sensory neuroanatomy of a skin-penetrating nematode parasitestrongyloides stercoralis. ii. labial...

Sensory Neuroanatomyof a Skin-Penetrating Nematode Parasite

Strongyloides stercoralis. II. Labialand Cephalic Neurons

A.E. FINE, F.T. ASHTON, V.M. BHOPALE, AND G.A. SCHAD*Department of Pathobiology, School of Veterinary Medicine, and NIH-IVEM Image

Processing Resource, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACTHost recognition, contact, and skin-penetration by Strongyloides stercoralis infective

larvae are crucially important behavioral functions mediating transition from free-living toparasitic life. The sensilla of the worm’s anterior tip presumably play an important role inthese processes. Besides the main chemosensilla, the amphids, which are of centralimportance, the larva has 16 putative mechanosensilla. There are six inner labial sensilla: twodorsal, two ventral, and two lateral. The two dorsal and ventral pairs are each innervated bytwo neurons, whereas each lateral sensillum is singly innervated. The six outer labial and fourcephalic sensilla are all singly innervated. All of these have the characteristics of mechanore-ceptors: they are closed to the external environment, and closely associated with the overlyingcuticle. Distally, their dendritic processes contain granular material and associated microtu-bules. With two exceptions, the relevant neuronal cell bodies lie in lateral ganglia adjacent tothe nerve ring, their positions remarkably similar to those of their homologues in thefree-living nematode, Caenorhabditis elegans. Cell bodies of two neuronal pairs, one of twodorsal inner labial neurons and one of two ventral inner labial neurons per side, are however,found far anterior to the remaining cell bodies. All labial and cephalic sensilla are apparentlymechanoreceptors, complementing the well-developed chemosensilla. Presumably infectivelarvae require touch and stretch receptors, not only to initiate skin penetration by findingirregularities as points of access, but also to bore through tissue to reach their ultimateenteral destination. J. Comp. Neurol. 389:212–223, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: Caenorhabditis elegans; dendrite; mechanoreceptor; three-dimensional

reconstructions; ultrastructure

Strongyloides stercoralis is an important nematode para-site of humans, other primates, and dogs. Host recognitionand subsequent skin-penetrating behavior are functions ofthe infective larva, the L3-stage (Schad, 1990). After aperiod of transformation in the skin, this same stagemigrates to the parasite’s ultimate predilection site, thesmall intestine. Thus, this stage is of particular parasito-logical interest.

Previously we described the two large lateral sensilla ofthe head of the larva, known as the amphids, and the 13neurons associated with each of them (Ashton et al., 1995;Ashton and Schad, 1996). These neurons, the tips of mostof which are exposed to the environment, are believed to bethe main chemoreceptors, and are thus probably directlyinvolved in the recognition of a suitable host by theinfective larva and in triggering its subsequent penetra-tion, migration, growth, and development. In addition to

the amphidial neurons, 16 labial and four cephalic neuronsare also part of the anterior neurosensory system of S.stercoralis. Although their precise role in the infectiveprocess is unknown, these mechanosensory neurons mostlikely play an important role in skin-penetration and thesubsequent migration of the infective larva.

The labial and cephalic neurons of S. stercoralis havehomologues in Caenorhabditis elegans, the much-studiedfree-living nematode (Ward et al., 1975; Ware et al., 1975;

Grant sponsor: NIH; Grant numbers: RR 02483, R01 A1 22662; Grantsponsor: Reserach Foundation of the University of Pennsylvania; Grantsponsor: the Upjohn Corporation.

*Correspondence to: G.A. Schad, Dept. of Pathobiology, School of Veteri-nary Medicine, University of Pennsylvania, Philadelphia, PA 19104.E-mail: [email protected]

Received 10 April 1997; Revised 31 July 1997; Accepted 4 August 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 389:212–223 (1997)

r 1997 WILEY-LISS, INC.

Chalfie and Thompson, 1979, 1982; Albert and Riddle,1983; Perkins et al., 1986). In this paper, we will comparethese neurons in the free-living infective stage of S.stercoralis with the corresponding neurons in the C.elegans dauer larva (Albert and Riddle, 1983; Ashton et al.,1995). We will also compare them with homologous, butincompletely described neurons in Ancylostoma duode-nale, a hookworm of humans. These three larval forms arestrikingly similar in both form and function (Hawdon andSchad, 1991; Hotez et al., 1993; Ashton and Schad, 1996).

We have examined the ultrastructure of the dendriticprocesses of the labial and cephalic neurons, and made athree-dimensional reconstruction of the anterior-most 10µm of the worm’s head, showing the relationship of thelabial and cephalic neurons with the cuticle, mouth andpharynx, and with the amphidial channels. In addition, wehave traced the labial and cephalic neurons to their cellbodies and made another three-dimensional reconstruc-tion showing the location of these cell bodies in relation tothe nerve ring. This reconstruction provided the basis for amap to be used in subsequent laser microbeam ablationstudies that will make it possible to identify the function(s)of the individual neurons (Bargmann and Avery, 1995).

