|dc.description.abstract||Channel catfish (Ictalurus punctatus), as a member of Siluriformes, has scaleless skin. However, it is hardy and resistant to many types of stresses including low oxygen, high density, handling, and infections from various pathogens. These characteristics make it a successful species for aquaculture. It is widely cultured in the world, especially in North America and Asia. As such, its genome research has been a priority of genetics research. Catfish is one of the aquaculture species on which the USDA NRSP-8 National Animal Genome Project Program has focused.
However, the evolutionary causes for the scaleless skin, and how the scaleless skin adapted to being resistant to disease infection is unknown. The catfish transcriptome has been well studied from various tissues, but the skin transcriptome has not been well characterized. The goals of this research are to conduct transcriptome analysis in the skin of scaleless catfish to generate the catfish skin transcriptome, and conduct comparative transcriptome analysis of scaled fish with scaleless fish to provide insights into the genomic causes for the evolutionary loss of scales, and for the compensatory expression of genes to account for the strong resistance against various infections without the physical protection of scales.
RNA-Seq was used to generate the short reads of the skin transcriptomes of channel catfish, common carp, and pleco. In all cases, hundreds of millions of short reads were generated, and they were then assembled into contigs for bioinformatic analysis. Annotation of the transcriptomes allowed identification of 20,474 genes from channel catfish skin, 25,136 from the common carp skin, and 21,105 from the pleco skin.
The interspecific comparative transcriptome analysis between the scaled common carp and scaleless channel catfish allowed identification of 836 genes that were expressed in the skin of common carp but not in the skin of channel catfish. This pool of the 836 genes should be of interest for the identification of the genes important for scale formation.
The vast majority of catfishes are scaleless, but some such as pleco has bony dermal plate type of scales. The scaled catfish should provide an ideal natural model for comparative studies as to what genes have been lost during evolution in scaleless catfishes. However, scale regeneration was not possible with pleco. I conducted scale regeneration experiments in common carp to identify differentially expressed genes during scale regeneration. Compared to the normal common carp skin, a total of 1,173 differentially expressed genes were identified during carp scale regeneration. Functional annotation indicated that the differentially expressed genes included genes regulating the immune response, metabolism, collagen trimer and binding, cellular cytoskeletal structure, calcium ion binding, and formation or development related genes in scale, bone, hair, tooth and fin, providing a pool of genes involved in scale formation.
Although the interspecific transcriptome subtraction and the scale regeneration experiments both allowed the identification of a fraction of the skin transcriptome to be considered for candidates of scale formation, the list of these pools of genes are yet too long (836 and 1,173, respectively). I further reasoned that the genes important for scale formation should satisfy both the conditions: 1) they should not be expressed in the scaleless catfish, and 2) they should be differentially expressed during scale regeneration. Therefore, a cross subtraction was conducted: coupling interspecific comparative transcriptome subtraction between common carp skin transcriptome and channel catfish skin transcriptome with the carp scale regeneration experiments. A total of 18 genes meeting the above two conditions were identified including 13 known genes and five unknown genes. Of the 13 known genes, 10 genes were up-regulated during scale regeneration, and three genes were down-regulated. The up-regulated genes include 14 kDa apolipoprotein, actinoporin-like protein, apolipoprotein A-I, apolipoprotein AIb1, lymphocyte antigen 6D, protein FAM133-like, protein GAPT-like, secretory calcium-binding phosphoprotein 7, si:dkey-22i16.3 (fa93e10), and uroplakin 2-like. Although their involvement in scale formation requires functional analysis, this pool of 18 genes is of great interest for additional analysis for their involvement in scale formation.
Although direct analysis of scale regeneration was not feasible with pleco, analysis of its skin transcriptome should still be useful to provide insights for the involved genes for scale formation. To obtain the differentially expressed genes between the channel catfish and pleco, RNA-Seq was conducted with pleco skin. Comparative transcriptome subtraction between the transcriptomes of pleco and channel catfish skin allowed identification of 704 genes that were expressed in the skin of scaled pleco but not in scaleless channel catfish.
Of the 18 genes that were only expressed in scaled carp skin but not in channel catfish skin and are differentially expressed genes during scale regeneration, three were expressed in scaled pleco skin, but not in channel catfish. These genes are lymphocyte antigen 6D, secretory calcium-binding phosphoprotein, and uncharacterized protein si:dkey-30j10.5. The sharing of these genes from both comparative subtraction analysis provided stronger evidence that these genes are likely involved in scale formation in teleosts. They may account, at least in part, for the evolutionary lack of scales in catfish. Future functional studies such as knock out experiments using CRISPR or its related technologies are warranted.
My dissertation work allowed generation and assembly of the transcriptomes from channel catfish, common carp, and pleco. Comparative transcriptome subtraction analysis, coupled to scale regeneration experiments, as demonstrated here, provided insights into genomic basis for the scaleless phenotype. My work also demonstrated the power of comparative transcriptome subtraction as an alternative for searching candidate genes of structural significance. Practically, the transcriptome resources I generated should be useful for various genome and transcriptome research, which would set the foundation for evolutionary analysis, as well as analysis for various biological and aquaculture-related traits. In addition, as the transcriptome resources accumulate from the scaled fishes and scaleless fishes, it will soon become possible to conduct comparative analysis as to what additional genes that are expressed in scaleless fishes, but not in scaled fishes, which provide the compensatory advantage allowing the scaleless fishes to be resistant against various infectious agents even though they lack the physical protection offered by scales.||en_US