Kinetic and molecular characterization of the gill (NA+, K+)-ATPase from Macrobrachium amazonicum inhabitants of the Mato Grosso Pantanal and Tocantins-Araguaia River Basin

• Grant number: 19/21899-8
• Support type: Regular Research Grants
• Duration: May 01, 2020 - April 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal researcher: Francisco de Assis Leone
• Grantee: Francisco de Assis Leone
• Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Assoc. researchers: Alessandra da Silva Augusto ; Carlos Frederico Leite Fontes ; Carlos Murilo Tenorio Maciel ; Cristiana Ramalho Maciel ; Daniela Pereira Garçon ; Fernando Luis Medina Mantelatto ; John Campbell McNamara ; Malson Neilson de Lucena ; Marcelo Rodrigues Pinto ; Rogério Oliveira Faleiros

Abstract

This project is a continuation of our research line which aims to systematically study the structural and kinetic properties of the (Na+, K+)-ATPase from the gill tissue of different species of decapod crustaceans, particularly crabs and shrimps, in order to establish a model for the adaptation of these organisms to environments of different salinities. The project involves the participation of an important network of researchers that will support the collection and analysis of samples collected in the different study areas.The maintenance of crustacean hemolymph osmolality is attributed to the (Na+, K+)- ATPase which suggests that the responses of these crustaceans involve the rapid activation of ion transport that constitutes the initial response before to any other physiological responses. Although data on molecular genetics and adult morphology support the existence of a single taxonomic entity, M. amazonicum, there is evidence from larval morphology, morphometry, reproductive performance, and ontogeny of physiological functions suggesting that physically separated M. amazonicum populations provisionally established in continental and coastal areas in different hydrogeographic regions of South America may have suffered incipient speciation.This project aims to evaluate the kinetic and molecular characteristics of the gill (Na+, K+)-ATPase from populations of M. amazonicum from the Mato Grosso Pantanal and from the Tocantins-Araguaia River Basin and to compare them with those of M. amazonicum originating from continental and coastal waters already characterized in our laboratory, in order to elucidate evolutionary patterns of these populations as to the biochemical and physiological mechanisms of geographically separated entities. To achieve our objectives, the following steps will be undertaken: 1) obtain gill membrane (Na+, K+)-ATPase-rich fractions from populations collected in different habitats; 2) PAGE electrophoresis and western blotting; 3) study of the effect of modulators on (Na+, K+)-ATPase and V(H+)-ATPase activities; 4) total RNA extraction from the gills; 5) analysis of (Na+, K+)-ATPase alpha subunit and V(H+)-ATPase B subunit mRNA expression; 6) effect of FXYD2 peptide on (Na+, K+)-ATPase activity; 7) measure of hemolymph osmolality and ionic concentration; 8) analyses of phosphoenzyme formation and decomposition; 9) mRNA reverse transcription; 10) next generation sequencing (RNAseq). (AU)