Localization and analysis of the cellular configuration of the larval pharyngeal sensilla.

(A) Depiction of the workflow for identification of larval pharyngeal sense organs and sensilla types. A previously generated scanning transmission EM (STEM) volume of a whole first instar Drosophila melanogaster larva (Peale et al. 2024) was used to localize all cells associated with the larval sensilla using CATMAID (Saalfeld et al. 2009). Smaller volumes were extracted from this volume to generate single organ or sensillum 3D volumes. These volumes were analyzed and manually reconstructed to describe and classify the types of pharyngeal sensilla and their corresponding sensory neurons. (B) Schematic drawing of the main external and pharyngeal organs of the larval head region. The projections via the main nerves into the subesophageal zone (SEZ) are visualized. 3D reconstructions of sensilla and sensory neurons were executed from the distal end to the ganglion. Axonal projections were not reconstructed. The numbers in the circles indicate the number of neurons in the respective ganglion. The pharynx (ocher), brain (grey), main external head organs and pharyngeal organs are highlighted. These organs are the dorsal organ (DO; purple), the terminal organ (TO; blue), the ventral organ (VO; light blue), the labial organ (LO; turquoise), the dorsal pharyngeal sensilla (DPS; yellow), the dorsal pharyngeal organ (DPO; orange); ventral pharyngeal sensilla (VPS; bright green) and posterior pharyngeal sensilla (PPS; red).

Abbreviations: D - dorsal; V - ventral; A - anterior, P - posterior, L1 – first instar, CB -central brain, SEZ – subesophageal zone, VNC – ventral nerve cord, AN – antennal nerve, MxN – maxillary nerve, LrN – labral nerve, LbN – labial nerve; CPS – cephalopharyngeal skeleton; DOG - dorsal organ ganglion; TOG - terminal organ ganglion; VOG - ventral organ ganglion; LOG - labial organ ganglion; DPSG - dorsal pharyngeal sensilla ganglion; DPOG - the dorsal pharyngeal organ ganglion; VPSG - ventral pharyngeal sensilla ganglion; PPSG - posterior pharyngeal sensilla ganglion

Structural properties of pharyngeal sensilla.

Abbreviations: VPS - ventral pharyngeal sensilla; DPS - dorsal pharyngeal sensilla; DPO - dorsal pharyngeal organ; PPS - posterior pharyngeal sensilla; P – papillum sensillum; Pmod – modified papillum sensillum; T – pit sensillum; H – hair sensillum; P/S – papilla/spot sensillum

+ = structure present; (+) = structure weakly present; - = structure not present.

Main cellular configuration of the ventral pharyngeal sensilla (VPS).

(A) 3D reconstruction of the sensory neurons innervating the VPS. The VPS is a bilaterally organized organ, but the left and right sides are fused. Only the reconstructions of the five sensilla on the left are shown. These are two papillum sensilla (P1 and P2), a modified papillum sensillum (Pmod), a hair-like sensillum (H1) and a pit sensillum (T1). Ultrastructural features of the outer and inner morphology were used to classify the different sensilla. These sensilla resemble canonical sensilla types that can also be found in the terminal organ (see Figure 1A), the main external gustatory organ. They display different features like a terminal pore (pit sensillum - T) or a sensillum shaft (modified papillum sensillum - Pmod; hair-like sensillum - H) or both (papillum sensillum - P). Mechanosensory cells are identified by their inner dendritic morphology, which contains a tubular body, a structure known to be important for mechanosensation. For a detailed description of the single VPS sensilla, see supplementary Figures 2 S1 – S5. Nomenclature for sensilla was adapted from previous work (Chu-Wang and Axtell 1972; Rist and Thum 2017). The number of neurons innervating the sensilla is indicated in brackets. (B) STEM section of the VPS showing the organs midline (dashed line). The sensilla of the right side are highlighted, and the innervating neurons are color-coded for their putative sensory function (purple - gustatory; green - mechanosensory). (C) 3D reconstruction of the outer morphology of the VPS. The position of the sensilla is indicated for the right side of the fused organ. Unlike the other pharyngeal sensilla (see Figure 3 and Figure 4), the VPS display prominent outer structures that extend into the pharyngeal cavity.

Abbreviations: d - dorsal; v - ventral; a - anterior, p - posterior, P – papillum sensillum, Pmod - papillum sensillum, H - hair sensillum, T - pit sensillum

Scale bars: (B) 1 µm

Main cellular configuration of the dorsal pharyngeal sensilla (DPS).

