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Molecular characterisation of, and changes in the mRNA expression of various Aquaporin Isoforms in the gills and air sacs of the Asian stinging catfish on exposure to terrestrial or high ammonia environment
Author
Sullivan, Kasthuri Silria
Supervisor
Chew, Shit Fun
Abstract
The Indian stinging catfish, Heteropneustes fossilis, a freshwater, amphibious and facultative air-breather that possesses an air sac, can tolerate drought and high environmental ammonia. Although regarded as an ureogenic organism previously, a recent study demonstrated that H. fossilis is non-ureogenic and actively excretes ammonia with the aid of Na+/K+- ATPase. Aquaporins (AQPs/Aqps) are integral membrane proteins that serve as channels for passage of water and small molecules like glycerol, urea and ammonia. While AQP1 serves mainly for cell volume regulation, AQP3 and AQP8 are important for ammonia transport. Thus, this study aimed to verify the presence of these three Aqps in the air sacs and gills of H. fossilis and their possible roles when H. fossilis was exposed to desiccation or ammonia loading. Using molecular cloning, the complete cDNA coding sequences of aqp1, aqp3 and aqp8 were obtained. The transcript levels of these isoforms in the gills and air sacs were measured using quantitative polymerase chain reaction after the fish was exposed to terrestrial condition for 1 day (d) or 2 d or 30 mmol l-1 NH4Cl condition for 1 d, 3 d or 6 d. The complete cDNA sequence of aqp1a (771 bp, 257 amino acids, ～27.15 kDa), aqp3a1 (870 bp, 290 amino acids, ～31.51 kDa) and aqp8a (774 bp, 258 amino acids, ~27.04 kDa) were obtained from both the gills and air sacs while aqp3a2 (870 bp, 290 amino acids, ～31.3 kDa) was obtained exclusively from the air sacs. The gills and air sacs had the highest transcript levels of aqp3a (~26,000 and ～10,000 copies per ng of total RNA, respectively). The transcript levels of aqp1a in the gills increased significantly after 1 d land (～1.6-fold) and 3 d ammonia loading (～2.3-fold) conditions compared to the control conditions. The transcript levels of aqp8a increased after 1 d land (～80-fold), 2 d land (～131-fold) and 1 d ammonia loading (～253-fold) conditions compared to the control conditions. However, the transcript levels of aqp3a in the gills decreased after 2 d land (～1.7-fold) and 1 d ammonia loading (～1.5-fold) conditions compared to the control conditions. Interestingly, there were no significant changes in the transcript levels of aqp1a and aqp8a in the air sacs except for a decrease of ～1.5-fold of aqp3a after 6 d ammonia loading condition. Both Aqp1a and Aqp8a seemed to play a more important role in ammonia excretion in the gills when the fish was exposed to ammonia loading or land conditions. On the other hand, the down-regulation of Aqp3a may serve to prevent a backflux of ammonia in fish exposed to ammonia loading condition or prevent desiccation when the fish was on land. The insignificant change in the mRNA expression of aqp1a in the air sacs of fish exposed to land or ammonia loading could be due to the presence of very high transcript levels of aqp1a in the air sacs than in gills. Thus, these three Aqps play different roles in the gills and air sacs of H. fossilis exposed to terrestrial or ammonia loading conditions.
Date Issued
2021
Call Number
QL637.9.S5 Sul
Project
RI 3/15 CSF
Date Submitted
2021
Grant ID
NIE Academic Research Fund (NIE AcRF)
Funding Agency
National Institute of Education, Singapore