Morphology of the aboral organ in Mnemiopsis leidyi

(A) Whole-body image of a 5-day-old M. leidyi cydippid larva in lateral view. The boxed region indicates the aboral organ (ao). Scale bar: 100 µm.

(B) Schematic diagram of the aboral view of a cydippid larva. The two-tone coloration represents the biradial symmetry of the body. For convenience, the body is divided into four quadrants, designated as the first (Q1) through fourth (Q4) quadrants. The two primary viewing angles are referred to as the sagittal plane (S) and the tentacular plane (T). Abbreviations: ao, aboral organ; cg, ciliated groove; cr, comb plates; tb, tentacle bulbs.

(C) Aboral views of the aboral organ highlighting its spatial organization. The left panel presents a schematic representation of the aboral organ, illustrating the four body quadrants (Q1–Q4), color-coded consistently with panel (B). The right panel shows a corresponding differential interference contrast (DIC) image from the same perspective. The region enclosed by the dotted line delineates the boundary of the aboral organ. The sagittal (S) and tentacular (T) arrows indicate the viewing directions corresponding to those planes. Scale bar: 50 µm. bal, balancer; cg, ciliated grooves; li, lithocyte.

(D) Lateral views of the aboral organ in two orthogonal planes captured using DIC microscopy. The left panel shows a lateral view in the sagittal (S) plane, while the right panel displays a view in the tentacular (T) plane. Balancer cilia (bal) and lithocytes (li) are enclosed within the dome (do). Scale bar: 50 µm. A–O indicates the aboral–oral body axis.

(E) Schematic representations of the aboral organ in sagittal and tentacular planes. The left panel shows a lateral schematic in the sagittal plane (S), and the right panel shows the same in the tentacular plane (T), corresponding to the views in panel (D). Colors match the quadrant scheme shown in panels (B) and (C).

(F) Example of cell tracing in CATMAID. The spherical objects indicate nuclear positions, while the lines represent the traced cell centers. Scale bar: 10 µm.

(G) Morphological rendering of cells composing the aboral organ displayed in aboral view (left pannel), sagittal plane view (middle panel) and tentacular plane view (right panel). Cells are color-coded according to quadrants. Lithocytes (li) are represented as three gray spheres, balancers (bal) are depicted as gray lines. Scale bar: 25 µm.

Organisation of the aboral synaptic nerve net

(A) Morphological rendering of the large aboral nerve-net neuron ANN_Q1-4 spanning all four quadrants, in aboral view (left), sagittal view (middle panel) and tentacular view (right panel). Spheres indicate the positions of nuclei.

(B) Morphological rendering of the two smaller ANN neurons ANN_Q1Q2 (pink) and ANN_Q3Q4 (orange), each spanning two quadrants, in aboral view (left), sagittal view (middle panel) and tentacular view (right panel). Spheres indicate the positions of nuclei.

(C) Number of mitochondria per cell for each cell type. The number of synapse-associated mitochondria is shown in green, the number of mitochondia outside synapses is shown in grey. N > 2 cells for each cell type except lithocytes. Abbreviations: bal, balancer; brg, bridge; lgc, large granular cell; cg, ciliated groove; dv, dense vesicle cells; imc, intra-multiciliated cells; la, lamellate bodies; li, lithocytes; pl, plumose; ef, epithelial floor cells.

(D) Position of mitochondria within ANN_Q1-4. Red indicates mitochondria associated with the presynaptic triad structures, yellow marks mitochondria containing synaptic vesicles but lacking a clearly defined presynaptic triad, blue represents mitochondria with unclear synaptic vesicles, and black denotes mitochondria where no synaptic vesicles were identified.

(E) Representative electron micrographs of presynaptic triad structures observed in the dataset and their schematic diagrams. The left diagram illustrates synaptic projections from the center ANN (ANN Q1-4) to the lateral ANN (ANN Q3Q4), while the right diagram shows synaptic projections from the lateral ANN (ANN Q1Q2) to the center ANN (ANN Q1-4). Abbreviations: mi, mitochondrion; er, endoplasmic reticulum; sv, synaptic vesicles. Scale bar: 500 nm.

(F) Position of synapses. Synapses from the center ANN to the lateral ANN are shown in magenta, while synapses from the lateral ANN to the center ANN are shown in cyan. Blue dots indicate the locations of autapses within ANN_Q1-4.

