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

Abstract

SummaryThe 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.

Gene expression profiles in Rana pirica tadpoles following exposure to a predation threat

“…The pirica protein was found to contain a zona pellucida domain similar to proteins that function to control water permeability; the pirica gene was shown to be expressed in the superficial epidermis of the tadpole skin [ 29 ]. We also demonstrated that water retention in the connective tissue and maintenance of a constant osmotic pressure were important factors for bulgy morph formation, supporting the interpretation that predator-induced expression of pirica in the skin causes retention of absorbed water [ 30 ]. The immuno-related proteins hyaluronic acid, histone H3 and 14-3-3 zeta were the most abundant constituents of the liquid aspirated from the connective tissue.…”

“…This gene showed opposite responses in tadpoles of the dragonfly and salamander treatment groups (Figure 8 b): a 6.6 fold increase was seen in the salamander treatment group, while a 6.5 fold increase occurred following the removal of the dragonfly larvae compared to control. In the salamander treatment, tadpoles maintain their osmotic pressure by uptake of Na and Cl ions to increase the amount of bodily fluids for bulgy morph formation [ 30 ]. Possibly, non-motile or sensory cilia may be involved in this process.…”

“…The immuno-related proteins hyaluronic acid, histone H3 and 14-3-3 zeta were the most abundant constituents of the liquid aspirated from the connective tissue. These findings suggested that formation of the bulgy morph might also require activation of the innate immune system [ 30 ].…”

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

“…The purifi ed fraction (0.22 g) diluted in a sample buffer containing SDS and 2-mercaptoethanol was boiled and electrophoresed on a 15% polyacrylamide gel. Mass spectrometric analysis and the pretreatment were performed as described previously ( 29 ). Briefl y, protein in the gel was detected by MS-compatible silver staining.…”

Identification of a Low Digestibility δ-Conglutin in Yellow Lupin (Lupinus luteus L.) Seed Meal for Atlantic Salmon (Salmo salar L.) by Coupling 2D-PAGE and Mass Spectrometry

“…Low digestibility proteins were analyzed and identified using a mass spectrometer, as previously described by Mori et al [31] . In brief, proteins (spots) were excised from the gel, destained, and the thiol groups of cysteines alkylated by iodoacetamide.…”