From Embryo to Adult: Anatomy and of a Leg Sensory Organ in...

THE JOURNAL OF COMPARATIVE NEUROLOGY 308~188-199 (1991)

From Embryo to Adult: Anatomy and Development of a Leg Sensory Organ in

Phormia regina Meigen (Insecta: Diptera). I. Anatomy and Physiology of a Larval

"Leg" Sensory Organ

R. LAKES-HARLAN, G.S. POLLACK, AND DJ. MERRITT Department of Biology, McGill University, Montreal, Quebec, H3A 1B1, Canada (RL-H., G.S.P.); Neurobiology Research Center, SUNY Albany, Albany, New York 12222 (D.J.M.)

ABSTRACT Neurons within the precursor of the adult leg, the imaginal disc, innervate a larval sense

organ, Keilin's organ. Electron microscopical investigations of first instar larvae show that five dendrites end at the organ: three insert at the bases of the three hairs of the organ and two end against the cuticle, without any apparent cuticular specialization. In third instar larvae, the imaginal leg discs invaginate into the body cavity, and only four of the dendrites (the outer segments of which become greatly elongated) remain in contact with Keilin's organ. The axons of the neurons that supply Keilin's organ project into a ventral neuropile region of the central nervous system, with a pattern that resembles the projections of other larval sensilla. Electrical activity can be recorded from neurons of the imaginal disc in response to mechanical stimulation.

Key words: ultrastructure, mechanoreceptor, imaginal disc, metamorphosis, blowfly

Flies undergo a complete metamorphosis. The larva that hatches from the egg differs markedly, both morphologi- cally and behaviorally, from the adult that emerges from the puparium. The different structures and lifestyles of these two developmental stages are served by at least partially different sets of peripheral sensory organs and central neurons and neural circuits. In flies and other holometabolous insects, conversion from larval to adult central nervous system involves death of some larval neurons, retention and remodeling of others, and birth of new adult neurons (Casaday and Camhi, '76; Levine and Truman, '82; Levine, '86; Breidbach, '87a,b; Truman and Bate, '88; Tix et al., '89a,b). The peripheral nervous system of the larva of Drosophila is established during embryogenesis (Campos-Ortega and Hartenstein, '85; Dambly- Chaudidre and Ghysen, '86; Ghysen et al., '86) and is nearly entirely discarded during the pupal period. During the pupal stage, most sensory structures of the adult are formed de novo in anlagen of adult structures, the imaginal discs, that are set aside during embryogenesis (Jan et al., '85; Lakes and Pollack, '90). The peripheral nervous system of the larva and adult are not, however, entirely indepen- dent of one another. Genetic studies have shown that the development of larval and adult sensory structures are a t least partly regulated by the same genes (Dambly-Chaudiere

et al., '88; Cohen et al., '891, and anatomical studies have shown that newly formed neurons of adult sensory structures often use preexisting larval nerves as conduits from the periphery into the central nervous system (Lakes et al., '89; Tix et al., '89a,b). In this paper and the following one (Lakes-Harlan et al., '911, we demonstrate a more direct link between the sensory structures of larval and adult flies; we show that some larval sensory neurons, which arise during embryogenesis, persist to the adult.

The neurons in question are a subset of those that are found in the imaginal leg discs. Unlike other imaginal discs, those that give rise to the legs contain neurons throughout larval life (Poodry and Schneiderman, '70; van Ruiten and Sprey, '74; Jan et al., '85; Lakes and Pollack, '90). Some of these are associated with a larval sensory organ found on

Accepted February 4,1991. Address reprint requests to Dr. Gerald Pollack, Department of Biology,

McGill University, 1205 Ave. Dr. Penfield, Montreal, Quebec, Canada H3A 1B1.

R. Lakes-Harlan is now at the I. Zoologisches Institut, Universitat Gottingen, Berliner Str. 28,3400 Gottingen, FRG.

D.J. Merritt is now at the Neuroscience and Behavior Program, Morrill Science Center, University of Massachusetts, Amherst, MA 01003.

Abbreviations used: anti-HRP, anti-horseradish peroxidase; rlc, receptor lymph cavity.

O 1991 WILEY-LISS, INC.

