Larval body organization and a spatial map of external and chordotonal sense organs.

A) Ventral view of an L3 Drosophila larva. The larval body is divided into the pseudocephalon (pce), three thoracic (T1-T3), and nine abdominal (A1-A9) segments. A’) Latero-frontal view of the pce. Major sensory organs of the head are bilaterally organized and consist of several sensilla: the dorsal organ (DO) and terminal organ (TO) on the tip of the head lobes; the ventral organ (VO) located between rows of cuticle hairs; the labial organ (LO) below the mouth opening. A’’) Lateral view of the last fused abdominal segments A8 and A9, called the anal division. Sensilla in this region are organized in terminal sensory cones (t1-t7). On the ventral side, the anal plate (ap) is visible; towards the posterior end, the posterior spiracles are located. B) Map of sensilla on pseudocephalon, thoracic and abdominal segments. We categorized types of sensilla as papilla (p)-, knob (k)-and hair (h) sensilla. The major head organs, the keilin’s organ (KO), the terminal sensory cones and the spiracle sense organ (sp) are formed by several sensilla. The organization of sensilla is bilateral. Sensilla have fixed positions on hemisegments; therefore, all segments exhibit a fixed sensillar pattern. We noticed varying arrangements for T1, T2-T3, and A1-A7, with a consistent sequence of sensilla in each configuration. The pseudocephalon and the anal division display a structural organization very different from the other segments. The schematic summarizes the result of our investigation and includes results from previous studies (Dambly-Chaudière and Ghysen, 1986; Green and Hartenstein, 1997). C) Map of neurons innervating the external sense organs and the chordotonal organs. Again, the results and names from previous studies (Dambly-Chaudière and Ghysen 1986; Green and Hartenstein 1997) were used but slightly modified to create a standardized nomenclature (see discussion). Scale bars: A) 500 µm. Abbreviations: TOG – terminal organ ganglion; DOG – dorsal organ ganglion; VOG – ventral organ ganglion; LOG – labial organ ganglion. For the nomenclature of neurons in (C), see discussion.

Ultrastructure of the dorsal organ – an overview.

A) 3D reconstruction of a representative dorsal organ of an L1 larva highlighting its position at the larval body (top) and its cell type organization. Color scheme: yellow-olfactory receptor neuron (ORN); – purple – chordotonal organ (ChO); blue – DOp3/5/6 cooling cell; orange – DOp3/5/6 warming cell; turquoise – DOp1/2/4 mechanosensory cell; red – DOp2/4 unknown sensory cell. Color code in (A) applies to all micrographs in Figures 2-6 B) SEM image depicting the outer morphology of the dorsal organ in an L3 larva. The dome is covered by multiple tiny perforations (pf). Three of the seven molting pores, which are traces of ecdysis, are visible. C) Reconstruction of the peripheral and olfactory sensilla of the DO from their sensory tip to their cell body in the DOG. D) L3: Cross section of the dome with tiny perforations and molting pores. The olfactory triplets are still visible, but dendritic branching is already present at this level. E) L1: longitudinal section of the dome with tiny perforations but absent molting pores, as ecdysis has not occurred yet. The olfactory dendrites are branched and spread throughout the dome. F) L3: ganglion of the dorsal organ, showing exemplary peripheral (p) and olfactory (asterisks) cell bodies G) L1: ganglion of the dorsal organ, showing exemplary peripheral (p) and olfactory (asterisks) cell bodies. Scale bars: (B) 2 μm; (D) 5 μm; (E) 2 μm; (F) 5 μm; (G) 10 μm. Abbreviations: d -dendritic branches; mp - molting pores; p –peripheral cell bodies; pf – perforations.

Summary of morphological features of head sensory organs of third instar Drosophila larvae *

Singh & Singh (1984) report the VO to consist of five sensilla ** adopted from Rist & Thum (2017)

Summary of morphological findings of this study for sensilla of thoracic and abdominal segments of first (L1) and third (L3) instar larvae.

1Hertweck (1931); 2 Lewis et al. (1978); 3 Lohs-Shardin et al. (1979); 4 Kankel et al. (1980); 5 Singh & Singh (1984); 6 Sato et al. (1985); 7 Campos-Ortega & Hartenstain (1985); 8 Dambly-Chaudiere & Ghysen (1986); 9 Hartenstein (1988); 10 Green & Hartenstein (1997); 11 Ju rgens (1987); abbreviations: t - terminal sensory cones, tb - tubular body.

