Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii

Abstract

Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na⁺/K⁺-ATPase (NKA), H⁺-ATPase (HAT), Na⁺/K⁺/2Cl^- cotransporter (NKCC), Na⁺/Cl^-/HCO[Formula: see text] cotransporter (NBC), Na⁺/H⁺ exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca⁺²-ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish, Cherax quadricarinatus, C. destructor and C. cainii, with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types.

Keywords: Carbonic anhydrase; Marron; Na+/K+-ATPase; Osmoregulatory genes; Redclaw; V-type H+-ATPase; Yabby.

Figure 1. Multiple alignments of amino acid sequences from three Cherax Carbonic anhydrase (CA) with other species.

Multiple alignments of amino acid sequences from three Cherax Carbonic anhydrase (CA) with other species. Colours indicate the similarity. The black-boxed indicate the predicted active-sites, Zinc-binding residues. (A) Multiple alignment of cytoplasmic CA (B) Multiple alignment of membrane associated CA, and (C) Multiple alignment of β-CA.

Figure 2. Phylogenetic relationships between the three Cherax crayfish with other organisms.

Bootstrapped Neighbour-joining tree showing the phylogenetic relationships between the three Cherax crayfish with other organisms on the basis of the amino acid sequences. Accession numbers are placed in brackets. (A) Phylogenetic tree of Carbonic Anhydrase proteins (CA) (B) Phylogenetic tree of Na⁺/K⁺-ATPase α-subunit (NKA) (C) Phylogenetic tree of V-type-H⁺-ATPase 116 kDa subunit a (HAT-A).

Figure 3. Multiple alignment of three Cherax Na⁺/K⁺-ATPase α-subunit amino acid sequences with other crustaceans.

Multiple alignment of three Cherax Na⁺/K⁺-ATPase α-subunit amino acid sequences with other crustaceans; Cherax quadricarinatus (), C. destructor (), C. cainii (), Homarus americanus (AAN17736), Callinectes sapidus (AAG47843), Paeneus monodon (ABD59803) and Portunus trituberculatus (AGF90965). Putative transmembrane domains are marked by black-boxed; likely ATP-binding site, by red-boxed; and phosphorylation site, by dashed-black boxed.

Figure 4. Multiple alignment of three Cherax V-type-H⁺-ATPase subunit-a amino acid sequences with other species.

Multiple alignment of three Cherax V-type- H⁺-ATPase 116 kDa subunit-a amino acid sequences with other closely related species. Putative transmembrane domains are marked by black-boxed.

Figure 5. Tissue-specific expression/occurrences of important genes associated with pH balance or ion-regulation in Redclaw crayfish (Cherax quadricarinatus).

(A) Presence-absence heat-map (B) Tissue-specific percentage of genes expressed (C) Maximum types of organs with % of genes expressed therein.