(A) 3D reconstruction of the sensory neurons innervating the DPS. The DPS is a bilaterally organized organ; only the reconstructions of the seven sensilla on the left are shown. These are two papilla/spot sensilla (P/S1 and P/S2) and five pit sensilla (T1 - T5). From the ganglion, the sensilla expand to an anterior and a posterior location. In the anterior group, we find three pit sensilla (T1, T2 and T3) and two papilla/spot sensilla (P/S1 and P/S2), the latter of which is located in a deep channel. The pit sensilla display an idiosyncrasy as they share their terminal pore into the pharyngeal lumen. The posterior group consists of two pit sensilla (T4 and T5), which share a terminal pore, too. Ultrastructural features of the outer and inner morphology were used to classify the sensilla. For a detailed description of the single DPS sensilla, see supplementary Figures 3 S1 – S7. The nomenclature for sensilla was adapted from previous work (Chu-Wang and Axtell 1972; Rist and Thum 2017). The number of neurons innervating the sensilla is indicated in brackets. (B and C) STEM section of the anterior group (B) and posterior group (C) of the DPS. The sensory neurons innervating the sensilla are highlighted and color-coded for their putative sensory function (purple - gustatory; green - mechanosensory).

Abbreviations: d - dorsal; v - ventral; a - anterior, p - posterior, P/S – papilla/spot sensillum, T - pit sensillum

Scale bars: (B) 1 µm; (C) 1 µm

Main cellular configuration of the dorsal pharyngeal organ (DPO) and posterior pharyngeal sensilla (PPS).

(A) 3D reconstruction of the sensory neurons innervating the DPO. The DPO is a bilaterally organized organ; only the reconstruction of the sensillum on the left is shown. This sensillum is a pit sensillum (T1) that is innervated by two sensory neurons. These are connected through a pore with the pharyngeal lumen. (B) STEM section of the DPO. The sensory neurons innervating the sensilla are highlighted and color-coded for their putative sensory function (purple - gustatory). (C) 3D reconstruction of the sensory neurons innervating the PPS. The PPS is a bilaterally organized organ; only the reconstructions of sensilla on the left are shown. These sensilla are pit sensilla (T1 and T2), innervated by three sensory neurons each. Their pores to the pharyngeal lumen are located in close proximity to each other. (D) STEM section of the PPS. The sensory neurons innervating the sensilla are highlighted and color-coded for their putative sensory function (purple - gustatory).

Ultrastructural features of the outer and inner morphology were used to classify the DPO and PPS sensilla. For a detailed description of DPO and PPS sensilla, see supplementary Figures 4 S1 – S3. The nomenclature for sensilla was adapted from previous work (Chu-Wang and Axtell 1972; Rist and Thum 2017).

Abbreviations: d - dorsal; v - ventral; a - anterior, p - posterior, T - pit sensillum

Scale bars: (B) 1 µm; (D) 1 µm

Neuronal composition and putative modality of larval head organs and their nerves.

Table updated from Python and Stocker (2002). This summary presents a synopsis of previously reported cell numbers and identities (Singh and Singh 1984; Campos-Ortega and Hartenstein 1985; Schmidt-Ott et al. 1994; Rist and Thum 2017; Richter et al. 2024), as well as the data presented in this work.

* In the VPS and DPS ganglia, there are three cell bodies of multidendritic neurons each, which in sum is in accordance with previously reported cell numbers. Multidendritic neurons (MDNs) have generally not been considered here; however, based on previously reported cell numbers in the head nerves (Miroschnikow et al., 2018), we propose that there are approximately 25 sensory MDNs (per side) innervating the head region through the MxN, AN, and PaN. Additionally, there are 36 sensory MDNs (in total) projecting through the larval vagus nerve (VN) and innervating the aorta, esophagus and gut.

Abbreviations: DO – dorsal organ; TO – terminal organ; TOdo – dorsal group of the terminal organ; TOdi – distal group of the terminal organ; VO – ventral organ; LO – labial organ; DPS – dorsal pharyngeal sensilla; DPO – dorsal pharyngeal organ; PPS – posterior pharyngeal sensilla; VPS – ventral pharyngeal sensilla; P/S – papilla/spot sensillum; ChO – chordotonal organ; OS – olfactory sensillum; TS – thermosensory sensillum; P – papillum sensillum; Pmod – modified papillum sensillum; T – pit sensillum; K – knob sensillum; DOG - dorsal organ ganglion; TOG - terminal organ ganglion; VOG - ventral organ ganglion; LOG - labial organ ganglion; DPSG – dorsal pharyngeal sensilla ganglion; DPOG – dorsal pharyngeal organ ganglion; PPSG – posterior pharyngeal sensilla ganglion; VPSG – ventral pharyngeal sensilla ganglion; CPS – cephalopharyngeal skeleton; Ph – Pharynx; Ext – external; AN – antennal nerve; MxN – maxillary nerve; LrN – labral nerve; LbN – labial nerve