Fig. 3

Serotonin immunoreactivity (5-HT-ir) in the trochophore and veliger larvae of Crassostrea gigas. Green—5-HT-ir; magenta—cilia, acetylated tubulin immunoreactivity. The apical pole is always upward; the ventral side is on the right. a The early trochophore at 20 hpf. The first two flask-shaped neurons (arrows, an) are located in the anterior extreme of the larval body. Each cell has a ciliated short dendrite (arrowheads). b D-hinge veliger at 36 hpf. 5-HT-ir cells comprise a compact apical organ (AO) and comprise flask-shaped and round cells each with thin neurites (arrowheads, n1) running to the velum. b1 two flask-shaped cells with thin neurites and neuropil (np). b2 single round cells (black asterisk). c The veliger stage at 52 hpf. Cells of the AO extend three long neurites to the velum (arrowheads, n1), anterior−dorsal (arrowheads, n2), and posterior−ventral (arrowheads, n3). Acetylated tubulin-ir reveals the digestive system and green autofluorescent particles are visible in the stomach (st) in b, c, d, e. insets: High magnification of the AO cell composition. c1 two flask-shaped and one round cells (black asterisk) (Z5), c2 third flask-shaped cells and second round cell (black asterisk), and neuropil (arrows, np) c3 merge picture. There are five cells: three flask-shaped cells and two round cells (black asterisk); d The middle veliger stage at 96 hpf. Two posterior−ventral neurites from cells of the AO run in parallel along the ventral part of the larval body organizing the ventral nerve cords (vnc). e The veliger at 9 dpf. Cerebral ganglia (CG) are located at the top, and pedal ganglia (PG) are detected in the middle of the ventral nerve cord. Small immunoreactive posterior neurons (pn) are located near the caudal end of each ventral cord. Neurites extending from the CG have multiple branches in the velum region. Note the solitary branch extending from the apical part of the ganglion (arrowhead) towards the dorsal edge of the velum. f The veliger at 15 dpf. Note the paired ventral nerve cords with two commissures (short arrows). Cerebral ganglia located on top of the ventral cords. The thickening of the upper portion of each ventral cord represents the anlagen of the pleural ganglia (PLG) and together with the CG they form a fused CG/PLG complex indicated as CPG. Right and left PG are located along the respective ventral cords at the middle of the foot. Posterior neurons are located caudally, and each sends an axon to the ipsilateral ventral cord. The velum is richly innervated by fibers extending from the CPG. Thin neurites extend to the foot, and digestive organs (mark as peripheral nerve system, PNS) extend from the PG region (arrowheads). insets: f1 High magnification of the CG/PLG region; f1 focus on CG and PLG parts of CPG, PG, ventral cord, and peripheral innervation. g Summary diagram of the ontogeny of the 5-HT-ir-containing structures in C. gigas. Only one lateral side is shown. Additional abbreviations: f – foot, m – mouth, pmo – presumptive mouth opening, pt. – prototroch, st – stomach. Scale bars =20 μm