Non-invasive sex genotyping of paiche Arapaima gigas by qPCR: An applied bioinformatic approach to identify sex differences

Abstract

Arapaima gigas, one of the largest freshwater fish in the world, is suffering from high fishing pressure and habitat loss, which threaten the conservation status of its natural populations. Of great cultural importance to Amazonian people, the paiche or pirarucu A. gigas is in high demand for its meat, ornamental uses and other byproducts such as scales. Aquaculture is a feasible solution to this dilemma. However, the fact that A. gigas presents no sexual dimorphism until it is 5 years old and its long period to sexual maturity are major obstacles for brood-stock management and fingerling production. Thus, the aim of this study was to develop a molecular tool for non-invasive genotypic sexing of paiche throughout its life cycle. We collected samples from gonads, fins and gill mucus of juvenile specimens from local facilities for histological and molecular analysis. Based on the recently available genome sequence of the paiche and making use of current NGS method, we implemented a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers. We found a Male-Specific Region (MSR), identified as MSR_3728, to be present only in males. Next, we designed two specific sets of primers on this region to identify genotypic sex by qPCR assays. Both primer sets, MSR_107 and MSR_129, detected males with 100% accuracy. Then we developed a duplex qPCR reaction for each primer set along a reference gene, analyzed the melting curves and detected males by observing two distinct peaks, one for MSR_107 or MSR_129 and one for the reference, while females only presented the reference peak. The same results were obtained for gonads, fins and interestingly, a non-invasive source from gill mucus samples. Finally, the gonads were evaluated histologically in a double-blind test, showing 100% accuracy with qPCR assay for identifying males and females. Data clearly demonstrated a novel pipeline approach for identifying DNA sex markers, followed by a quick, non-invasive, cost-effective duplex qPCR method for sexing A. gigas. These results may be valuable to efficient paiche aquaculture and conservation studies, helping to reduce the fishing pressure on its natural populations. Highlights of the manuscript- Implementation of a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers. - Finding of a Male-Specific Region (MSR), present only in males. - Development of a duplex qPCR to identify genotypic sex through non-invasive sampling.