ANATOMY AND PHYSIOLOGY OF KEILIN'S ORGAN 189

discs of third instar larvae, together with the attached central nervous system, were dissected and transferred to a drop of saline (Normann and Duve, '69). The hypodermal stalk, through which the dendrites extend from the disc to Keilin's organ, was placed in a pipette filled with 10% NiC1,. After 4 hours at 4"C, the dye was precipitated with rubeanic acid (5 ml of a saturated solution in 100% ethanol diluted in 100 ml saline) and fixed with Carnoy's I1 fixative. The preparations were silver-intensified (Bacon and Altman, '77) and mounted as wholemounts. Selected preparations were later embedded in soft Spurr's resin and sectioned (6 pm). Sections were counterstained with methylene blue and embedded in Permount.

each of the thoracic hemisegments, Keilin's organ (Keilin, '15). Keilin's organ has been used as a morphological marker in developmental and genetic studies to determine the effect of mutations on peripheral sensory neurons (Hartenstein and Campos-Ortega, '86) and on the central nervous system (Ghysen et al., '85). However, little is known about its anatomy (Peters, '61) and function. In the present paper, we show that each Keilin's organ is inner- vated by sensory neurons with cell bodies in the leg imaginal disc of the corresponding hemisegment. We present anatomical and physiological evidence that the Keilin's orgadeg disc complex serves sensory functions throughout larval life. In the accompanying paper (Lakes-Harlan et al., '91), we follow the neurons associated with Keilin's organ through development and show that some of them persist from the embryo to the adult.

MATERIALS AND METHODS Animals

The blowfly Phormia regina was reared at 25"C, with a 16 hour: 8 hour light: dark cycle, and freshly laid eggs were collected and placed on artificial medium (modified after Hill et al., '47). The first instar larvae hatch ca. 22 hours after egg-laying. The three larval instars take approximately 213 hours until pupariation, and the imagos emerge after about 129 hours of pupal development.

Scanning electron microscopy (SEMI Third instar larvae were sonicated in 0.3% Triton X,

blotted dry, and fixed by immersion in acidified dimethoxy- propane (Bjerke et al., "79). Adult flies were fixed by injection with alcoholic Bouin's fixative. Following fixation, both larvae and adults were sonicated for several seconds in amyl acetate, dehydrated in ethanol, air-dried, sputter-coated, and examined with an IS1 scanning electron microscope.

Transmission electron microscopy (TEM) Eight Keilin's organs were analyzed: three from two first

instar larvae (15 hours after hatching) and the remainder from three third instar larvae (168 hours after hatching). One of the organs from a first instar, and two from third instars, were reconstructed from serial sections. The larvae were fixed by injection of ice-cold 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The ventral cuticle was dissected in fresh fixative and transferred to a fresh solution of fixative for 2-3 hours. Specimens were postfixed with 1.5% OsO, in the same buffer for 1 hour on ice, dehydrated, and embedded in Spurr's resin (Spun, '69). Thin sections were placed on 0.5-0.75% Formvar support film, stained with 2% aqueous uranyl acetate (60°C, 1 hour) and Reynolds lead citrate (Reynolds, '63), and viewed with a Philips EM 410 transmission electron microscope.

Immunocytochemistry The sensory neurons of imaginal leg discs were visualized

with an anti-horseradish peroxidase antibody (anti-HRP), which binds to the surface of insect neurons (Jan and Jan, '82). The antibody was visualized by the PAPDAB proce- dure (for details of the protocol, see Lakes and Pollack, '90).

Central projections For analysis of the central projections of the sensory

neurons that innervate Keilin's organ, the imaginal leg

Physiology The ventral cuticle of the pro- and mesothorax was

dissected from third instar larvae, together with the attached imaginal discs, and was pinned out, exterior upper- most, in a drop of saline. The imaginal discs were arranged so that their proximal ends protruded from under the piece of cuticle. The stump of the severed nerve that connected one of the discs to the central nervous system was drawn into a suction electrode. Potentials were amplified (Grass P15, 1 , 0 0 0 ~ ) and stored on tape (Vetter Model D) for later analysis. Mechanical stimuli were applied by touching or stroking the cuticle in the region of Keilin's organ with a needle or by tapping the stage on which the preparation was mounted.