Synaptic connectivity of the balancer and and bridge cells with the ANN.

(A) Number of synaptic inputs from the ANNs (ANN_Q1-4: blue, ANN_Q1Q2: orange, ANN_Q3Q4: pink) to each cell type. Abbreviations: bal, balancer; brg, bridge; lgc, large granular cell; cg, ciliated groove; dv, dense vesicle cell; imc, intra-multiciliated cell; la, lamellate body; li, lithocyte; pl, plumose cell; ef, epithelial floor cell.

(B) Morphological rendering of balancer cells, with each cell color-coded by quadrant: Q1 (yellow), Q2 (blue), Q3 (orange), and Q4 (magenta). Each sphere represents the position of an individual nucleus. The fine projections extending from the cells represent traced trajectories that follow the cell body and continue into a single cilium from the point where it emerges. Views are shown from three orthogonal perspectives: aboral view (left), sagittal plane (middle), and tentacular plane (right). The spatial arrangement highlights the quadrant-specific organization of balancer clusters within the aboral organ. Scale bar: 25 µm.

(C) Morphological rendering of bridge cells spanning the Q1Q2 and Q3Q4 quadrants. The Q1Q2-side bridge cells (8 cells) are shown in blue, while the Q3Q4-side bridge cells (6 cells) are shown in xxx color. The morphology of individual bridge cells extending across opposite quadrant regions is depicted. The spheres represent the positions of individual nuclei. The left panel shows an aboral view of the aboral organ, the middle panel presents a sagittal plane view, and the right panel provides a tentacular plane view.

(D) Mitochondrial localization within bridge cells and associated presynaptic triad structures. Red indicates mitochondria associated with presynaptic triad structures, yellow marks mitochondria containing synaptic vesicles but lacking a clearly defined presynaptic triad, blue represents mitochondria with unclear synaptic vesicles, and black denotes mitochondria where no synaptic vesicles were identified. The left panel shows a dorsal view of the aboral organ, the middle panel presents a sagittal plane view, and the right panel provides a tentacular plane view.

(E) Synaptic connections from ANN neurons to balancer ciliated cells. The positions of synapses from ANNs to balancers (magenta) are indicated. The left panel presents an aboral view, while the right panel shows a lateral view and a tentacular plane view. Balancer ciliated cells are depicted in light gray.

(F) Synaptic connections between ANNs and bridge cells. The positions of synapses from ANNs to bridge cells (magenta) and from bridge cells to ANNs (light blue) are indicated. The left panel shows an aboral view, while the right panel presents a tentacular plane lateral view. Bridge cells are shown in light gray.

(G) Connectivity matrix of the gravity-sensing neural circuit. Columns represent presynaptic cell groups, while rows represent postsynaptic cell groups. The numbers and varying shades of blue correspond to the number of synapses.

(H) Complete connectivity map of the gravity-sensing neural circuit. Cells belonging to the same group are enclosed in hexagons, and the number of cells is added to their labels. The thickness of the arrows and the numerical values indicate the number of synapses. ANN Q1-4 are shown in blue, ANN Q1Q2 in orange, ANNs in Q3Q4 in pink, balancer cell groups in gray, and bridge cell groups in yellow.

Analysis of ciliary beating, arrests and re-beat across balancers in the four quadrants

(A) Schematic diagram of the differential interference contrast (DIC) microscopy setup used to image the movement of balancer cilia. The microscope was tilted 90 degrees so that the stage was positioned vertically. We used a 40x objective lens and a monochrome CMOS camera sensitive to near-infrared (NIR) light that was synchronized with an 850 nm strobe light source.

(B) Representative DIC images of the aboral organ viewed along the sagittal (left) and tentacular (right) planes. Arrowheads indicate the balancer cilia selected for kymograph-based analysis in each orientation. Scale bar: 25 µm.

(C) Representative kymographs showing arrest and re-beat events of balancer cilia. Left and right balancer cilia were simultaneously recorded in either the sagittal (left) or tentacular plane (right). Arrowheads mark the onset of arrest (open) and re-beat (filled); horizontal arrows represent 1-second intervals.

(D) Boxplots showing time differences in the onset of arrest (white boxes) and re-beat (grey boxes) between left and right balancer cilia. Colors of the box outlines indicate the imaging plane: sagittal (blue) or tentacular (orange). Each dot represents a single larva.

