Larval metamorphosis in four bivalve species in response to NMDA receptor ligands: The NMDA receptor pathway as potential regulator of bivalve transition to spat

Over the last three decades, a variety of chemical inducers or disruptors for settlement and metamorphosis have been identified for species of clams, oysters, scallops, and mussels. Identifying inducers is important in shellfish aquaculture for increasing metamorphosis rates, and hence, spat (seed) production; however, it also has wider applications, such as in the development of marine anti-fouling agents. Previously, we have suggested the potential importance of an N-Methyl-D-aspartate (NMDA) receptor pathway in regulating bivalve metamorphosis based on preliminary work in Crassostrea gigas. In this study, we further this work by assessing the ability of several known vertebrate NMDA receptor antagonists (MK-801, ifenprodil and chlorpromazine) to induce metamorphosis in a range of bivalve species. In two oyster species (Crassostrea virginica, C. gigas) and two clam species (Mercenaria mercenaria, Mya arenaria) all three NMDA receptor antagonists induced metamorphosis, but with variability in optimal concentrations and effectiveness. Specifically, both clam species were highly responsive to the NMDA receptor antagonists, with M. mercenaria displaying up to 100% induction with both ifenprodil at 10⁻⁵ M and 10⁻⁶ M and chlorpromazine at 10⁻⁷ M to 10⁻⁵ M. There is very limited prior work on successful induction using neuroactive compounds for metamorphosis in the hard-shell clam, M. mercenaria, and no prior published work for the soft-shell clam, M. arenaria. Epinephrine and levodopa (L-DOPA), two widely used and effective inducers in Crassostrea species, also induced metamorphosis in both clam species with epinephrine at 10⁻⁴ M resulting in 44% and 75% metamorphosis and L-DOPA at 10⁻⁵ M resulting in 67% and 98% for M. arenaria and M. mercenaria, respectively. Co-exposures of L-DOPA or epinephrine in conjunction with NMDA receptor antagonists were conducted to identify putative interactions between the proposed NMDA receptor pathway and the dopaminergic or adrenergic pathways by which L-DOPA and epinephrine are believed to operate. In general, co-exposures demonstrated induction of metamorphosis similar or higher than the most effective single treatments. In experiments conducted in 2018 for both oyster species, epinephrine at 10⁻⁴ M in conjunction with ifenprodil at 10⁻⁶ M, significantly inhibited metamorphosis down to 4% from 12%. The percentage of larvae that underwent metamorphosis was similarly low in response to ifenprodil alone, 2% to 18% metamorphosis, whereas epinephrine alone had a positive inducing effect, 34% to 74% metamorphosis. Results are discussed in relation to the NMDA receptor pathway that appears to be acting independently of previously believed dopaminergic or adrenergic pathways in bivalves.