Estudo da composição e das atividades biológicas dos microrganismos isolados do ninho de espuma do anuro Adenomera hylaedactyla

Abstract

Many groups of amphibians lay their eggs in foam nests. Th ese struc tures are for m e d during amplexing and resist degradation due to their composition rich in surfactant proteins, carbohydrates and lectins. Foam nests provide shelter for deposited eggs and young larvae, protection against ultraviolet rays, desiccat ion effec ts, predators in addition to providing oxygen and food reserve. Currently is known that the defense against many pathogens in the adult individual comes from their microbiota. However, little is known about the microbiota present in the nests dur ing their juvenile sta g e . This work search to isolate and identify cultivable bacteria from the foam nest of the anuran Adenomera hylaedactyla , in order to evaluate the ecological importance of these microorganisms and their biological activities, as well as their biotechnologi c a l potential. The nests were collected in São Gonçalo do Amarante CE during the rainy season, in additional samples of the adjacent soil. Then, the samples were diluted and plated in ATGE and BDA medium and maintened for 48/168 h at 30ºC. The bacterial an d fungal morphotypes were isolated and stored. Enzymatic tests were carried out on ATGE + starch (0.1% w v) for amylases, ATGE + skimmed milk (1% w v) and ATGE + gelatin (3% w v) to search for proteases with different cleavage sites. Lugol was added t o det e c t amylase activity and a 4.1M ammonium sulfate ((NH4) 2SO4) solution for gelatinase activity. For the antifungal assay, the fungus Fusarium sp was grown in BDA medium and on the fifth day the bacteria and negative control E. coli ATCC 2592 2) were t rans f e rred in a log phase to the plate. The results were observed by inhibiting the growth of the fungus on the plate. For antibacterial tests, the antibiogram was performed with standardized samples of the supernatant from the bacterial cultures of the ne st, u s ing the bacteria Escherichia coli (ATCC 25922) and Bacillus subtilis (ATCC 6633) as target. The genomic DNA of the nest isolates was extracted by thermolysis or CTAB 2X and the 16S rRNA gene was amplified using primers 27F and 1525R or 63F a nd 1389R and s e nt for sequencing. The sequences were treated using the Geneious Prime software and the genera identified from the alignment with the database. 17 fungi and 21 bacteria were isolated from the adjacent soil, in addition to 7 fungi an d 30 bact eria in t he n e s t samples. The bacterial isolates of the foam nest were predominantly Gram negative from the Proteobacteria phylum, and there was a greater abundance of the Enterobacter , Bacillus and Serratia genera. 8 (27%) of foam nest isolates w ere posit ive for a myla s e s and 19 (63%) positive for proteases with different cleavage sites, compared to the adjacent soil that presented 5 (24%) for amylases and 8 (38%) for proteases. 9 (30%) of the nest isolates showed antifungal activity on the growth of the fu ngus Fusa rium s p . No significant antibacterial activity has been reported. The present work provided information on the cultivable microorganisms of the foam nest of the species A. hylaedactyla , highlighting its importance in the defense against p athogens and biote chno l o gical potential for the production of hydrolytic enzymes.