(E) Bar plots showing ciliary beat frequency (CBF) dynamics of left and right balancers (top and bottom, respectively) recorded from the sagittal (left) or the tentacular (right) planes. Positive and negative values represent opposite sides within each plane.

(F) Mean rolling Pearson correlation of left–right balancer CBF calculated with a 20-frame window. Each dot represents an individual larva. Balancers on the tentacular plane showed significantly higher correlation than those on the sagittal plane (t-test, p = 0.014).

Comparison of Neural Circuits Regulating Ciliary Movement

(A) Neurons are represented by circles, color-coded to indicate analogous functions in ciliary control. Ciliated cells are shown in gray. Synapses are indicated by arrows, with magenta representing synapses that induce ciliary arrest and blue representing synapses that induce ciliary re-beat or an increase in ciliary beating frequency. (Left) Neural circuit of the M. leidyi gravi-sensory organ. Bridge cells (yellow squares) are suggested to be electrically coupled (indicated by yellow zigzag lines), implying a potential involvement in feedback mechanisms between neurons and ciliated cells. (Right) Neural circuit regulating prototroch ciliary movement in P. dumerilii. Serotonergic neurons (Ser-h1 neurons, blue) activate ciliary movement, while a cholinergic neuron (MC neuron, magenta) induces ciliary arrest.

Cell-type composition of the ctenophore aboral organ

Morphological rendering of skeletonised cell types, (A) balancer cells (B) bridge cells, (C) large granular cells, (D) ciliated groove cells, (E) dense vesicle cells, (F) dome cells, (G) intra-multiciliated cells, (H) lamellate bodies, (I) lithocytes, (J) plumose cells, (K) aboral nerve-net neurons (L) epithelial floor cells.

3D reconstruction of all cells comprising the aboral organ

(A) Morphological rendering of skeletonised cells in the ctenophore aboral organ. Each color represents a distinct cell type. The balancer cells are shown in orange. Some cell groups lack distinguishing subcellular features required for identification and are shown in white.

Ultrastructural features and synaptic organization of the ANN, in comparison to the subepithelial nerve net (SNN)

(A) Registration and annotation of synapses in the actual cellular traces. Pink dots indicate the approximate center of each individual cell, marked within its plasma membrane boundary. Mitochondria involved in synaptic contacts are labeled with orange dots. Red arrows point from these mitochondria to the presynaptic cell body, marking presynaptic structures. Cyan arrows point from the orange-labeled mitochondria to the corresponding postsynaptic cell.

(B) Electron microscopy (EM) images showing representative presynaptic structures. The left panel shows a monadic synapse, while the right panel shows a polyadic (byadic) synapse, in which a single presynaptic site contacts two postsynaptic cells.

(C) Nuclei within two different types of ANN cells are shown in purple in a single EM section. On the left, the ANN_Q1-4 cell extends widely across the aboral organ and appears as a continuous structure in 3D, despite having nuclei located distantly from each other. In contrast, ANN_Q1Q2 (right) has two adjacent nuclei.

(D) Three-dimensional reconstruction of the subepithelial nerve net (SNN). The SNN extends broadly, enveloping the aboral organ. Some portions of the network remain as unconnected fragments due to incomplete reconstruction.

(E) Morphological comparison between the ANN in EM images. The SNN shows a “beads-on-a-string” morphology, with discrete bead-like swellings connected by faint electron-dense fibrous structures.

(F) Three-dimensional reconstruction of the SNN and ANNs.

(G) Morphological comparison between the SNN in EM images. The ANN exhibits a more diffuse distribution, often appearing to interdigitate between surrounding cells. Relatively large mitochondria are frequently observed in the ANN processes.

Full synaptic connectome of the ctenophore apical organ

Full synaptic graph showing individual cells as nodes connected by synaptic contacts. Arrows point from the presynaptic to the postsynaptic cell. Edge thickness is proportional to the number of synapses.

Ciliary beat frequency (CBF) dynamics of balancer cells in additional samples

Bar plots showing CBF dynamics of left and right balancers (top and bottom panels, respectively) recorded from the sagittal (A–C) and tentacular (D–F) planes. Positive and negative values indicate opposing sides within each imaging plane. These data correspond to additional samples not shown in the main figure and support the results presented in Figure 4E.