Ultrastructure of the dorsal organ – olfactory sensilla

A) 3D reconstruction of a dorsal organ of an L1 larva showing its position at the larval body (top) and one exemplary triplet of olfactory receptor neurons (ORNs) (yellow, bottom). The color code in A applies to all micrographs in this Figure. B) L1: A longitudinal section through the base of the dorsal organ showing seven olfactory triplets bathed in the outer sensillum lymph (osl). Peripheral sensilla (DOp) 1, 2 and 6 are visible C) L3: A cross section through the base of the dorsal organ showing seven olfactory triplets and all peripheral sensilla 1 to 6. D) L1: olfactory triplet at the level of the inner sensillum lymph (isl) cavity. E) L3: olfactory triplets further proximal than in C. F) L1: dendritic inner segments of the olfactory triplet bathed in the isl. The thecogen support cell is highly lamellated (lm). G) L1: longitudinal section through an olfactory sensillum from dendritic branching (b) in the dome through osl where the dendrite is enclosed by a dendritic sheath (ds) to the ciliary constriction (cc) at the transition from the outer to the inner dendritic segment inside the isl cavity. Scale bars: (B) 2 μm; (C) 5 μm; (D) 1 μm; (E) 5 μm; (F) 1 μm; (G) 5 μm. Abbreviations: DOp1-p6 – peripheral sensilla 1 to 6; b – (dendritic) branching; cc - ciliary constriction; d - dendrite; ds - dendritic sheath; inner sensillum lymph – isl; lc - lymph cavity; lm – lamellation; osl – outer sensillum lymph; ORN – olfactory receptor neuron.

Ultrastructure of the dorsal organ – peripheral sensilla 1, 2 and 4.

A) 3D reconstruction of a dorsal organ of an L1 larva showing its position at the larval body (top) and the peripheral sensilla DOp1/2/4 and their innervating neurons DOp1/2/4-A and DOp2/4-B (bottom). The color code in A applies to all micrographs in this Figure. B) DOp1 at the sensory tip with the sensory dendrite Dop1A containing a tubular body (tb). The dendrite is enclosed by a dendritic sheath (ds). C) DOp1A at the level of the ciliary constriction (cc) inside the inner sensillum lymph (isl) cavity. D) DOp2 at the sensory tip with ds, the dendrite of DOp2B does not contain a tb in contrast to DOp2A. D’) DOp2A at the sensory tip with tb. E) DOp2A and DOp2B at the level of the cc inside the isl cavity. F) DOp4 at the sensory tip with ds. DOp4A contains a tb, whereas DOp4B does not. G) DOp4A and DOp4B at the level of cc bathed in the isl. Scale bars: (B) 1 μm; (C) 1 μm (D) 1 μm; (E) 1 μm; (F) 1 μm; (G) 1 μm. Abbreviations: tb - tubular body; ds - dendritic sheath; cc - ciliary construction; isl - inner sensillum lymph.

Ultrastructure of the dorsal organ – peripheral sensilla 3, 5 and 6.

A) 3D reconstruction of a dorsal organ of an L1 larva showing its position at the larval body (top) and the peripheral sensilla DOp3/5/6, which exhibit the same morphology (bottom). DOp3/5/6 are innervated by their corresponding neurons DOp3/5/6-A and DOp3/5/6-B. The color code in A applies to all micrographs in this Figure. B) DOp3B is enclosed by a dendritic sheath (ds), which is connected to the epicuticle. C) DOp3 further proximal with DOp3A enclosed by a dendritic sheath and a thecogen cell. D-G) DOp3A enters the inner sensillum lymph (isl) cavity formed by the thecogen cell; the ds is no longer visible. The dendrite is transforming from the outer to the inner dendritic segment through a ciliary constriction (cc). The same is true for the dendrite of DOp3B, but it is not projecting through the ds towards the cuticle. Instead, it forms a lamellated bulbous structure within the lymph cavity, the dendritic bulb (db). D) longitudinal section of peripheral sensillum DOp5 at the level of the isl cavity, which is formed by thecogen cell. E) Longitudinal section of DOp3 at the level of the isl cavity. DOp3B is enters the cavity proximally, where it transforms from an enlarged inner dendritic segment (ids) to a ciliary outer dendritic segment (ods). The cilium forms a lamellated db inside the cavity. DOp3A appears rather inconspicuously but protrudes towards the cuticle in contrast to DOp3B. F) Close-up view of the isl cavity in D), the db of DOp5B is visible. DOp5A is projecting inside the ds towards the cuticle. G) L3: peripheral sensillum DOp3 at the ciliary constriction. Both ids are heavily swollen. Scale bars: (B) 0.5 μm; (C) 0.5 μm; (D) 5 μm; (E) 2 μm; (F) 1 μm; (G) 2 μm. Abbreviations: db - dendritic bulb; ds - dendritic sheath; cc - ciliary constriction; isl - inner sensillum lymph.

