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

Abstract

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, and central mitochondria-rich (CMRA) and peripheral mitochondria-poor areas (PMPA). CMRA and ventral half-lamella exhibited characteristics important to 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 to PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whilst the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than LpRh-2 in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than 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 due to the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly-directed ammonia gradient. Novel findings suggest a role for the coxal gland in excretion and maintaining hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between 2 halves of a branchial lamella may be physiologically beneficial in reducing backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.