RESULTS The anatomy of Keilin's organ

The following description of Keilin's organ is derived from examination of the first and third larval instars. Some aspects of the fine structure and innervation of the organ change during larval development. Where appropriate, we indicate stage-specific findings below.

Cuticular structures. A pair of Keilin's organs is found near the ventral midline in each of the thoracic segments (Fig. 1). Each organ consists of a triplet of short (ca. 5 pm) hairs (Fig. lb). The hairs are situated in a depression at the top of a circular mound that has a diameter of ca. 4 pm. Two small depressions occur, one near the medial edge of the mound and the other 4-5 pm posterior to the mound (Fig. lb). Each Keilin's organ is flanked by two campaniform sensilla and a sensillum basiconicum (not shown).

The hairs of first instar larvae have an aporous cuticular layer and their interiors are filled with electron-lucent material (Fig. 2a). In third instar larvae, the hairs are similarly without apparent pores but their interiors are composed of cuticle (Fig. 2b). The hairs do not have membranous sockets; their cuticle layer is continuous with that of the surface of the dome in which they reside. Fibrous structures extend from the interiors of the hairs into the outer receptor lymph cavity (rlc; Fig. 2d).

In third instar larvae, a structure similar to a rlc is visible posterior-medial to Keilin's organ (Figs. 2c, 5a). It contains what appears to be the same electron-lucent material as does the rlc associated with hairs, as well as cuticular fibers and other particles, but no dendrites can be seen. Some of the particles resemble material of the sheath that sur- rounds each of the dendrites of Keilin's organ (see below). The rlc constricts approximately 7 pm proximal of Keilin's organ and proceeds into the hypodermal stalk.

190 R. LAKES-HARLAN ET AL.

Fig. 1. Scanning electron micrographs of the third instar larva of Phormia regina. a: Ventral view of the prothoracic segment (Tl) and the head. The square indicates the position of Keilin’s organ. b: Keilin’s organ of the prothoracic segment. Two small depressions (arrows) are visible. T2: mesothoracic segment. Scales: a, 20 pm; b, 3 pm

Sensory neurons. General pattern of innervation. There are five sensory

neurons associated with Keilin’s organ in first instar larvae. In third instars, only four dendritic profiles can be found near the organ. In embryos and in young larvae, the somata of the sensory neurons lie near the cuticle at the base of the organ. Early in the third larval instar, the leg imaginal discs invaginate into the body cavity, and these neurons, as well as a few others, become incorporated into the disc. The disc remains connected to Keilin’s organ by a hypodermal stalk, which contains the now elongated dendrites of the sensory cells. As the stalk increases in length throughout larval development, so do the dendrites; by the late third instar, they can exceed 1 mm in length. A more detailed description of the incorporation of these neurons into the invaginating disc is given in the following paper (Lakes-Harlan et al., ’91).

Fine structure -First instar larva. Three dendrites insert eccentrically at the bases of the three hairs (Figs. 3, 6). Their outer dendritic segments extend within the rlc, which is bordered by cuticle and, more proximally, by the auxiliary cells. Each dendrite is surrounded by a thick electron-dense sheath (Fig. 3c). At the tip of each dendrite, a tubular body is formed from microtubules (Fig. 3c). The dendrites extend basally (8 pm) until they leave the rlc and become enveloped by auxiliary cells (Fig. 4c-e). As the dendrites travel through the rlc, they vary greatly in shape and diameter. Many infoldings and short branches occur (Figs. 3, 4b). Ciliary structures are found in the dendrites after they have entered the epidermal cell layer (Fig. 4fJ.

One of the remaining two dendrites (d4) has a structure generally similar to that described above. However, this fourth dendrite ends not at the base of a hair, but instead terminates medially at the wall of the depression containing the three hairs (Fig. 3a,d). The tubular body of d4 is less well developed than are those of the three hair-associated dendrites .

The fifth dendrite (d5) terminates posterior of the depression in which the hairs reside. It is smaller in diameter than

the others, is highly infolded, and has no detectable tubular body (Figs. 3b, 4a, 6).