Ultrastructure of the dorsal organ – the chordotonal organ.

A) 3D reconstruction of a dorsal organ of an L1 larva showing the position of the chordotonal organ dCh1A at the larval body (top) and its cellular organization (bottom). The color code in A applies to all micrographs in this Figure. B) Longitudinal section of dCh1A at left DO (white box) within the whole larval volume. C) Cross section of dCh1A at right DO (white box) within the whole larval volume. D) Presentation of the white box shown in B at higher magnification. The dendrite (purple) is inserted into the scolopale made of the scolopale rods (sr). The dendritic inner segment (dis) and the cilium with a ciliary dilation (cd) are visible. The cilium is inserted into a cap at the distal end. E) Presentation of the white box shown in C at higher magnification. The sr enclose the inner sensillum lymph (isl), in which the cilium is bathed. F) Dis of dCh1A with striated ciliary rootlet (ro). G) The dCh1A is also present in L3 larvae (white arrow). Scale bars: (B) 5 μm; (C) 5 μm; (D) 2 μm; (E) 1 μm; (F) 1 μm; (G) 5 μm. Abbreviations: cd – ciliary dilation; ChO – chordotonal organ; dis – dendritic inner segment; isl - inner sensillum lymph; ro - ciliary rootlet; sr - scolopale rods.

Ultrastructure of the dorsal organ – the sensilla and associated support cells.

A) Overview of the dorsal organ proximal to the sensory dome (to the upper left corner). B) Schematic drawing of A) defining all olfactory and peripheral sensilla and their associated support cells. All sensilla possess their individual set of support cells. The thecogen cells form an inner sensillum lymph space for each individual sensillum. The trichogen and tormogen cells wrap around the thecogen cell and contribute to the sensillar integrity. In addition, the olfactory support cells build up a common outer sensillum lymph space (asterisk). Abbreviations: DOp1-p6 – dorsal organ peripheral sensilla 1 to 6; Scale bar: 2 μm.

Ultrastructure of the ventral organ.

A) 3D reconstruction of the ventral organ of an L1 larva (bottom). The outline shows the position of this organ on the larval head (top). B, D, E: (FIB)-SEM images of L3 larvae; C, F, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) Electron micrograph of the ventral organ in L3 larva consisting of four sensilla, which were named VO1-VO4. VO1 is arched outwards like a dome, whereas VO2-4 lie in small depressions. C) Cross section of the ventral organ in an L1 larva. VO1/3/4 possess a tubular body each (tb; white arrowheads). VO2 consists of a cuticle tube (ct) with a terminal pore. C’) tubular body of VO3. C’’) tubular body of VO1. D) Cross section through the base of VO4 in an L3 larva. The dendrite with tb and dendritic sheath (ds) is anchored in the cuticle by electron-dense material. A putative molting pore (mp) can be observed. E) VO2 in an L3 larva. The pore channel is streaked by fine dendritic branches (b). F) Cross section through the ventral organ further proximal than in C). G) Schematic drawing of F), showing the associated sensory and support cells. H and I) Micrograph (I) and schematic drawing (H) of the ventral organ further proximal than F) at the level of the ganglion, defining the sensory cells and the associated support cells. The two dendrites of VO2 become clearly visible when the pore enters a greater cavity formed by the thecogen cell. The dendrites then penetrate the thecogen cell, which exhibits a particular appearance like the support cells of the pit sensilla of the terminal organ (not shown). Those cells appear to be electron-lucent (asterisks), and their mitochondria exhibit a tubular (white arrowhead) instead of a cristae type (black arrowhead). Scale bars: (B) 1 μm; (C) 1 μm, inlets 0.5 μm; (D) 0.5 μm; (E) 0.5 μm; (F) 1 μm; (G) 2 μm; (H) 2 μm. Abbreviations: b – dendritic branches; ds - dendritic sheath; ct - cuticle tube; mp – molting pore; tb - tubular body.

