RESEARCH ARTICLE Ammonia excretion and acid–base regulation in the American horseshoe crab, Limulus polyphemus Stephanie Hans 1 , Alex R. Quijada-Rodriguez 1 , Garett J. P. Allen 1 , Horst Onken 2 , Jason R. Treberg 1,3 and Dirk Weihrauch 1, * 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, 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. KEY WORDS: Ussing chamber, Gills, Rh-proteins, Carbonic anhydrase INTRODUCTION Amino acid-catabolizing organisms produce toxic nitrogenous waste products that must be eliminated via excretion strategies such as ammonotelism, where ammonia is the dominant excretory product (Wright, 1995). Compared with other nitrogenous waste products, ammonia is energetically beneficial as it can be released as is and does not require additional energy for conversion into its less toxic counterparts, such as urea or uric acid. Aquatic animals (excluding mammals and elasmobranchs) commonly exhibit ammonotelism because of abundant water availability for continuous excretion, preventing toxic build-ups (Larsen et al., 2014). Ammonia exists in both gaseous (NH 3 ) and ionic (NH 4 + ; ammonium) forms, and the relationship between the two is depicted in Eqn 1: NH 3 þ H 2 O $ NH 4 þ þ OH  : ð1Þ Aquatic animals may experience excess extracellular ammonia (in this study, ammonia refers to the sum of NH 3 and NH 4 + ) whilst burying or upon emersion, when excretion is impaired (Weihrauch et al., 1999). Increased concentration of circulating ammonia can cause numerous deleterious effects, such as acid–base imbalance (Goldsmith and Hilton, 1992; Wilson and Taylor, 1992), ionoregulatory disruption (Young-Lai et al., 1991) and neurotoxicity (Butterworth, 2002; Marcaida et al., 1992). Several key proteins have been suggested to influence ammonia excretion of invertebrate species, including Na + /K + -ATPase (NKA), V-type H + - ATPase (HAT) and glycosylated Rhesus proteins (Rh-proteins) (Chasiotis et al., 2016; Larsen et al., 2014; Masui et al., 2002; Pitts et al., 2014; Quijada-Rodriguez et al., 2015; Weihrauch et al., 1998, 2012). Mounting evidence suggests that ammonia excretion and acid– base regulation of several invertebrate species are intricately linked, possibly because of the acidic and basic forms of ammonia (NH 4 + and NH 3 , respectively) and the sharing of key transporters such as NKA, HAT and Rh-proteins (Fehsenfeld and Weihrauch, 2016a). This notion has been encouraged by investigations concluding that anterior and posterior gills of Carcinus maenas, the green shore crab, have similar capacities for ammonia and H + -equivalent excretion (e.g. Fehsenfeld and Weihrauch, 2013). Although extensive studies have focused on ammonia excretory mechanisms of crustaceans and teleost fishes, there has yet to be an equivalent study on chelicerates. This is likely because the vast majority of this subphylum are terrestrial arachnids, which excrete guanine as their dominant nitrogenous waste product because of environmental water constraints (Larsen et al., 2014). Xiphosura, which includes the American horseshoe crab, Limulus polyphemus (Linnaeus 1758), is an exception to the terrestrial chelicerates in terms of lifestyle. Limulus polyphemus has remained morphologically unchanged for over 200 million years (Avise et al., 1994) and is currently widespread along the east coast of the USA, and Mexico (Shuster, Received 19 October 2016; Accepted 10 January 2018 1 Department of Biological Sciences, University of Manitoba, Winnipeg, MB, R3T2N2, Canada. 2 Department of Biological Sciences, Wagner College, Staten Island, NY 10309, USA. 3 Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, R3T2N2, Canada. *Author for correspondence (Dirk.Weihrauch@umanitoba.ca) D.W., 0000-0002-3218-9093 1 © 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb151894. doi:10.1242/jeb.151894 Journal of Experimental Biology