The auxiliary cells surround the inner segments of the dendrites and do not extend to the hairs. Each dendrite is enveloped separately (Fig. 4c-e). The innermost auxiliary cell, the thecogen cell, bears microvilli facing the rlc.

Fine structure -Third instar larva. In contrast to the first instar larva, only four dendrites terminate at Keilin’s organ in the third instar larva. The “missing” dendrite is the one that, in the first instar, terminates posterior of the depression that houses the hairs (Fig. 5a, arrow). The remaining four dendrites can be found at the same positions that they occupy in the first instar. The three dendrites associated with the hairs terminate at channels or pores at the bases of each of the hairs (Fig. 54 ; these are probably “moulting channels,” which have been described for other insect sensilla (Schmidt and Gnatzy, ’71). Close to the termination site, an eccentric tubular body (approximately 1 pm long) can be found. The tip of the dendrite dilates to a diameter of about 2 Fm and contains membranous vesicles 50-130 nm in diameter (Fig. 5c).

The dense dendritic sheath at the very distal tip is homogeneous and 100 nm thick. At approximately 1-2 pm proximal of the tip of the dendrites (at the level of the tubular body), the form of the sheath changes. In cross section, it now appears to consist of small particles arranged around the exterior of each dendrite (Fig. 5d). Oblique sections (not shown) reveal that these are, in fact, longitudinally oriented rods 22 nm in diameter.

The fourth dendrite terminates at the wall of the depression, at approximately the same position at which a shallow, smaller depression can be seen externally in scanning electron micrographs (Fig. lb). Like the dendrites associated with hairs, this dendrite contains bundles of microtubules (which form a small tubular body) and a thickened dendritic sheath that changes, more proximally, to longitudinal rods.

About 6 km below Keilin’s organ, all four dendrites bend caudally and travel obliquely through the cuticle. The

ANATOMY AND PHYSIOLOGY OF KEILIN’S ORGAN 191

Fig. 2. Transmission electron micrographs of the cuticular parts of Keilin’s organ. a: Section of a hair in a first instar larva. b: Oblique section of two hairs (hl , h2) in a third instar larva. c: Receptor lymph cavity without a dendrite posterior-medial of the hairs of Keilin’s organ

in third instar larva. For an overview, see Figure 5a. d: Fibrous structures (fs) at the base of a hair in a third instar larvae. cu: cuticle; d, dendrites; rlc: receptor lymph cavity; p: electron dense particles. Scales: a, 100 nm; b,c, 500 nm; d, 300 nm.

dendrites are irregular in shape here, with many deep invaginations and branches (Fig. 5d). The channel in which the dendrites traverse the cuticle, an extension of the rlc, continues through the hypodermal stalk and is continuous with the peripodial cavity of the imaginal leg disc. Unlike the situation in typical rlc’s, where the lumen in which the dendrites lie is delineated by cuticle and the auxiliary cells of the sensillum (Zacharuk, ’85), the channel of the hypoder-

mal stalk is lined by the epidermal cells that compose the inner surface of the stalk. The stalk begins at the extension of the cuticle into the body cavity. The dendrites proceed within the stalk (Fig. 5d) over a distance of more than 1 mm to their cell bodies in the epithelial cell layer of the disc (Lakes and Pollack, ’90). Within the stalk, the transverse profiles of the dendrites are irregular but approximately oval and again undergo considerable invagination and


Fig. 3. Transmission electron micrographs of the dendrites in first instar larva. a: Overview of the arrangement of the dendrites at Keilin’s organ. b The distal tip of the fifth dendrite (d5). c: Tip of a dendrite inserting a t the base of a hair. d Oblique section through the fourth

dendrite. cu: cuticle; d: dendrite; ds: dendritic sheath; ep: epidermal cells; mt: microtubules; rlc: receptor lymph cavity; tb: tubular body. Scale: a, 1 wm; M, 250 nm.

branching. There are consistently four larger processes (ca. 0.7 p,m x 1.3 pm) in third instar larvae, which can easily be distinguished from the much smaller diameter ( < 0.4 p,m) branches. It is possible that one of the smaller processes may correspond to the fifth dendrite seen in first instar larvae. In anti-HRP-labeled preparations of third instar larvae, five dendrites can be seen in the peripodial cavity of the imaginal disc. Four dendrites enter the stalk in all preparations, whereas the fifth is only rarely seen to proceed to the beginning of the hypodermal stalk. As the dendrites travel through the stalk, they remain clad in their

dendritic sheaths. There is a more or less regular layer of microtubules just inside the cell membrane, and additional microtubules are dispersed within the interior of the dendrite (Fig. 5d). No other organelles are seen.