Ultrastructure of the labial organ.

A) 3D reconstruction of the labial organ of an L1 larva (bottom). The outline shows the position of this organ on the larval head (top). B, C, D: (FIB)-SEM images of L3 larvae; E, F, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) SEM image of the lower larval head with left and right labial organ (LO). C) SEM image of the LO in an L3 larva. The LO is composed of two sensilla, here named LO1 and LO2. LO1 forms a pore in the center of a small socket on the bottom of a cylindrical cuticle depression. LO2 protrudes peg-shaped from the cuticle; its outer cuticle structure appears rough. D) Cross section through the base of LO1 and LO2. Here, putative molting pores (mp) are visible. LO2 is enclosed by a socket septum (ss). E) Cross section of the base of LO1 and LO2. LO1 contains a tubular body and is enclosed by a dendritic sheath. The knob of LO2 is mainly formed by the epicuticle and partially by the exocuticle at the base (asterisk). F) Cross section proximal of E). LO1 and LO2 are enclosed by a dendritic sheath. In LO2, the tubular body is visible. The whole knob-shaped structure is held in place by the socket septum. G) Cross section proximal of F). Dendrite of LO1 has entered the inner sensillum lymph (isl) cavity. The ciliary constriction (cc) is visible at the transition between the inner and outer segment. LO2 is still enclosed by the dendritic sheath. H) Cross section through the labial organ showing the enveloping support cells. I) Schematic drawing of (H): The dendrites are surrounded by their sensillar support cells, the thecogen (which forms the dendritic sheath), the trichogen and the tormogen cell. Scale bars: (B) 10 μm; (C) 1 μm; (D) 1 μm; (E) 1 μm; (F) 1 μm; (G) 1 μm; (H) 2 μm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; mp – molting pore; ss - socket septum; tb - tubular body.

Ultrastructure of the papilla sensillum.

A) 3D reconstruction of a papilla sensillum of an L1 larva (bottom). The outline shows the distribution of this sensillum type on the thoracic and abdominal hemisegments (top). B, D, F: (FIB)-SEM images of L3 larvae; C, E, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) Electron micrographs of a papilla sensillum in an L3 larva. The sensillum lays in a cuticular depression with a visible molting pore (mp). C) Longitudinal section through a papilla sensillum showing typical features of a mechanoreceptive sensillum. The base is formed by the dendrite, and the tubular body (tb) is enclosed by a dendritic sheath (ds), which is formed by the thecogen cell. The dendrite tip is anchored in the cuticle by the socket septum (ss). Further proximal, the dendrite enters the inner sensillum lymph (isl) cavity and transitions from the outer to the inner dendritic segment at the ciliary constriction (cc). D and E) Longitudinal section through the base of a papilla sensillum in third (D) and L1 larva (E). A molting pore is visible in (D), whereas it is missing in (E). F) Untypical papilla sensillum, with a short hair-like protuberance. G) Sensory tip of abdominal papilla sensillum p6 with two dendrites, one without a tubular body (white arrowhead). G’) Sensory tip of abdominal papilla sensillum p5, also called slit papilla sensillum. The tubular body is oval-shaped and appears more electron-lucent than canonical tubular bodies. G’’) Sensory tip of thoracic papilla sensillum px with an electron-lucent and oval-shaped tubular body. G’’’) Sensory tip of thoracic papilla sensillum py with very electron-dense tubular body. H) Electron micrograph of sensory and support cells at the level of ciliary constriction. I) Schematic drawing of (H) highlighting the sensillar support cells: the thecogen, the trichogen and the tormogen cell. Scale bars: (B) 1 µm; (C) 0.5 µm; (D) 1 µm; (E) 1 µm; (F) 0.5 µm; (G) 0.5 µm; (H) 1 µm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; mp – molting pore; ss - socket septum; tb - tubular body.

Ultrastructure of the hair sensillum.

