Characterization of two novel ammonia transporters, HIAT1α and HIAT1ß, in the American Horseshoe Crab, Limulus polyphemus.

The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In the current study, two isoforms of a novel ammonia transporter, LpHIAT1α and LpHIAT1ß, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other, and 77-86% amino acid homology to HIAT1 found in fish and crustaceans. In L. polyphemus, both isoforms were expressed in the gills, coxal glands, and brain. Slightly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and brain compared to the LpHIAT1ß isoform. A functional expression analysis of LpHIAT1α and LpHIAT1ß in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine when compared to controls, indicating an ammonia excretory function of the proteins. Exposure to elevated environmental ammonia (HEA, 1 mmol l-1 NH4Cl) caused an increase in mRNA expression of LpHIAT1ß in the ion-conductive ventral gill half. High mRNA expression of both isoforms in the brain, and the observation that LpHIAT1α and LpHIAT1ß likely mediate cellular ammonia excretion, suggests that these highly conserved ammonia transporters have an important housekeeping function in cellular ammonia elimination.

Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.