Identification and characterisation of functional aquaporin water channel (Anomala cuprea DRIP) in a coleopteran insect

Abstract

Summary Water transport across the plasma membrane depends on the presence of the water channel aquaporin (AQP), which mediates the bulk movement of water through osmotic and pressure gradients. In terrestrial insects, which are solid/plant feeders, the entrance and exit of water is primarily executed along the alimentary tract, where the hindgut, particularly the rectum, is the major site of water conservation. A cDNA encoding the homologue of the water-specific Drosophila AQP (Drosophila integral protein: DRIP) was identified through the RT-PCR of RNA isolated from the rectum of the cupreous chafer larvae, Anomala cuprea, a humus and plant root feeder. This gene (Anocu AQP1) has a predicted molecular mass of 26.471 kDa similar to the DRIP clade of insect AQPs characterised from caterpillars, flies and several liquid-feeding insects. When expressed in Xenopus laevis oocytes, Anocu AQP1 showed the hallmarks of aquaporin-mediated water transport but no glycerol nor urea permeability, and the reversible inhibition of elevated water transport through 1 mM HgCl2. This is the first experimental demonstration of the presence of a water-specific AQP, namely DRIP, in the Coleoptera. The genome of the model beetle, Tribolium castaneum, contains six putative AQP sequences, one of which (Trica-1a, XP_972862) showed the highest similarity to Anocu AQP1 (~60% amino acid identity). Anocu AQP1 is predominantly expressed in the rectum. Using a specific antibody raised against DRIP in the silkworm, Bombyx mori (AQP-Bom1), Anocu AQP1 was localised to the apical plasma membrane of rectal epithelial cells, and lacking in the midgut and gastric caecal epithelia. Based on the BeetleBase prediction, there are three putative AQPs (Trica-3a, 3b, 3c: XP_970728, 970912, 970791) that are homologous to B. mori aquaglyceroporin (AQP-Bom2 [GLP]). The immunocytochemical studies using the specific anti-peptide antibody against AQP-Bom2 revealed the presence of the GLP homologue at the apical plasma membrane of enterocytes in the midgut and gastric caeca. Thus, DRIP (Anocu AQP1) and the putative GLP share epithelial fluid-transporting roles along the alimentary tract in cupreous chafer larvae.