A) 3D reconstruction of a hair sensillum of an L1 larva (bottom). The outline shows the distribution of this sensillum type on the thoracic and abdominal hemisegments (top). B, D, F: (FIB)-SEM images of L3 larvae; C, E, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B/B’) Electron micrographs of hair sensilla in L3 larvae. Hair sensilla can be branched (B’) or unbranched (B). A molting pore is visible on the hair in B. C) Longitudinal section through a hair sensillum showing typical features of a mechanoreceptive sensillum (see (E) for details). The base is formed by the dendrite (green), and the tubular body (tb) is enclosed by a dendritic sheath (ds), which is formed by the thecogen cell. The dendrite tip is anchored in the cuticle by the socket septum (ss). The hair is devoid of dendrites. D) Longitudinal section through the base of a hair with a putative molting pore (mp). E) and G) Longitudinal section through the sensory dendrite. The dendritic outer segment enters the lymph cavity and tapers off, reaching the ciliary constriction from where the inner dendritic segment begins. The dendrite is enclosed by the thecogen cell (the), trichogen cell (tri) and tormogen cell (tor). F) Mechanoreceptive region of the dendrite with a tubular body surrounded by a dendritic sheath and enclosed by a septum socket. H) Electron micrograph of sensory and support cells at the level of ciliary constriction. I) schematic drawing of (H) highlighting the sensillar support cells: the thecogen, the trichogen and the tormogen cell. Scale bars: (B) 2 and (B’) 1 µm, respectively; (C) 1 µm; (D) 1 µm; (E) 1 µm; (F) 1 µm; (G) 1 µm; (H) 1 µm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; mp – molting pore; ss - socket septum; tb - tubular body; the – thecogen cell; to – tormogen cell; tri – trichogen cell.

Ultrastructure of the double hair organ.

A) 3D reconstruction of a double hair organ of an L1 larva (bottom). The outline shows the distribution of this organ on the abdominal hemisegments (top). B: SEM image of an L3 larva; C-H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) SEM image of a double hair organ, which consists of a long hair and a short bristle. C) Longitudinal section through a double hair organ showing a dendrite at the base of the hair and a dendrite with a tubular body at the base of the bristle. D) Dendrites at the base of the hair and bristle, both enclosed by a dendritic sheath (ds), and both containing a tubular body but of different appearance. The dendrite at the hair is surrounded by a socket septum. E) ssTEM further proximal of D: the dendrite of the hair is enclosed by a dendritic sheath and support cells. The dendrites of the bristle are entering an inner sensillum lymph (isl) cavity and are in the transition from outer to inner segment with associated ciliary constrictions (cc). F) ssTEM further proximal of E): The dendrite of the hair is entering a lymph cavity and is in the transition from outer to inner segment with an associated ciliary constriction. The outer dendritic segments of the bristle-associated sensory cells are visible. G) The dendrites of a double hair organ at the level of the isl cavity: individual variations within one animal can occur; in this case, all three dendrites share the same set of support cells, forming one united sensillum. H) Section through a double hair organ at the level of isl cavity. The enveloping tormogen, trichogen and thecogen cells can be seen. I) Schematic drawing of H, defining the sensory cells and the associated tormogen, trichogen and thecogen cells. Scale bars: (B) 1 μm; (C) 1 μm; (D) 1 μm; (E) 2 μm; (F) 2 μm; (G) 0.5 μm; (H) 1 μm. Abbreviations: cc - ciliary constriction; ds – dendritic sheath; isl – inner sensillum lymph; tb – tubular body; ss - socket septum

Ultrastructure of the knob sensillum.

A) 3D reconstruction of a knob sensillum of an L1 larva (bottom). The outline shows the distribution of this sensillum type on the thoracic hemisegments (top). B, D, F: (FIB)-SEM images of L3 larvae; C, E, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) SEM image of a knob sensillum. Externally, a knob-shaped sensillum shaft is visible that is sunken into a deep and steep cavity. C) Longitudinal section through the knob sensillum. The dendrite of one sensory cell protrudes into the knob (orange) and bulges out at the end. Two other dendrites, one with (green) and one without (red) a tubular body (tb), end at the base of the shaft. D) and E) Close-up view of a cross section through all three dendrites at the base of the shaft in third (D) and L1 (E) larvae, respectively. The dendrites are enclosed by a common dendritic sheath (ds). In L3, we see a putative molting pore (mp). F) Cross section through the three dendrites at the level of the ciliary constriction (cc). The common thecogen cell forms an inner sensillum lymph (isl) cavity. G) Longitudinal section showing all three dendrites from the base of the shaft to their transition from dendritic outer to dendritic inner segment at the cc inside the isl. H) Cross section with all three dendrites bathed in the isl cavity and enclosed by the tormogen, trichogen and thecogen cell. I) Schematic drawing of (H), defining the sensory cells and the associated tormogen, trichogen and thecogen cells. Scale bars: (B) 0.5 μm; (C) 1 μm; (D) 1 μm; (E) 1 μm; (F) 1 μm; (G) 1 μm; (H) 1 μm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; mp – molting pore; tb – tubular body.

