The Use of MHC I Molecular Marker in The Selection of Catfish (Clarias sp.) Resistance to Aeromonas hydrophila Infection

Abstract

High demand of catfish (Clarias sp.) in the market encourages the people to continuously increase the production. One of the threats to fail the harvest is motile aeromonad septicemia (MAS) disease caused by Aeromonas hydrophila infection. MAS could cause high mortality in catfish. Several approaches can be applied to overcome MAS disease ; one is farming of catfish that genetically resists to A. hydrophila infection. Application of molecular marker assisted selection could accelerate the process to obtain disease-resistant fish broodstock. The aims of this study were to: 1) obtain a major histocompatibility complex (MHC) I marker in the marker based selection to generate A. hydrophila-resistant sangkuriang catfish; 2) evaluate the inheritance of MHC I marker in F1 generation, and 3) Determine the growth and resistance of the F1 against A. hydrophila infection. In the first experiment, a total of 200 sangkuriang catfish (average body weight: 60±5 g) were challenged by intramuscular injecting 0.1 mL/fish with A. hydrophila at a dose of 105 cfu/fish. The results showed the viability of fish was 54% (14 fish survived without injure, and 94 fish recovered from injured). PCR analysis with specific primers for MHC I was used to distinguish the survived and dead fish after challenge test. Seven set primers were designed based on database gene sequences of MHC I of Clarias gariepinus. Genomic DNA was extracted from caudal fin tissue of living and the dead fish, and then used as template in PCR amplification. The PCR results using a set of primer (ClMHAh-01) showed specific DNA bands of approximately 300, 500 and 930 bp in the survived fish. PCR product was then sequenced to confirm and analyze homology of MHC I sequence of sangkuriang catfish and the database. The results of sequence analysis using the basic local alignment search tools showed that the nucleotide sequence of those PCR products had similarity of 69-88% with MHC I of C. gariepinus. As conclusion, MHC I could be used as a molecular marker of A. hydrophila resistance catfish. In the second experiment, a total of 8 pairs of parent those carries the MHC I and one pair of control without MHC I were mated to produce F1 generation. Those crosses were female R and male R (abbreviated: RxR), LxL, RxL, and LxR, and each was replicated twice (two brood pairs per crossing). R fishes are the fish that survived without injury, while L fishes are the fish that injured and survived when they are challenged with A. hydrophila. Female and male fish without MHC I marker were mated to produce control fish (C). Results of PCR analysis showed that the percentage of F1 progenies from R x R broods carrying MHC I marker was 83.4%, while C fish was only 25%. This indicated that MHC I marker was inherited to F1 offsprings. Furthermore, results from the challenge test by intramuscular injection of 0.1 mL A. hydrophila (105 cfu/fish) showed that the survival of F1 fish from R and L broods (76.8-87.0%) was 2.21 times higher than that of C fish. Percentage of R category offspring from R and L broods was 64.0- 77.2%, and the L category was 9.2-18.8%, while R and L categories offspring from control were 4.0% and 33.2%, respectively. Blood profiles and antibody titer were in line with high resistance of F1 offspring from R and L broods against A. hydrophila infection. Thus, the resistance to A. hydrophila infection can be passed on to F1. Furthermore, rearing of fish for 95 days in aquarium showed that body weight growth was not different in all crosses. With high survival rate, catfish having MHC I marker is potentially to posses high farming productivity. As conclusion, the results of study indicated that A. hydrophila-resistance catfish can be produced by MHC I marker based selection. Further research is needed to evaluate the performance of F1 catfish farming, and the stability of marker inheritance to next generations.