The innervation of Keilin’s organ is summarized in Figure 6.

Central projections. The central projections of the neurons of Keilin’s organ were visualized by backfilling the cells via the hypodermal stalk. The axons of these cells terminate at the ventral margin of the neuropile in the central nervous system (Fig. 7). Within each thoracic


Fig. 4. Transmission electron micrographs of the dendrites in first instar larva. a: Low-power view of the five dendrites (d, d5) in a common receptor lymph cavity (rlc) before entering the epidermal cell layer. b: Section through a dendrite showing the numerous invaginations and the dendritic sheath (ds). c: Oblique section through a

dendrite entering the epidermal cell layer. d: Dendrite surrounded by auxiliary cells (al, a2) within the epidermis. e: Overview of the dendrites in the epidermis (ep). fi Ciliary region (c) of a dendrite. cu: cuticle. Scales: a,e, 1 pm; b,f, 300 nm; c,d, 500 nm.


Fig. 5. Transmission electron micrographs of the dendrites in a third instar larva. a: Low-power view of the dendrites (d, d4) at Keilin’s organ. The arrow points to the receptor lymph cavity without a dendrite (compare Fig. 2dJ. b Cross section through the dendrites within their cuticular extension (CUJ traversing the epidermal cell layer (epJ. c: Tip

neuromere, the future leg neuropile is evident as a lateral extension of the main neuropile. The neurons of Keilin’s organ bypass this region (Fig. 7) and project more medially. The main orientation of the central projection region is longitudinal, with short (5-20 pm) lateral and medial side branches (Fig. 7). Three of the cells have indistinguishable projections, but a fourth projects more medially than do the others (Fig. 7b,c). The backfills also reveal two motor neurons whose axons run through the imaginal disc. Their cell bodies are situated laterally and their dendritic arboriza- tions extend in the dorsal part of the neuropile.

Physiology of the sensory neurons of the imaginal disc

Action potentials can be recorded from the distal stump of the nerve that connects the leg disc to the central nervous system in response to gently touching or stroking the cuticle in the region of Keilin’s organ with a needle or to

of a dendrite ending at the base of a hair. d Cross section through a dendrite with an invagination (it within the hypodermal stalk. ds: dendritic sheath; fs: fibrous structure; mc: moulting channel; mt: microtubules; tb: tubular body; v: vesicle. Scales: a,b, 2 km; c,d, 300 nm.

tapping the stage on which the preparation is mounted (Fig. 8).

The disc is connected to the periphery by a tracheal process as well as by the hypodermal stalk. A nerve that runs along the tracheal process carries the axons of a few larval motor neurons and may also contain sensory processes. Responses to mechanical stimulation can be recorded from the nerve between the disc and the central nervous system when the tracheal process is severed and the disc is left connected to the cuticle only by the hypodermal stalk. Under these conditions, the recorded neurons can only be those in the imaginal disc.

DISCUSSION Organization of neurons and auxiliary cells In typical type I sensilla of insects (i.e., those that consist

of both neurons and a cuticular specialization), the somata

ANATOMY AND PHYSIOLOGY OF KEILIN'S ORGAN 195

Fig. 6. Semischematic drawing of the anatomy of the Keilin's organ. The drawing is derived from sections of first instar larva, except for the cuticle-filled hairs, which are only found in third instar larva. th, tr: thecogen and trichogen cells; c: ciliary structure; d: dendrites-the

of the neurons are situated near the cuticle, within tens of microns of their associated cuticular apparatus (McIver, '85). The dendrites of the sensory cells are specialized into inner and outer segments, with the former ensheathed by, usually, three concentrically arranged auxiliary cells and the latter by a dendritic sheath that continues proximally to the level of the auxiliary cells. This situation applies to Keilin's organ in first instar larvae. However, during the third instar, the somata of the neurons invaginate into the body cavity along with the rest of the imaginal disc, resulting in pronounced elongation of the dendritic outer segments, which now span the length of the hypodermal stalk, still clad in their dendritic sheaths. The proximal portion of the sheath is somewhat unusual in that it

drawing shows one of the three dendrites that terminate at the hairs and two that do not; ds: dendritic sheath; en: endocuticle; ep: epidermal cell; ex: exocuticle; f: folds of the dendrites; rlc: receptor lymph cavity; tb: tubular body.

