The widespread relationship between salt excreting structures (e.g., salt glands) and marine life strongly suggests that the ability to regulate salt balance has been crucial during the transition to marine life in tetrapods. Elevated natremia (plasma sodium) recorded in several marine snakes species suggests that the development of a tolerance toward hypernatremia, in addition to salt gland development, has been a critical feature in the evolution of marine snakes. However, data from intermediate stage (species lacking salt glands but occasionally using salty environments) are lacking to draw a comprehensive picture of the evolution of an euryhaline physiology in these organisms. In this study, we assessed natremia of free-ranging Dice snakes (Natrix tessellata, a predominantly fresh water natricine lacking salt glands) from a coastal population in Bulgaria. Our results show that coastal N. tessellata can display hypernatremia (up to 195.5 mmol.l⁻¹) without any apparent effect on several physiological and behavioural traits (e.g., hematocrit, body condition, foraging). More generally, a review of natremia in species situated along a continuum of habitat use between fresh- and seawater shows that snake species display a concomitant tolerance toward hypernatremia, even in species lacking salt glands. Collectively, these data suggest that a physiological tolerance toward hypernatremia has been critical during the evolution of an euryhaline physiology, and may well have preceded the evolution of salt glands.

Snakes rely heavily on chemical cues when foraging, searching for mates, etc. Snakes’ sex attractiveness pheromones comprise mainly heavy, semi-volatile compounds such as ketones. Here we investigated the composition of skin secretions of adult Vipera ammodytes (Linnaeus, 1758) individuals. The samples were analyzed by gas chromatography/mass spectrometry and the identification of the compounds was performed using commercial mass spectral libraries and retention times. The relative concentrations of all detected compounds were tested for significant differences between (1) male vs. female live individuals, (2) shed skin vs. live individuals, and (3) pre-reproductive vs. reproductive live individuals. We detected fifty-nine compounds of which six were ketones. Two ketones (2-pentacosanone and 2-heptacosanone) were present in many of the samples and thus may have an important role in the V. ammodytes chemical communication. We did not find significant differences between the relative concentrations of the compounds between male and female individuals (only three compounds are exceptions). Significant differences were found between extracts from shed skins and live individuals and between live pre-reproductive individuals and live reproductive individuals. The results of the study suggest that chemical communication in V. ammodytes involves less compounds in comparison to the known literature data for other species.

Dice snakes (Natrix tessellata, Laurenti, 1768) inhabit oligohaline and brackish waters along the Bulgarian Black Sea coast, where they often forage at sea. Under these conditions, this species should tolerate highly variable blood plasma sodium concentrations for extended time periods, but the effect of high sodium concentrations to blood cell morphology and physiology is largely unknown. In this experiment, we placed adult dice snakes in waters with different salinity for 84 h, representing the three typical saline concentrations in which dice snakes live and forage: 0.2 ‰ NaCl, fresh water, 16 ‰ NaCl, Black Sea water and 36 ‰ NaCl, World Ocean water. We collected blood through cardiocentesis after exposure to each treatment and used Giemsa's solution for staining the formed elements. We registered numerous nuclear abnormalities (nuclear buds, lobes and blebs) in the blood cells of all investigated specimens. In the snakes placed in the waters with high saline concentration (16 and 36 ‰ NaCl) we found numerous cells with ruptured membranes. These snakes also showed increased mitochondrial activity compared to those in fresh water (1.85 and 2.53 times higher, respectively). Our data suggest that even though dice snakes show a remarkable tolerance to waters with increased salinity, prolonged exposure to it induces significant physiological stress in N. tessellata, which poses a clear limit for their ability to stay in the sea for a long time.