BackgroundCatfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing.Methodology/Principal FindingsA total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen (double haploid female) allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination.Conclusion/SignificanceWe provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish.

Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

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