consists of longitudinally orientated rods. It is unlikely, however, that this form of the sheath is directly related to the elongation of the outer segments, because similar rods are observed surrounding the outer segments of dendrites of the nearby sensillum basiconicum, which are not unduly elongated (unpublished results).

A consequence of the elongation of the outer segments is that the transducing region, the distal tip (in which the presumed transducing structure, the tubular body, is found) is placed at some distance from the auxiliary cells that surround the inner segment. We presume that these cells, like the somata of the neurons, are incorporated in the imaginal disc. In their ultrastructural study of imaginal discs, van Ruiten and Sprey ('74) described inner dendritic

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ANATOMY AND PHYSIOLOGY OF KEXLIN'S ORGAN

segments surrounded by auxiliary cells. Tix et al. ('89a) found that neurons in the disc are closely associated with other cells, probably the auxiliary cells. The unusually long distance between the auxiliary cells and the transducing region is of possible functional importance because, in more typical sensilla, these cells regulate the ionic composition of the fluid in the outer receptor lymph cavity, which, in turn, influences the generation of receptor potentials (Thurm, '74).

From their terminations at the cuticle to their somata and auxiliary cells, the elongated dendrites associated with Keilin's organ lie in the fluid-filled peripodial cavity (and its extension, the lumen of the hypodermal stalk), which is bounded by epidermal cells at the prospective outer surface of the leg. This situation is reminiscent of that which occurs in sensilla of hemimetabolous insects during the apolytic phase of molting (Gnatzy and Romer, '80; Gnatzy, '84). During this phase, the dendrites elongate and remain attached to the cuticle of the old sense organ, ensuring that they remain functional up to the time of ecdysis (Gnatzy and Tautz, '77). At the same time, one of the auxiliary cells, the thecogen cell, secretes a dendritic sheath around the lengthening dendrites. The epidermis pulls away from the cuticle, thus opening the receptor lymph cavity to the newly formed exuvial space, and the epidermal cells proliferate. This is normally followed by secretion of a new sense organ and cuticle before the old cuticle is shed at ecdysis. In third instar fly larvae, the lumen of the hypodermal stalk and the peripodial cavity, which develop during disc invagination, are analogous to the exuvial space that appears during apolysis, and the dendrites of the neurons of Keilin's organ elongate to span this space, similar to the elongation seen in molting hemimetabolous insects. Keilin's organ is believed to be the sensory remnant of the distal portion of a vestigial larval leg (Keilin, '15; Lakes-Harlan et al. '91). Extension of the parallels observed at the level of sensory structures to the developing leg as a whole leads to the suggestion that the process of disc invagination may be a highly modified form of hemimetabolous molting, whereby the normal sequence of apolysis and cell proliferation is restricted to the cells of the disc such that growth is directed internally, resulting in formation of the developing leg (i.e., the imaginal disc) within the thorax of the larva.

Function of Keilin's organ Hafez ('50) and Benz ('56) concluded from behavioral

experiments that Keilin's organ detects humidity. They

197

Fig. 7. Central projections of the sensory neurons innervating Keilin's organ in the third instar larva and of two motor neurons whose axons run through the imaginal leg disc. Drawings on the left are ventral views of wholemounts; those on the right are of parasagittal sections. a: Low-magnification view of larval nervous system. The arrow marks the site at which NiCl, was applied to the nerves leaving the mesothoracic leg disc. br: brain hemispheres; ID: imaginal disc; c b cell body layer; np: neuropil; th-ah: thoracic-abdominal neuromeres. b Higher-magnification views of central projections shown in a. Four neuronal somata were labeled in the disc (not shown), as well as two motor neurons (mot) whose axons travel through the disc. The sensory neurons (sens) arborize near the midline, which is indicated by the dashed line in the drawing on the left. The parasagittal sections drawn to the right were taken from the positions indicated by the arrows. The broad arrows in the drawings of the sections indicate branches of the filled neurons. lnp: leg neuropil; It: longitudinal tract. c: Central projection from a prothoracic disc, in which only the four sensory neurons were filled.