MATERIALS AND METHODS

The same specimens that were used previously to recon-struct the amphidial neurons (Ashton et al., 1995) wereused for the present study. Therefore, methods of fixation,staining, and sectioning were the same. Briefly, third-stage larvae were fixed, using a procedure modified fromJones and ApGwynn (1991) that combines microwaveradiation with conventional fixatives. Larvae were fixed in4% glutaraldehyde in 0.1 M cacodylate buffer, pH 6.8,postfixed in 1% OsO4 in the same buffer, followed by en blocstaining in 1% uranyl acetate in water. At each stepmicrowave radiation was used to enhance the fixation.After fixation the specimens were dehydrated in acetoneand embedded in Araldite 502: DDSA (Polysciences, Inc.,Warrington, PA).

Serial sections were cut on a Sorvall MT-2 ultramicro-tome fitted with a diamond knife, and were mounted onslotted, Formvar-covered grids. The sections were stainedin 1% uranyl acetate in methanol, followed by Reynold’s

lead citrate. Two worms were sectioned serially well pastthe nerve ring (650–700 sections), with loss of only anoccasional single section. Two more complete sets of sec-tions through the amphids (60–65 sections), as well as anumber of less complete series were also cut.

Electron microscopy

Although the sets of micrographs used in our previousstudy (Ashton et al., 1995) formed the basis for the presentreconstructions, because the pathways of the labial andcephalic neurons are tortuous, many regions were rephoto-graphed to clarify the identities of individual neurons.Sections were photographed in a JEOL JEM-4000EXoperated at 300 KV, or in a JEOL JEM 200A operated at150 KV.

The labial and cephalic neurons curve inward at themost anterior part of the nose of the third stage larva of S.stercoralis and are therefore seen in oblique view intransverse sections. To study the ultrastructure of theseneurons, several sets of serial transverse sections approxi-mately 100 nm thick were examined. These sectionscontained the anterior-most 8–10 µm of the larva. Toexamine the dendritic tips of the outer labial and cephalicneurons in particular, the unique unlimited tilt capabilityof the JEM 4000EX microscope in the IVEM and Biomedi-cal Image Analysis Resource, Department of Biology, Uni-versity of Pennsylvania was utilized, allowing tilt anglesgreater than 60°, where necessary, to obtain transverseimages of the dendritic processes.

Computer imaging

To make the three-dimensional reconstructions of theanterior end of the larva, the contours of the cuticle, buccal

Fig. 1. Scanning electron micrograph of a third-stage larva ofStrongyloides stercoralis. Except for the mouth (M) the only openingsin the cuticle are the amphids (A). One of the two prominent laterallabia (L) is indicated. The notch (N) does not lead to an opening (seetext). Scale bar 5 1 µm.

Abbreviations

A amphidCEP cephalic neuronCu cuticleD dorsal. Suffix to a neuron’s name to indicate dorsal member

of a neuron classILL inner lateral labial neuronIL1 inner labial neuron 1IL2 inner labial neuron 2L labiaM mouth openingN notchOLL outer lateral labial neuronOLQ outer labial quadrant neuronS stomaSh sheath cellSJ the self-junction formed by the socket cell to form the end of

the channelSo socket cellTAM tubule associated materialV ventral. Suffix to a neuron’s name to indicate ventral mem-

ber of a neuron class

S. STERCORALIS LABIAL AND CEPHALIC NEUROANATOMY 213

cavity, and pharynx, the amphidial channels, and of thedendrites of the labial and cephalic neurons were tracedusing the program 3DED (Young et al., 1987) running onan IBM XT personal computer. The contour data weretransferred to a Silicon Graphics Iris Indigo workstation,where the surface renderings were generated by usingSYNU (SYNthetic Universe), a suite of programs withwhich three-dimensional data sets may be easily andusefully manipulated (Hessler et al., 1992). The reconstruc-tion of the anterior of the larva was generated by usingevery section, whereas the cell bodies of these neuronswere reconstructed by using every third section. Thislatter reconstruction is so long and thin that it is impracti-cal to use it directly. Therefore drawings made from thereconstruction are presented in the text.

Nomenclature and description of a typicalnematode sensillum

We have adopted the system of nomenclature, with onlyslight modification, used for C. elegans (White et al., 1986;

Sulston et al., 1988). In this system, neurons are namedwith three letters, or two letters and a number. Positionaldescriptors, D and V for dorsal and ventral and L and R forleft and right, are added to the name to indicate specificmembers of a class of cells. Strongyloides stercoralis hasalmost the same complement of labial and cephalic neu-rons as does C. elegans, so most of these can be namedaccordingly. Thus the cephalic neurons are named CEP,and the four outer labial quadrant neurons OLQ. Thepaired dorsal and ventral inner labial neuronal classes areeach constituted of two neurons, named IL1 and IL2. Asthe lateral inner labial neurons are not double, the C.elegans name for the unpaired outer labial neurons (OLL)was modified: the inner labial neurons are called ILL.

