Endoscopy and Cannulation as Non-Invasive Tools to Identify Sex and Monitor Reproductive Development in Arapaima gigas

AbstractArapaima 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.

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Assessing the sex-related genomic composition difference using a K-mer-based approach: a case of study in Arapaima gigas (Pirarucu)

10.1101/2020.03.29.014647 ◽

2020 ◽

Author(s):

R.L.D Cavalcante ◽

J.M. Ortega ◽

J.E.S Souza ◽

T. Sakamoto

Keyword(s):

Fish Farming ◽

Non Invasive ◽

Large Size ◽

Arapaima Gigas ◽

Genome Features ◽

Invasive Method ◽

Genetic Mechanisms ◽

Opposite Sex ◽

Determination System ◽

Sex Determination System

ABSTRACTArapaima gigas is one of the largest freshwater bony fish in the world, in which adults could weigh 200 kilograms and measure 3 meters in length. Due to its large size and its low-fat meat, Arapaima gigas has quickly become a species of special interest in fish-farming. One challenge faced during their production is the lack of an efficient sexing methodology, since their sexual maturation occurs late (around the third to the fifth year) and the genetic mechanisms linked to their sex determination system are not known yet. For a more sustainable management, it is of paramount importance to seek an effective and non-invasive method to differentiate sexually juvenile individuals of Arapaima gigas. For this, the establishment of genetic markers associated with sexual differentiation would be an advantageous tool. In this study, we proposed a k-mer based approach to identify genome features with sex-determining properties. For this purpose, we used genomic data from four adult representatives of Arapaima gigas, two males and two females, and counted the k-mers comprising them. As result, we found k-mers from repetitive regions with high difference and disproportion in the count among individuals of the opposite sex. These differences in the k-mer-based genomic composition indicate the existence of genetic factors involved in the sexing of individuals in Arapaima gigas.

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

Aquaculture ◽

10.1016/j.aquaculture.2021.737388 ◽

2022 ◽

Vol 546 ◽

pp. 737388

Author(s):

Edgar A. López-Landavery ◽

Guillermo A. Corona-Herrera ◽

Luis E. Santos-Rojas ◽

Nadhia M. Herrera-Castillo ◽

Tomás H. Delgadin ◽

...

Keyword(s):

Sex Differences ◽

Non Invasive ◽

Arapaima Gigas ◽

Bioinformatic Approach

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X-ray microtomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100107058 ◽

1988 ◽

Vol 46 ◽

pp. 998-999

Author(s):

H.W. Deckman ◽

B.F. Flannery ◽

J.H. Dunsmuir ◽

K.D' Amico

Keyword(s):

Computed Tomography ◽

Internal Structure ◽

Imaging Technique ◽

Three Dimensional ◽

Tissue Structure ◽

Individual Element ◽

X Ray ◽

Non Invasive ◽

Panoramic Imaging ◽

Three Dimensional Images

We have developed a new X-ray microscope which produces complete three dimensional images of samples. The microscope operates by performing X-ray tomography with unprecedented resolution. Tomography is a non-invasive imaging technique that creates maps of the internal structure of samples from measurement of the attenuation of penetrating radiation. As conventionally practiced in medical Computed Tomography (CT), radiologists produce maps of bone and tissue structure in several planar sections that reveal features with 1mm resolution and 1% contrast. Microtomography extends the capability of CT in several ways. First, the resolution which approaches one micron, is one thousand times higher than that of the medical CT. Second, our approach acquires and analyses the data in a panoramic imaging format that directly produces three-dimensional maps in a series of contiguous stacked planes. Typical maps available today consist of three hundred planar sections each containing 512x512 pixels. Finally, and perhaps of most import scientifically, microtomography using a synchrotron X-ray source, allows us to generate maps of individual element.

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