Fig. 8. Examples of responses of neurons of the imaginal leg disc to mechanical stimulation (all from the same preparation). The nerve connecting the disc with the central nervous system was severed and a suction electrode was placed on the distal stump. The horizontal bar under each trace indicates when gentle pressure was applied to the cuticle in the region of Keilin's organ. Time scale at lower right: 100 mseconds.

found that the ability of larvae of Musca and Drosophila, respectively, to discriminate between humidity levels was impaired when the ventral thoracic region (Hafez) or Keilin's organ (Benz) was covered with petroleum jelly. Altner and Loftus ('85) concluded that a particular morphological class of sensilla, those with short hairs or pegs having neither pores nor flexible sockets, was most closely associated with hygroreception. The hairs of Keilin's organ fall within this category. In typical hygroreceptors, the


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dendrites of the sensory receptors terminate as a tightly packed triad within the hairs (Altner and Loftus, '85; Steinbrecht, '89). This is not the case in Keilin's organ.

Another possibility, which we favor, is that Keilin's organ is mechanoreceptive in function. Our ultrastructural findings are consistent with this interpretation. Tubular bodies, which we find in all of the hair-associated dendrites and in one of the remaining two dendrites, are hallmarks of mechanoreceptive neurons. The cuticular extensions into the rlc that we have observed may be analogous to the suspension fibers that are often found extending interiorly from the bases of known mechanoreceptive hairs (Gaffal et al., '75). Their close association with the dendritic sheath at the tip of the dendrite suggests that they transmit or resist movement of this region. This is common in the mechanoreceptive sense organs of adult flies (e.g., Memitt, '87). The site of termination of the dendrites, eccentrically at the bases of the hairs, is typical for known mechanosensory hair sensilla and serves to convert angular deflections of the hair to changes in length and/or tension of the dendrite. The hairs of Keilin's organ differ from those of most known mechanoreceptive hairs in that they lack morphological specializations to facilitate displacement of the hair, such as membranous sockets. However, previous work on dipteran larvae has found that sockets are often less highly specialized than they are in adults (Chu-Wang and Axtell, '721, presumably because the softer cuticle of the larval body wall is inherently more malleable than is the rigid, heavily sclerotized cuticle of adults. The location of Keilin's organ, on the ventral surface, also ensures that it will experience the presumably large forces associated with crawling. Thus, additional mechanical specializations for flexibility may not be necessary. Our electrophysiological recordings show that at least some of the neurons of the disc respond with action potentials to mechanical stimulation of the cuticle.

If Keilin's organ is mechanoreceptive, then how can the behavioral results of Hafez ('50) and Benz ('56) be ex- plained? One possibility is that their treatments with petroleum jelly covered not only Keilin's organ but also other nearby sensilla, e.g., the sensillum basiconicum found just lateral to each Keilin's organ. Hartenstein ('88) found that the structural features of sensilla basiconica of Droso- phila larvae resemble those identified by Altner and Loftus ('85) as associated with hygroreception.

The role of the imaginal discs in development of the adult is well known, but discs have not, to our knowledge, previously been proposed to play a role in larval life. Our results suggest that some cells of the imaginal discs, namely, the neurons that supply Keilin's organ and per- haps the remaining neurons as well, do function throughout larval life as sensory receptors.

ACKNOWLEDGMENTS We thank Ms. G. Kenner for expert help with the

transmission electron microscopy, Ms. S. Beilin de Weitzner for excellent technical assistance, and Dr. P. Lasko for helpful comments on the manuscript. This study was funded by the Natural Sciences and Engineering Research Council of Canada (G.S.P.), the Faculty of Graduate Stud- ies, McGill University (R.L.-H.), and an NIH Jacob Javits Neuroscience Investigator Award to R.K. Murphey (D.J.M.).

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