Abstract
Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.
Highlights
Metamorphosis in bivalve molluscs includes the loss of larval organs such as the velum, the development of adult gills, the production of adult shell and in species such as the Pacific oyster Crassostrea gigas, resorption of the larval foot (Kennedy et al, 1996)
Given that NMDA receptor pathways have been largely overlooked in invertebrate developmental biology research, these findings provide fundamental information about the role of previously unexplored NMDA receptors and its potential link with the catecholamine pathways during Pacific oyster metamorphosis
The NR1 subunit homolog CgNR1 coding sequence is 2883 bp long and comparison to the genome sequences showed that the CDS comprises of 17 exons encoding for a 960 aa long protein
Summary
Metamorphosis in bivalve molluscs includes the loss of larval organs such as the velum, the development of adult gills, the production of adult shell and in species such as the Pacific oyster Crassostrea gigas, resorption of the larval foot (Kennedy et al, 1996). We recently successfully induced metamorphosis in two oyster and two clam species by exposing competent larvae to two selective vertebrate N-Methyl-D-aspartate (NMDA) receptor antagonists, the channel blocker MK-801 and a non-competitive negative allosteric NMDA receptor modulator ifenprodil, which was the first evidence that an NMDA receptor pathway is involved in regulating bivalve metamorphosis (Vogeler et al, 2018, 2019). Ligand-gated ion channel NMDA receptors are members of the glutamate receptor family, allowing the flow of positively charged ions such as Ca2þ, Naþ, Kþ through synaptic cell membranes (for review see (Traynelis et al, 2010)). Unique to NMDA receptors is a voltage dependent block inside the channel pore by extracellular Mg2þ ions, which for successful ion gating requires dislodgment, usually caused by depolarisation of the cell membrane through rapid activation of nearby
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