Gene Expression and Development of the Air-breathing Organs (ABO) in Asian Catfish and Transfer of Putative ABO Transgenes into Channel Catfish (Ictalurus punctatus)

Abstract

Tra catfish (Pangasianodon hypophthalmus) and bighead catfish (Clarias macrocephalus) are two freshwater air-breathing Asian species that belong to the order Siluriformes. Tra catfish have a swimbladder, whereas bighead catfish have both gills and modified gill structures that serve as an air-breathing organ (ABO) to allow aerial breathing. These two species are excellent models for investigating the development of the accessory air-breathing organ in teleosts. On the other hand, channel catfish (Ictalurus punctatus), one of the major aquaculture species in the United States, breathes only with gills and have no air-breathing ability. They easily die of low oxygen in water resulting into a massive economic loss. In this study, larvae of tra and bighead catfish were exposed to various hypoxic environment including atmospheric air for bighead catfish larvae. Early life stages of P. hypophthalmus from 5 to 10 days post fertilization (dpf) were identified as the developmental time points for the swimbladder and air-breathing ability. Likewise, for C. macrocephalus, 3, 5, 13, 14, 16, 17 and 26 dpf were selected as important time points for the formation of ABO. Tra catfish larvae of 11 dpf demonstrated 100 % survival at 0 mg/L of dissolved oxygen whereas bighead catfish larvae showed fully functional air-breathing at 26 dpf when exposed to the atmospheric air. The advancement of the ability to survive in hypoxia along with air-breathing function was consistent with the structural development of the ABO of the corresponding species. Previously conducted gene expression profiles of the two species were correlated with the gradual development of the ABOs during the early stages life stages to reveal the potential association of these genes with air-breathing function and formation of the ABO. Hypoxia challenge coupled with histological and anatomical observation of tra catfish revealed the critical time points for the development of air-breathing function and the swimbladder. Comparative genomic analysis between channel catfish and tra catfish along with channel catfish and bighead catfish, identified species-specific genes for tra and bighead and RNA-seq analysis based on the transition towards survival during anoxia identified the potential candidate genes for air-breathing ability in tra and bighead catfish. From this list, Grp, Cx3cl1 and Hrg were identified as the most likely contributors to the formation of swimbladder and air-breathing in tra catfish whereas in bighead catfish, Fras1 along with Mb, Ngb and Hbae were found to play key roles in air-breathing. Cx3cl1 was chosen to detect its tissue-specific expression with in situ hybridization in tra catfish larvae. The mRNA of Cx3cl1 was detected in the esophageal tissue of 1 dpf larvae and significantly increased at 2 dpf through the entire swimbladder region. During the later stages such as 5 to 6 dpf, the expression was found to decrease gradually and detected only in the mid portion and posterior end of the swimbladder. Previously conducted histological examination also detected the presence of swimbladder at 6 dpf in tra catfish larvae. These results provide further evidence that Cx3cl1 has a role in the structural development of the swimbladder and air-breathing in tra catfish. Since channel catfish (Ictalurus punctatus) breathes only with gills and cannot breathe in the air, to modify their response to hypoxia and to improve survival, Grp, Hrg and Cx3cl1 transgenes isolated from tra catfish and Fras1 transgene from bighead catfish, were knocked in into channel catfish genome through CRISPR/Cas9. The transgenes were driven by their native promoters. Different integration rates of these four transgenes were obtained at different life stages of transgenic channel catfish. However, significant differences in integration rate were observed only among the live fingerlings from the four types of transgenic groups (P < 0.05). The highest integration rate was observed in the Grp transgenic fingerlings (7.32 %). The integration rates were found to be negatively correlated with the size of the transgene constructs. Precise integration of ABO transgenes in channel catfish genome was confirmed by sequencing as well as the insertion of the complete sequence of Grp and Cx3cl1 transgene in several individuals despite the large size of the transgene. Individuals transgenic for Hrg and Cx3cl1 had significantly lower mean body weight (35.53 to 36.02 % for Hrg and 22.25 to 25.65 % for Cx3cl1) than the corresponding non-transgenics. However, Grp had no pleiotropic effect on growth. Although not statistically significant (P>0.05), Fras1 transgenics had observed body weight 22.24 to 23.92 % larger than the non-transgenics. Transgenic individuals demonstrated more vulnerability to enteric septicemia of catfish (ESC) but more resistance to Ichthyophthirius multifiliis otherwise known as ‘Ich’. Alterations in the morphology of the swimbladder of a sample of Grp transgenic individuals supports the hypothesis of the involvement of this gene in ABO development and structure. Optimization of sgRNA design to increase activity and lower off-target effects of CRISPR/Cas9 should be examined in future transgenic research. The study enhances the knowledge base concerning the adaptation of aquatic organisms to hypoxia, the formation and gradual development of air-breathing organs (ABO) as well as preliminary insights into the production of ABO transgenic channel catfish capable of surviving in low oxygen water.