Histological and MS spectrometric analyses of the modified tissue of bulgy form tadpoles induced by salamander predation

The rapid induction of a defensive morphology by a prey species in face of a predation risk is an intriguing in ecological context; however, the physiological mechanisms that underlie this phenotypic plasticity remain uncertain. Here we investigated the phenotypic changes shown by Rana pirica tadpoles in response to a predation threat by larvae of the salamander Hynobius retardatus. One such response is the bulgy morph phenotype, a relatively rapid swelling in size by the tadpoles that begins within 4 days and reaches a maximum at 8 to 10 days. We found that although the total volume of bodily fluid increased significantly (P<0.01) in bulgy morph tadpoles, osmotic pressure was maintained at the same level as control tadpoles by a significant increase (P<0.01) in Na and Cl ion concentrations. In our previous report, we identified a novel frog gene named pirica that affects the waterproofing of the skin membrane in tadpoles. Our results support the hypothesis that predator-induced expression of pirica on the skin membrane causes retention of absorbed water. Midline sections of bulgy morph tadpoles showed the presence of swollen connective tissue beneath the skin that was sparsely composed of cells containing hyaluronic acid. Mass spectrographic (LC-MS/MS) analysis identified histone H3 and 14-3-3 zeta as the most abundant constituents in the liquid aspirated from the connective tissue of bulgy tadpoles. Immunohistochemistry using antibodies against these proteins showed the presence of non-chromatin associated histone H3 in the swollen connective tissue. Histones and 14-3-3 proteins are also involved in antimicrobial activity and secretion of antibacterial proteins, respectively. Bulgy tadpoles have a larger surface area than controls, and their skin often has bite wounds inflicted by the larval salamanders. Thus, formation of the bulgy morph may also require and be supported by activation of innate immune systems.