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Biol Open
2012 Apr 15;14:308-17. doi: 10.1242/bio2012604.
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Histological and MS spectrometric analyses of the modified tissue of bulgy form tadpoles induced by salamander predation.
Mori T
,
Kitani Y
,
Ogihara J
,
Sugiyama M
,
Yamamoto G
,
Kishida O
,
Nishimura K
.
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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.
Fig. 1. Comparative morphologies of control and the bulgy morph R. pirica tadpoles.
(a–c) Tadpoles are shown in side view (a), front view (b) and top view (c); the control tadpole is the upper specimen in (a) and the left specimen in (b) and (c), the bulgy morph tadpole is the lower specimen in (a) and the right specimen in (b) and (c). (d–k) Schematic diagrams illustrating the center marker method for ensuring preparation of midline sections. The tadpole is positioned over the midline of the center marker (d,e,f). (i) illustrates a section cut along the line a-a′ of the tadpole (g) and of the center marker (h). (j) illustrates an oblique section along the line b-b′ of the tadpole (g) and of the center marker (in h). (k) illustrates a midline section along the line e–f of the tadpole (g) and of the center marker (h). (l–o) Hematoxylin-eosin stained sections of control and bulgy morph tadpoles. Images of the head end of control (l) and bulgy morph (m) tadpoles, and their parietal regions (n, o, respectively).
Fig. 2.
Sections through bulgy morph tadpoles.
The relative position of each image on the section is identified by the region highlighted in red in the superimposed line drawing. (a,b) Mallory-Azan staining of the parietal region. In (a), a typical region of the parietal surface is shown, while (b) shows the structure of a deeper part of the parietal region. bl, basal lamella; fb, fibroblast; cell, connective tissue cell; pg, pigment granule; se, squamous epithelium.
Fig. 3. Sections through bulgy morph tadpoles and alcian blue staining of the parietal region.
(a) Section stained at pH2.5, (b) section stained at pH1.0, (c) section stained at pH2.5 after hyaluronidase treatment. Ct, cartilage.
fig. 4. Effect of a predator on bodily fluid volumes and osmotic pressures in tadpoles.
Tadpoles were homogenized using a Polytron (Kinematica) and the homogenate centrifuged at 15,000 rpm for 30 min at 4°C. The total volume of supernatant from each tadpole was measured using a micropipette (n = 24 control (C); n = 25 bulgy morph (S) stimulated by predator); osmolarity was measured using the supernatant (n = 14 control; n = 15 bulgy morph). Vertical bars represent standard errors, and * indicates a significant difference by Student's t test (p<0.01).
Fig. 5. Changes in Na, K, and Cl ion concentrations in tadpoles exposed to a predator.
Tadpoles were homogenized using a Polytron (Kinematica) and the homogenate was centrifuged at 15,000â rpm for 30â min at 4°C. Na, K, Cl ion concentrations were measured in the supernatants of control (C) and bulgy morph (S) tadpoles (C, nâ=â16; S, nâ=â21); the data are expressed as values relative to control levels. Vertical bars represent standard errors, and * indicates a significant difference by Student's t test (p<0.01).
Fig. 6. Immunohistochemical staining of midline sections.
Sections were incubated with anti-histone H3 antibody (a,b,c) or anti-14-3-3 zeta antibody (d,e,f). (a,d) Parietal surface of bulgy morph tadpole. (b,e) Deeper structures of the parietal region of the bulgy morph tadpole. (c,f) Surface and deeper structure of parietal region of control tadpoles. Arrows indicate stained proteins, the superimposed circular images show HE staining of material stained by each antibody.
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