmunization with crude venom and Freund's adjuvant. Methods: In the current work, we analyzed the ascorbyl palmitate liquid crystal structure with snake protein or PLA2, the carrier charge capacity, and we evaluated the immune response induced by the enzyme P9a(Cdt-PLA2) formulated in a nanostructure using CpG-ODN, determining the titer of IgG antibodies. BALB/c mice were subcutaneously immunized on days 0, 15 and 30 with P9a(Cdt-PLA2)/CpG-ODN/Coa-ASC16 or P9a(Cdt-PLA2)/Freund's adjuvant (complete first and incomplete-booster). On day 48 the mice were sacrificed. The neutralization ability of antibodies from animals immunized with P9a(Cdt-PLA2)/CpG-ODN/Coa-ASC16 or P9a(Cdt-PLA2)/Freund's adjuvant was tested against PLA2 activity and venom lethality. Results: In both groups of immunized mice, the antibody titers in blood samples at the assayed time were high (approximately 1×105). The antibodies were able to neutralize P9a(Cdt-PLA2) activity in vitro and lethality in vivo. Microscopic analysis showed that P9a(Cdt-PLA2)/CpG-ODN/Coa-ASC16 produces minimal damage at injection sites compared with Freund's adjuvant. Conclusion: The Coa-ASC16/CpG-ODN formulation shows promise as a safe and effective adjuvant against crotalic PLA2, inducing a strong humoral response and reducing local tissue damage compared with Freund's adjuvant.