Ultrastructure of the keilin’s organ.

A) 3D reconstruction of a keilin’s organ of an L1 larva (bottom). The outline shows the distribution of this sensillum type on the thoracic hemisegments (top). B, D, F: (FIB)-SEM images of L3 larvae; C, E, G, H: ssTEM images of L1 larvae. The color code in A applies to all micrographs in this Figure. B) SEM image of a keilin’s organ consisting of three external hairs. C) Cross section of keilin’s organ with three hairs of hair-like sensilla and one dendrite of a papilla-like sensillum without a hair. D) A dendrite with a tubular body (tb) and a putative molting pore (mp) is visible at the base of the left hair. The dendrite is surrounded by a dendritic sheath and enclosed by a septum socket (ss). E) The keilin’s organ dendrites have visible tubular bodies but absent molting pores. The dendrites are surrounded by a dendritic sheath and enclosed by a septum socket. Three hair-like and two papilla-like sensilla can be observed. F) Keilin’s organ dendrites with visible tubular bodies. In L3 larvae, only one papilla-like sensillum is abundant. G) The dendrite of the keilin’s organ further proximal than E), with ciliary constrictions (cc) at the transition from dendritic outer to dendritic inner segment. Dendrites are surrounded by thecogen cells, which form an inner sensillum lymph (isl) cavity in this region. H) Cross section further proximal than G). The enveloping tormogen, trichogen and thecogen cells can be seen. I) Schematic drawing of (H), defining the sensory cells and the associated tormogen, trichogen and thecogen cells. Scale bars: (B) 1 μm; (C) 1 μm; (D) 1 μm; (E) 1 μm; (F) 1 μm; (G) 2 μm; (H) 2 μm. Abbreviations: cc - ciliary constriction; dd – dendritic degeneration; ds - dendritic sheath; isl – inner sensillum lymph; mp – molting pore; ss - socket septum; tb – tubular body.

Ultrastructure of the hair sensillum at the terminal sensory cones.

A) 3D reconstruction of a hair sensillum at the terminal segment of an L1 larva (bottom). The outline shows the distribution of this sensillum type on the last fused hemisegments (top). B, C: ssTEM images of an L1 larva. The color code in A applies to all micrographs in this Figure. B) Longitudinal section through the hair sensillum. A sensory hair is sitting on top of a cone-like structure with more (non-innervated) hairs and bristles. A dendrite with a tubular body (tb) ends at the base of the sensory hair. It is surrounded by a dendritic sheath (ds) and anchored in the cuticle by a septum socket (ss). C) Longitudinal section further proximal than B) at the level of the ciliary constriction (cc). The inner sensillum lymph (isl) cavity and the support cells can be observed. D)Schematic drawing of (C), defining the sensory cell and the associated tormogen, trichogen and thecogen cells. Scale bars: B) 2 μm; (C) 1 μm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; ss - socket septum; tb – tubular body.

Ultrastructure of the knob sensillum at the terminal sensory cones.

