Synchrotron microtomography applied to the volumetric analysis of internal structures of Thoropa miliaris tadpoles

Amphibians are models for studying applied ecological issues such as habitat loss, pollution, disease, and global climate change due to their sensitivity and vulnerability to changes in the environment. Developmental series of amphibians are informative about their biology, and X-ray based 3D reconstruction holds promise for quantifying morphological changes during growth—some with a direct impact on the possibility of an experimental investigation on several of the ecological topics listed above. However, 3D resolution and discrimination of their soft tissues have been difficult with traditional X-ray computed tomography, without time-consuming contrast staining. Tomographic data were initially performed (pre-processing and reconstruction) using the open-source software tool SYRMEP Tomo Project. Data processing and analysis of the reconstructed tomography volumes were conducted using the segmentation semi-automatic settings of the software Avizo Fire 8, which provide information about each investigated tissues, organs or bone elements. Hence, volumetric analyses were carried out to quantify the development of structures in different tadpole developmental stages. Our work shows that synchrotron X-ray microtomography using phase-contrast mode resolves the edges of the internal tissues (as well as overall tadpole morphology), facilitating the segmentation of the investigated tissues. Reconstruction algorithms and segmentation software played an important role in the qualitative and quantitative analysis of each target structure of the Thoropa miliaris tadpole at different stages of development, providing information on volume, shape and length. The use of the synchrotron X-ray microtomography setup of the SYRMEP beamline of Elettra Synchrotron, in phase-contrast mode, allows access to volumetric data for bone formation, eye development, nervous system and notochordal changes during the development (ontogeny) of tadpoles of a cycloramphid frog Thoropa miliaris. As key elements in the normal development of these and any other frog tadpole, the application of such a comparative ontogenetic study, may hold interest to researchers in experimental and environmental disciplines.

The relationship between pond habitat depth and functional tadpole diversity in an agricultural landscape

One of the most important goals of biodiversity studies is to identify which characteristics of local habitats act as filters that determine the diversity of functional traits along environmental gradients. In this study, we investigated the relationship between the environmental variables of ponds and the functional trait diversity distribution of anuran tadpoles in an agricultural area in southeastern Brazil. Our results show that the functional trait diversity of frog tadpoles has a bell-curve-shaped relationship with the depths of ponds inserted in a pasture matrix. Because we are witnessing increasing human pressure on land use, simple acts (e.g. maintaining reproductive habitats with medium depth) can be the first steps towards preserving the diversity of Neotropical frog tadpole traits in agricultural landscapes.

The effects of two fish predators on Wood Frog (Lithobates sylvaticus) tadpoles in a subarctic wetland: Hudson Bay Lowlands, Canada

Fish can have strong predatory impacts on aquatic food webs. Indeed, fish are known to have strong effects on amphibians, with some species being excluded from communities where fish are present. Most research with amphibians and fish has focused on lower latitudes and very little is known of amphibian–fish interactions at higher latitudes. Therefore, we conducted an enclosure experiment in a subarctic natural wetland to examine the predatory effects of two species of fish, brook sticklebacks (Culaea inconstans (Cuvier, 1829)) and ninespine sticklebacks (Pungitius pungitius (L., 1758)), on the survival and growth of Wood Frogs (Lithobates sylvaticus (LeConte, 1825)). We found no significant difference in survival and size at metamorphosis among the two fish species treatments and fish-free treatments. We found that individuals from fish-free treatments metamorphosed earlier than those from either fish species present treatment. Our work suggests that stickleback fish predation may not have a major impact on Wood Frog tadpole survival and growth in a subarctic wetland. Sticklebacks may still have an impact on earlier developmental stages of Wood Frogs. This work begins to fill an important gap in potential factors that may impact larval amphibian survival and growth at higher latitudes.

Prolactin inhibits corticoid-induced differentiation of active Na+ transport across cultured frog tadpole skin

Active Na+ transport differentiates in larval bullfrog skin cultured with corticoids. After 2 wk in culture, the epidermis became positive against human blood group antigen A, the marker for the adult-type cells of the epidermis, but was negative to the antibody against the acetylcholine receptor, the marker for the larval-type epidermis. Amiloride (10(-5) M) did not inhibit the differentiation of active Na+ transport. On the other hand, in skin cultured with prolactin (2 micrograms/ml), the epidermis remained negative against antigen A and positive against acetylcholine receptor, and the differentiation of active Na+ transport was inhibited. Thyroid hormone did not antagonize the inhibitory action of prolactin on this transport differentiation. Prolactin affected the basal cells of the larval epidermis and inhibited development of corticoid-induced adult features in the epidermis.

Low-affinity mixed acetylcholine-responsive receptors at the apical membrane of frog tadpole skin

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin through nonselective cation channels. Acetylcholine (ACh) stimulates Isc like amiloride, although the response is more prolonged. Pretreatment with ACh markedly suppressed amiloride stimulation of Isc; amiloride pretreatment also suppressed ACh stimulation. Half-maximal stimulation of Isc by ACh occurred at 347 microM. Stimulation by ACh was inhibited by both d-tubocurarine [dissociation constant (Kd) = 57 microM] and atropine (Kd = 49 microM). The specific nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium and the specific muscarinic agonist oxotremorine-M both stimulated Isc and were blocked by either atropine or d-tubocurarine. Reciprocal desensitization and blocker cross-reactivity suggest that ACh activates the same population of receptors as amiloride. This ACh-responsive receptor has characteristics of both nicotinic and muscarinic receptors found in other tissues.

Calcium channel blockers inhibit amiloride-stimulated short-circuit current in frog tadpole skin

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin. The time course and concentration response to amiloride and the effects of calcium channel blockers on Isc were studied with larval frog skin mounted in modified Ussing chambers. The amiloride (1 mM) transient was markedly blunted if the skin was previously exposed to low amiloride (0.01-0.1 mM) concentrations. The calcium channel blockers verapamil, nitrendipine, diltiazem, W-7, and lanthanum all blocked the amiloride transient. Diltiazem itself caused a rapid transient in Isc, indicating that it may be a partial agonist. These data suggest that the amiloride-stimulated cation channels rapidly desensitize in a manner similar to the acetylcholine receptor. The decline in Isc after amiloride stimulation could be caused by amiloride block of the open channel. Blockade of amiloride stimulation by well-known calcium channel blockers suggests that these larval cation channels may have some characteristics in common with calcium channels.