SUMMARYMany amphibians lay their eggs in foam nests, which allow the eggs to be deposited out of the water. Analysis of some of these foam nests has revealed that they are a rich source of proteins with unusual primary structures and remarkable surfactant activity, named ranaspumins. The aim of this work was to study the foam nests of the frog Leptodactylus vastus in order to obtain information regarding their composition and function and to improve the understanding of ranaspumins, which are probably a novel class of surfactant proteins. Analyses of the foam fluid composition showed proteins and carbohydrates that presumably are responsible for providing nutrients for the developing tadpoles. Investigation of the function of foam fluid in chemical defence revealed no significant biological activity that could be associated with recognized defence compounds. However, foam fluid presented UV absorbance, suggesting a role in protection against sun damage, which is considered to be one of the possible causes of recently reported amphibian population declines. The foam nests do not prevent the colonization of microorganisms, such as the observed bacterial community of predominantly Gram-positive bacilli. L. vastus foam fluid shows a strong surfactant activity that was associated with their proteins and this activity seems to be due mainly to a protein named Lv-ranaspumin. This protein was isolated by ion-exchange chromatography and found to be a 20 kDa monomeric molecule with the following Nterminal sequence: FLEGFLVPKVVPGPTAALLKKALDD. This protein did not show any match to known proteins or structures, which suggests that it belongs to a new class of surfactant protein.

Spores of Bacillus subtilis LAMI008 were entrapped in 3-mm chitosan beads and cross-linked with 0.3% glutaraldehyde for n-hexadecane biodegradation and biosurfactant recovery. When exposed to nutrients, the spores generated vegetative cells without morphological alterations as revealed by atomic force microscopy. The entrapped cells degraded almost 100% of 1% of n-hexadecane in medium supplemented with 1% glucose and produce biosurfactant within 48 h, as well as free cells. The number of viable cells inside the beads was maintained throughout the n-hexadecane degradation process and the released biosurfactant was not used as a carbon source. Entrapment of bacterial spores in chitosan beads overcomes problems with stability, storage, and long term cell viability encountered with vegetative cells. This approach can potentially be utilized for biodegradation of complex compounds by entrapping spores of different species of bacteria.

Breeding by releasing eggs into stable biofoams (“foam nests”) is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv‐ranaspumin (Lv‐RSN‐1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv‐RSN‐1 by de novo sequencing with mass‐spectrometry and determined the high‐resolution X‐ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α‐helices and two small antiparallel β‐strands. Lv‐RSN‐1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β‐sheet. It possesses intrinsic surface‐active properties, reducing the surface tension of water from 73 to 61 mN m−1 (15 μg mL−1). Lv‐RSN‐1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.

Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

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