Figure 1.
Anatomical overview of L. satsuma.
A: Colony of L. satsuma attached on a whale bone in an aquarium. B: A worm removed from a tube shows four body regions. C: Dorsal (upper) and ventral (lower) views. Scale bar: B: 1 mm. ob, obturaculum; op, opisthosome; ten, tentacular region; tr, trunk; vcf, ventral ciliated field; ves, vestimental region; vg, vestimental groove.
Figure 2.
Brain and ventral nerve cord of the tentacular and vestimental regions in L. satsuma.
A–C: Z-projection images of anti-SYNORF1 and anti-serotonin immunoreactivities showing Anti-SYNORF1 positive neuropils and serotonergic neurons of the brain and the ventral nerve cord. D–F: Z-projection images of anti-SYNORF1 and anti-serotonin immunoreactivities at the anterior end of the central nervous system. A pair of prominent neuropils is connected through the anterior (arrows) and posterior (arrowheads) connectives. G–I: Three-dimensional reconstructions of the central nervous system. Ventrolateral view (G) and dorsolateral view (H) views show that anti-SYNORF1 positive neuropils in the brain are thickened dorsally. Serotonergic neurons are located on the ventral and lateral sides of the anti-SYNORF1 positive neuropils. I: Dorsal view of the brain: anterior is up. The anterior (arrows) and posterior (arrowheads) anti-SYNORF1 positive signals connect a pair of neuropils. J, K: Expression patterns of elav (J) and syt (K) showing the distribution of neural cell bodies. L: Section through the posterior end of the brain neuropils. Dorsal is up. Double stain of syt (brown) and elav (yellow) shows the overlapped expression patterns of these neuron marker genes. The neuropils are encapsulated by neurons. M−O: Cross sections of brain. Positions are shown in D. The cell bodies cover the neuropils. Scale bars: 100 µm. br, brain; vcf, ventral ciliated field.
Figure 3.
Ventral and dorsal nerve cords in the vestimental region.
A–C, E: Anti-acetylated tubulin and anti-serotonin immunoreactivities. Serotonergic neural cell bodies are located on the lateral side of the giant axon (asterisks), and they project their axons medially (arrows). Anti-serotonin immunoreactivity was found in the dorsal nerve cords (arrowheads). D: Expression of syt was detected in the ventral nerve cord. F: Double-stain against anti-acetylated tubulin (green) and anti-FMRFamide (magenta) antibodies shows the presence of FMRFamidergic neurons in the ventral nerve cord. Scale bars = 100 µm. vcf, ventral ciliated field; vg, vestimental groove.
Figure 4.
Ventral nerve cord in L. satsuma.
A–C: Double stain against SYNORF1 (green) and anti-serotonin (magenta) immunoreactivities from the posterior end of the vestimental region to the anterior end of the trunk shows localizations of neuropil and serotonergic neurons of the ventral nerve cord. D–F: Double stain with anti-acetylated tubulin (green) and DAPI (blue) of the anterior part of the ventral nerve cord in the trunk shows the metameric organization of the nerve cord. The anterior is shown on the left. Yellow arrowheads indicate commissures. G: Ventral view of whole-mount image of elav expression showing the repeated pattern of neurons (arrowheads). The anterior is on the left. H–J: Sectioned images of syt (H, I) and elav (J) expressions. Neurons are located on the lateral side of the ventral nerve cord. Asterisks indicate the position of giant axon. K−N: Sections of the ventral nerve cord at positions of ganglia (K, L) and of inter ganglia (M, N) O–Q: Anti-acetylated tubulin (green) and anti-serotonin (magenta) immunoreactivities in a cross section of the ventral nerve cord show ventrally located serotonergic neurons and dorsally located axons. R–T: Double stain against SYNORF1 (green) and anti-serotonin (magenta) immunoreactivities in the opisthosome shows that the localization of serotonergic neurons in the ventral nervous system of the opisthosome. U: Cross section of the opisthosome shows syt expression in the ventral nerve cord. Scale bars: A–H, O–T = 100 µm; I−N, Q = 50 µm.
Figure 5.
Dorsal nerve cord in L. satsuma.
A, B: Immunoreactivities against anti-acetylated tubulin antibody show that the dorsal nerve cord is continuous from the vestimental region to the trunk. The cord branches in the anterior half of the vestimental region. C–H: Double stain against anti-acetylated tubulin (green) and anti-serotonin (magenta) antibodies shows that the dorsal nerve cord of the trunk is a serotonergic and non-metameric cord. C–E: Confocal laser scanning microscopy of the whole animal. F–H: Cross section of the trunk. I, J: Cross section of the vestimentum. No axon of the dorsal nerve cord was detected by HE staining. K, L: Cross section of the trunk showing the thin fiber of the dorsal nerve cord. Scale bars: A, B = 200 µm; C–H = 100 µm; I–L = 50 µm.
Figure 6.
Peripheral sensory neurons in the trunk.
A–C: Immunoreactivity against anti-acetylated tubulin antibody (green) and DAPI staining (blue) of the posterior part of the trunk shows the immunoreactive cell masses on the lateral side of the trunk. D: SEM images of the lateral side of the trunk showing the number of pyriform glands in the trunk (arrowheads). E, F: High-magnification images of a pyriform gland show a pore of the gland (arrowhead) and two cilia near the pore (arrows). G: A HE-stained section of a pyriform gland. Arrowhead indicates a pore of the gland. H–J: Anti-acetylated tubulin immunoreactivity for a cross-section of the pyriform gland showing two immunoreactive cells (arrows) near the pore of the gland (arrowhead). Scale bars: A–D = 200 µm; E = 10 µm; F = 1 µm; G = 50 µm; H–J = 20 µm. vnc, ventral nerve cord.