Development and application of a mini-QuEChERS method for the determination of pesticide residues in anuran adipose tissues.

The expansion of monocultures to regions close to conservation areas has put biodiversity at risk, mainly due to the intense use of pesticides. Anurans are highly susceptible to pesticides and may be a biological marker in the contamination of an area. However, methods for determining pesticides in anurans are incipient. In this work, a miniaturized QuEChERS method was developed for the extraction of atrazine, chlorpyrifos, α- and ß-endosulfan, α-, ß-, θ- and ζ-cypermethrin in anuran adipose tissues. The method was optimized for the tissue sample size scale according to sample mass availability. Extracting solvent and adsorbents for the clean-up step was evaluated, achieving recoveries next to 100% with acetonitrile and without a clean-up step. The mini-QuEChERS method, using 500 mg of adipose tissue, 50 mg of NaCl and 200 mg of MgSO4, 100 µL of ultrapure water, and 1.50 mL of acetonitrile with no purification step, followed by high-performance liquid chromatography analysis and photodiode array detection was validated following the European Community guidelines. The methodology showed a moderate matrix effect for some pesticides, which was corrected using the matrix-matched calibration. The limits of quantification for the pesticide residues in adipose tissues ranged from 10 to 75 µg kg-1. Pesticide recoveries ranged from 74% to 115%, and repeatability and within-lab reproducibility showed relative standard deviations < 11%. The mini-QuEChERS method was applied to extract pesticide residues from the adipose tissues of two species of anurans: Leptodactylus macrosternum and Scinax x-signatus. 25% of samples were positive, detecting endosulfan and chlorpyriphos, confirmed by liquid chromatography coupled to tandem mass spectrometry. The mini-QuEChERS was a simple, economical, and eco-friendly method for extracting pesticide residues in anuran adipose tissue samples.

Frog Foam Nest Protein Diversity and Synthesis.

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.

Note on the paleobiogeography of Compsognathidae (Dinosauria: Theropoda) and its paleoecological implications.

The paleobiogeography of the theropod clade Compsognathidae is here reaccessed in order to test the hypothesis of this taxon being adapted specifically to inhabit semi-arid environments. Data about localities where these fossils were collected and their paleoenvironments were gathered from the literature. Compsognathids seem to be found especially in sedimentary deposits known as Fossil Lagerstätten, which were formed under a set of specific conditions that allowed the preservation of the fragile bone remains of these animals. This bias limits an accurate analysis of the historical and/or ecological paleobiogeography of this taxon. Actually, it is possible that compsognathids had an almost worldwide distribution during the Mesozoic Era. Their occurrence in Lower Cretaceous rocks of China suggests that they also inhabited environments with moist conditions instead of being restricted to semi-arid to arid environments.

Novel surfactant proteins are involved in the structure and stability of foam nests from the frog Leptodactylus vastus.

Many 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 N-terminal 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.