Investigation of genetic mechanisms associated with intermuscular bone formation pathways in Tambaqui (Colossoma macropomum)

Author(s)

Emily Bronze

• Document type: Master's Dissertation
• Press: Botucatu. 2021-01-26.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2020-11-27
• Advisor: Danillo {UNESP] Pinhal

Abstract

Several species of fish with importance in aquaculture worldwide have intermuscular bones (IBs) or spines, as they are popularly known. These IBs are a commercially undesirable characteristic because they reduce the palatability of the fish, requiring their removal for production of the fillet, which results in economic and freshness impacts of the product. Tambaqui, Colossoma macropomum, is a neotropical sweet species of the order Characiformes that has great potential for aquaculture, but which has the disadvantage of generating IBs. Recently, in a cultivated population of tambaqui, individuals with absence of IBs have been reported, suggesting that genetic and/or epigenetic variants are associated with the generation of the mutant phenotype. The objective of this work was to investigate the molecular mechanisms associated with the formation of intermuscular bones, in order to contribute to the generation of animals with greater added economic value. For this purpose, the expression of coding genes and non-coding genes associated with the pathways of IBs formation was analyzed, in addition to the temporal characterization of the formation of IBs during development. Based on the literature, three candidate genes were initially selected for study in the intramembranous ossification pathways: bmp4, runx2 and mstn. These were evaluated for expression in the growth interval that comprises from the beginning until the complete formation of the IBs, in lengths 18,20,23 and 26mm. Initially, the spawning was carried out with the matrices that present the desired mutant phenotype (without IBs) and a control group (with IBs), followed by the cultivation of fingerlings and their monitoring to determine the time of ossification beginning of the IBs or absence of these. Subsequently, tissue was collected from the region close to the caudal peduncle followed by RNA extraction and cDNA synthesis of these individuals for subsequent analysis of gene expression profiles by qPCR. Concomitantly, the fixation and diaphanization of fingerlings at different stages of development was carried out for the morphological characterization of IBs. The total RNA was isolated from the mutant and control matrices for large-scale sequencing of miRNAs (miRNA-Seq). As a result, it was found that the IBs in tambaqui are formed from a total length of 15 mm, prior to previously reported. The comparative analysis of bmp4 expression revealed a significant difference between mutants and control, with a decrease in the expression of the mutante groups, mainly in the lengths considered to be active bone mineralization (i.e., 20 and 23mm). Runx2b also showed a significant difference between mutants and control, but the peak of decreased expression occurred in animals with 20mm in length. On the other hand, mstn showed a significant increase in expression in mutants without IBs compared to control animals, suggesting possible involvement of these genes in the pathways associated with the formation of IBS. MiRNA-seq revealed the expression of miRNAs important for the regulation of bone formation pathways, and others potentially active in the regulation of the three coding genes analyzed in RT-qPCR, in addition to the detection of new miRNAs exclusively expressed only in mutant tambaquis without IBs. This is the first study to evaluate the expression of genes involved in bone formation in neotropical species, whose pecimens originated from the mating of mutant matrices without IBs. Additionally, it is the first analysis of the complete microRNAoma to investigate the mutant phenotype without intermuscular bones. These unprecedented data provide subsidies for future work to produce tambaqui strains without IBs, with great potential for improving productivity in aquaculture. (AU)

• FAPESP's process: 18/11795-8 - Functional characterization of microRNAs and target genes associated with the formation of intermuscular bones in tambaqui (Colossoma macropomum)
• Grantee: Emily Bronze dos Santos
• Support Opportunities: Scholarships in Brazil - Master