Fig. 5

Double staining of 5-HT/FMRFa-ir, VAChT/FMRFa-ir, and VAChT/5-HT-ir nervous elements combined with tubulin immunoreactivity of ciliary structures in Crassostrea gigas larvae. Neurotransmitter co-localization appears white in all pictures. a and b Side and front views, respectively, of the alternative expression of FMRFa-ir and 5-HT-ir within early neurons at the 28-hpf trochophore stage. In the apical organ (AO), 5-HT-ir neurons are surrounded by FMRFa-ir neurons. Arrows point to the growth cone of the right dorsal cell (gc). c and d Alternative expression of FMRFa-ir and 5-HT-ir in cells of the AO/cerebral ganglia complex (AO/CG) at the 96-hpf stage. Only FMRFa-ir cells are present in the pedal ganglia (PG) anlagen. Arrows point to the peripheral process from the PG to the velum, and an arrowhead marks the process (n) from the AO/CG to the velum. e Alternative expression of VAChT-ir and FMRFa-ir within the early neurons (dn, vn, pn, and AO) in the 44-hpf veliger. e1 VAChT-ir cells (asterisks) are surrounded by FMRFa-ir cells in the AO. e2 A posterior VAChT-ir cell (pn) sends a process along the FMRFa-ir fiber; an asterisk marks the posterior cell nucleus. f and g Side and front views in the 7-dpf veliger stage demonstrate the presence of both VAChT-ir and FMRFa-ir elements in the AO/CG complex, PG, and posterior neurons (pn). Partial co-localization occurs in the AO/CG neuropil and in the ventral nerve cords. f1 and f2 Micrograph demonstrating alternative VAChT-ir and FMRFa-ir expression in PG and AO/CG neurons, their partial co-localization within the AO/CG and the PG neuropil, and the processes of the ventral nerve cords (vnc). f3 A VAChT-ir process runs from the AO/CG to the velum (arrowhead) and from the PG to the foot anlagen (arrow). h Alternative expression of VAChT-ir and 5-HT in the 44-hpf veliger stage. The AO contains both VAChT-ir and 5-HT-ir neurons, while a posterior neuron (pn) exhibits VAChT-ir only. i In the 92-hpf veliger stage, VAChT and 5-HT are alternatively seen within the cell bodies of the AO/CG and are partly co-localized in the neuropil. i1 Magnification of 5-HT-ir and VAChT-ir neurons in PG. A 5-HT-ir process runs along the VAChT-ir fiber; asterisks mark the cell bodies. j In the 7-dpf veliger stage, both VAChT-ir and 5-HT-ir fibers emanating from the AO/CG run to the velum (arrowheads). inset: Alternative expression of VAChT-ir and 5-HT-ir within the AO/CG cell bodies and their partial co-localization within the neuropil. j1 5-HT-ir within the PG appears to be adjacent to a VAChT-ir nerve bundle in the ventral nerve cord. An asterisk marks the body of the posterior VAChT-ir cell (pn). k Summary diagram of the ontogeny of the VAChT-ir, 5-HT-ir, and FMRFamide-ir-containing structures in C. gigas. Only one lateral side is shown. Additional abbreviations: a – anus, m – mouth, pt. – prototroph. Scale bars =20 μm