A) 3D reconstruction of a knob sensillum at the terminal sensory cone t3 in an L1 larva (bottom). The outline shows the distribution of knob sensilla on the last fused hemisegments (top). C-H: ssTEM images of an L1 larva. The color code in A applies to all micrographs in this Figure. B) 3D reconstruction of the outer appearance of t3. Inside the cone, a knob-shaped sensillum shaft is visible that is sunken into a deep and steep cavity. The cone is surrounded by (non-sensory) hairs and bristles. C) and D) Longitudinal section through the knob in the cone t3. The dendrite of one sensory cell protrudes into the shaft and bulges out at the end (orange). Two other dendrites end at the base of the shaft, one containing a tubular body (green), the other not (red, arrowhead). All dendrites are enclosed by a dendritic sheath (ds). Knob sensilla innervated by three sensory cells are found in t2 and t3. E) Longitudinal section through the knob sensillum of cone t5. Only two dendrites are abundant, one protruding into the sensillum shaft (orange) and the other containing a tubular body (green). Further proximal, the dendrites are bathed inside the inner sensillum lymph (isl) at the level of the ciliary constriction (cc). Knob sensilla innervated by only two sensory cells are found in cones t1, t5 and t7. F) Longitudinal section through the three dendrites of t3 at the level of the ciliary constriction. The common thecogen cell (the) forms an isl cavity. The thecogen cell is enclosed by the trichogen cell (tri) and the tormogen (tor) cell. G) Knob sensillum of the terminal organ with only one abundant dendrite (orange) that protrudes into the knob. H) Longitudinal section of t3 proximal of D) with all three dendrites sharing a common dendritic sheath, enclosed by the tormogen, trichogen and thecogen cell. I) Schematic drawing of (H), defining the sensory cells and the associated tormogen, trichogen and thecogen cells. Scale bars: (C) 2 μm; (D) 2 μm; (E) 2 μm; (F) 1 μm; (G) 1 μm; (H) 1 μm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; tb – tubular body; the – thecogen cell; to – tormogen cell; tri – trichogen cell.

Ultrastructure of the spiracle sense organ.

A) 3D reconstruction of the spiracle sense organ (sp) at the terminal segment of an L1 larva (bottom). The outline shows the position of this organ on the last fused hemisegments (top). B, C: ssTEM images of an L1 larva. The color code in A applies to all micrographs in this Figure. B) Longitudinal section through a posterior spiracle. The main tracheal tube (tr) and two spiracular glands (sg) are visible. C) Longitudinal section of the tip of the spiracle. Two sensilla of the spiracle sense organ can be observed. Furthermore, a spiracular gland and a wax pore (wp) associated with another spiracular gland are visible. The glands secrete wax around the tracheal valve to prevent moisture ingress. D) Close-up view of the mechanoreceptive region of a spiracle sensillum near the tracheal valve. The dendrite contains a tubular body (tb) at the tip and is enclosed by a typical dendritic sheath (ds). E), Longitudinal section of the tip of the spiracle further proximal than (B) showing the two sensilla inside the inner sensillum lymph (isl) with their associated support cells. The ciliary constriction (cc) is visible for one sensillum. F) schematic drawing of (E), defining the sensory cells and the associated tormogen, trichogen and thecogen cells. Furthermore, the tracheal and gland cells are shown. Scale bars: (B) 10 μm; (C) 5 μm; (D) 2 μm; (E) 5 μm. Abbreviations: cc - ciliary constriction; ds - dendritic sheath; isl – inner sensillum lymph; sg – spiracular glands; sp – spiracle; tb – tubular body; tr – trachea.

Block embedding versus minimal embedding technique.

A) In conventional block- embedding, the sample is embedded in a bulk of resin. A’) Block-embedded larval head: Frontal view of the block surface imaged with a stereomicroscope. The contours of the cuticle are complex to identify. Therefore, the block leads to extensive processing prior to FIB-SEM: excessive resin must be cut away to expose the region of interest to the ion and the electron beam. A’’) Block surface seen through the SEM. Visible are the trenches left by FIB slicing of the regions of interest. B) In contrast, minimal embedding preserves the topology of the sample because only a thin layer of resin covers the sample (B’). Samples can be glued directly to a SEM stub without prior cutting. Body parts and sensilla are visible by SEM (B’’) and can be directly targeted by the FIB. Scale bars: A’), A’’): 200 µm; B’): 100 µm B’’): 20 µm.

SEM image of the larval head seen from above.

The terminal organ (TO) and the dorsal organ (DO) are visible. Numbers define the peripheral DO sensilla which lie in a circle around the dome structure.

3D reconstruction of terminal sensory cone 1 (t1).

The reconstruction shows the outer appearance of t1 in an L1 larva. Inside the cone, we find a knob sensillum and a hair sensillum. In addition, one papilla sensillum sits at the base of the cone. The hair and the papilla sensillum are both innervated by one sensory cell containing a tubular body (tb). The knob sensillum is innervated by two sensory cells, one containing a tb that ends at the base of the knob and one without a tb that protrudes into the knob (asterisk).

Mechanotransduction in a hair sensillum.

Shown is a ssTEM image of a typical hair sensillum in an L1 larva. When force (F) is applied to either side of the hair, it is transmitted to the tubular body (tb) by the hair base and the dendritic sheath. Inside the tb body, specialized channels ensure mechanotransduction.