Molecular characterization of branchial aquaporin 1aa and effects of seawater acclimation, emersion or ammonia exposure on its mRNA expression in the gills, gut, kidney and skin of the freshwater climbing perch, Anabas testudineus

Abstract

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.

Figure 1. Molecular characterization of aquaporin 1aa (Aqp1aa) from the gills of Anabas testudineus.

Multiple amino acid alignment of Aqp1aa from the gills of A. testudineus, with five other known Aqp1/Aqp1a from Sparus aurata (seabream Aqp1a; ABM26907.1), Takifugu obscurus (pufferfish Aqp1; ADG86337.1), Protopterus annectens (lungfish Aqp1; BAI48049.1), Xenopus laevis (frog AQP1; NP_001085391.1), and Homo sapiens (human AQP1; CAQ51480.2). Identical amino acids are indicated by shaded residues. Substrate discrimination sites at the aromatic/arginine (ar/R) constriction are indicated with arrows. Central pore-lining residues are indicated with open triangles. The binding site for AQP1-inhibitor HgCl₂ is indicated by an asterisk. The Asn-Pro-Ala (NPA) motifs are underlined. P denotes phosphorylation sites and N denotes N-glycosylation sites. The predicted transmembrane domains (TM) are underlined. The transmembrane domains of Aqp1 of A. testudineus were predicted using MEMSATS & MEMSAT-SVA provided by PSIPRED protein structure prediction server.

Figure 2. Phylogenetic analysis of aquaporin 1aa (Aqp1aa) of Anabas testudineus.

The phylogenetic tree illustrates the relationship between Aqp1aa of A. testudineus and AQP1/Aqp1 of selected vertebrates.

Figure 3. Tissue expression of aquaporin 1aa (aqp1aa) of Anabas testudineus in freshwater.

Tissue expression of aqp1aa was examined in gills, accessory breathing organs (ABO), brain, liver, kidney, anterior gut (AG), posterior gut (PG) and skin of A. testudineus kept in freshwater.

Figure 4. Aquaporin 1aa (aqp1aa) mRNA expression in Anabas testudineus kept in freshwater or seawater.

Absolute quantification (×10² copies of transcript per ng cDNA; N = 5) of aqp1aa mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of A. testudineus kept in freshwater (FW) or exposed to seawater (SW; salinity 30) for 1 or 6 days after a progressive increase in salinity. Results represent means ± S.E.M. Means not sharing the same letter are significantly different (P<0.05).

Figure 5. Aquaporin 1aa (aqp1aa) mRNA expression in Anabas testudineus kept in freshwater or exposed to terrestrial conditions.

Absolute quantification (×10² copies of transcript per ng cDNA; N = 5) of aqp1aa mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of A. testudineus kept in freshwater (FW) or exposed to terrestrial conditions for 1 day. Results represent means ± S.E.M. ^*Significantly different from the FW control (P<0.05).

Figure 6. Aquaporin 1aa (aqp1aa) mRNA expression in Anabas testudineus kept in freshwater or exposed to ammonia.

Absolute quantification (×10² copies of transcript per ng cDNA; N = 5) of aqp1aa mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of A. testudineus kept in freshwater (FW) or exposed to 100 mmol l⁻¹ NH₄Cl for 1 or 6 days. Results represent means ± S.E.M. Means not sharing the same letter are significantly different (P<0.05).