Jose A. Domingos

Whole‐genome shotgun sequence assembly enables rapid gene characterization in the tropical fish barramundi, Lates calcarifer

[Extract] Source description: Barramundi, Lates calcarifer, is a commercially important fish farmed throughout Australia and South-East Asia. Despite an increasing availability of genetic resources for the species (e.g. microsatellite and SNP markers, linkage and BAC-based maps and transcriptomic assemblies), the complete characterization of genes is still reliant on laborious molecular methods (e.g. genome walking/RACE PCR cloning/Sanger sequencing).

A robust flow-cytometric protocol for assessing growth rate of hatchery-reared barramundi Lates calcarifer larvae

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

Sex-specific dmrt1 and cyp19a1 methylation and alternative splicing in gonads of the protandrous hermaphrodite barramundi

Epigenetics is involved in sex differentiation of gonochoristic and hermaphroditic fish species, whereby two genes dmrt1 (pro-male) and cyp19a1 (pro-female) are known to play major roles. Barramundi, Lates calcarifer, is an important tropical aquaculture species that undergo natural and permanent male to female sex change, a process for which the exact underlying molecular mechanisms are still unknown. To elucidate whether DNA methylation is involved in sex control of barramundi, a next-generation bisulfite amplicon sequencing approach was used to target 146 CpG sites within proximal promoters and first exons of seven sex-related genes (dmrt1, cyp19a1, amh, foxl2, nr5a2, sox8 and sox9) of 24 testis and 18 ovaries of captive and wild adult barramundi. Moreover, comparative expression profiles of the key dmrt1 and cyp19a1 genes were further investigated using RT-qPCR and Sanger sequencing approaches, whereas expression levels of remaining targeted genes were based on available literature for the species. Results showed that cyp19a1 and amh were more methylated in males, whereas dmrt1 and nr5a2 were more methylated in females (P < 0.001), with no gender differences found for foxl2, sox8 or sox9 genes (P > 0.05). Sex-biased promoter DNA methylation was inversely related to gene expression only for dmrt1 and nr5a2, and directly related to amh expression, whereas no differences in cyp19a1 expression were found between testes and ovaries. Notably, unique sex-specific alternative splicing of dmrt1 and cyp19a1 were discovered, whereby males lacked the full-length aromatase coding cyp19a1 mRNA due to partial or total exon splicing, and females lacked the dmrt1 exon containing the DM-domain sequence. This study advances the current knowledge aiming to elucidate the genetic mechanisms within male and female gonads of this large protandrous hermaphrodite by providing the first evidence of epigenetics and alternative splicing simultaneously affecting key genes (cyp19a1 and dmrt1) central to sex differentiation pathways.

Ruler Detection for Automatic Scaling of Fish Images

Fast and low-cost image collection and processing is often required in aquaculture farms for quality/size attributes and breeding programs. For example, the absolute physical dimensions of fish (in millimeters or inches) could be estimated from electronic images. The absolute scale of the photographed fish is often unknown or requires additional hardware, data-collection and/or management overheads. One cost and time effective solution is to capture the absolute scale (in pixels-per-millimeter or dots-per-inch) by including a measuring ruler in the photographed scene. To assist that type of workflow, this paper presents a relatively simple image-processing algorithm that automatically located a sufficiently large section of the ruler in a given image. The algorithm utilized the Fast Fourier Transform and was designed to be free from adjustable parameters and therefore did not require training. The algorithm was tested on 445 images of Barramundi (Asian sea bass, Lates calcarifer), where a millimeter-graded ruler was included in each image. The algorithm achieved precision of 98% (on the original, 10, 20, 70, 80 90 degree rotated images) and 95-96% on 40, 50, 60 degree rotated images. The test Barramundi images were released to public domain (on this publication) via https://github.com/dmitryako/BarraRulerDataset445.