The typical sensillum of a nematode is constituted of aring-like socket cell that anchors the sensillum into thebody wall and surrounds the tip of the particular sensil-lum’s dendritic process. Seated against and extendingsomewhat into the circular opening of the socket cellposteriorly is the sheath cell. The goblet-shaped sheath

Fig. 2. Transverse section through a third stage larva of S. stercoralis approximately 1.7 µm from theanterior end. The amphids (A), the main chemosensory sensilla, are found lateral to the stoma (S). Thenotches (N), dorsal and ventral, appear to anchor the stoma to the cuticle (Cu). The labial and cephalicdendrites are indicated (see list for abbreviations). Scale bar 5 0.5 µm.

214 A.E. FINE ET AL.

Fig. 3. Three stereo images of a reconstruction made with thesurface-rendering program SYNU of the labial and cephalic dendritesin the anterior of a third stage larva. The central stoma is in red; it isflanked by the amphidial channels in blue-grey. Each class of den-drites is assigned a color: inner labials: blue (IL1: dark blue, IL2: lightblue), OLQ: lavender, CEP: yellow, ILL: dark green, OLL: light green.The cuticle is rendered highly translucent. a: Apical view of theanterior end, showing the relationships of the dendrites to the centrallaterally compressed stoma and the amphidial channels. The tips ofthe ILL and OLL dendrites project forward into the lateral lobes of thelips where they are closely associated. Posteriad, they diverge, ILL

running close to the stoma, while OLL lies ventral to the amphidialchannel. The flattened sensory ends of the CEP dendrites on the leftare evident. b: Dorsal view of the reconstruction; anterior is up. Thedifference in lengths of the IL1D and IL2D dendrites is evident. Thepaths of the ILL and OLL dendrites are also clearly seen in this viewThe OLQ dendrites are seen in close proximity to the CEP dendrites. c:Slightly oblique view of the anterior end. On the left, the ends of OLLand ILL are seen just anterior to the opening of the amphidial channelthrough the cuticle. The flattened sensory ends of the CEP dendritescan be seen in this view, as well. Magnification approximately20,000X.

cell forms the channel within which are found one or moredendritic processes. The dendritic processes are sensorycilia, and, depending on the kind of sensillum, the sensorycilia may have highly modified tips: button-like, flattened,angular, etc. However, through most of its length, eachkind of process is, in fact, cilium-like (see Wright, 1980;Ashton and Schad, 1996).

RESULTS

Neuronal complement

Scanning electron micrographs of the anterior end of theL3 larva of Strongyloides stercoralis (Fig. 1) show a centralmouth with two tripartite lateral labia, each consisting of aprominent medial lobe and less prominent dorsal andventral lobes. The only openings (aside from the mouth)evident in these images are the bilaterally situated am-phidial pores (A); there are no openings on the labiathemselves. (When the dorsal and ventral notches seenbetween the labia are examined in sections, they clearly donot lead to openings; Ashton et al., 1995).

Figure 2 is a transmission electron micrograph of atransverse section just posterior to the amphidial pores,approximately 1.7 µm from the anterior tip (‘‘nose’’) of theworm. At this level the mouth cavity or stoma (S) is adorso-ventral slit. The notches referred to above are againseen above and below the stoma. They appear to becuticular anchor points for the mouth cavity. The openingsof the amphidial channels, showing a prominent cuticularlining, are situated anterior to the level at which the tips ofthe amphidial neurons are seen. The single sensory end-ings of the cephalic neurons (CEP) are peripherally situ-ated, one in each of the four quadrants of the section, withthe outer labials (i.e., outer labial quadrant neurons(OLQ)) just dorsal or ventral to them depending on thequadrant. The single outer lateral labial neurons (OLL)are found just ventral to each amphid, while the innerlateral labial neurons, also single (ILL), are found medialto them, close to the stoma. Dorsally, on each side, thepaired double inner labial neurons, IL1 and IL2, are seen.

IL1, which extends anteriorly to its ending just under thecuticle, is sectioned at the level of its axoneme, while onlythe very tip of IL2 appears at this level. Ventrally, only IL1is seen on each side; IL2 does not extend into this slightlyoblique section. The ultrastructural details of each class ofneurons is described subsequently.

Three-dimensional configuration of thedendritic processes in the nose of the larva

To show the relationships of the inner labial, outerlabial, and cephalic dendritic processes to each other andto the other major structures in the nose of the third stagelarva, a three-dimensional reconstruction was made. Threeviews of this reconstruction, presented as stereo pairs, areshown in Figure 3. Figure 3a shows a direct apical view.The centrally situated mouth opening, shown in red, issurrounded by the tissues of the buccal tube, whichbecomes laterally compressed as it extends posteriad. Alsoextending posteriad, lateral to the buccal tube, are the twoamphidial channels, shown in blue-grey. The amphidialdendritic processes contained within are not shown (seeAshton et al., 1995; Ashton and Schad, 1996). The den-dritic processes of the inner labial neurons (ILL, darkgreen, OLL, light green) extend into the protruding mediallobes of each lateral lip, and therefore extend forward tothe level of the mouth opening, as is apparent in thesestereo views. The tips of these neurons lie in close proxim-ity; the neurons then diverge and follow separate pathsposteriad. The inner labial dendrite (ILL) extends poste-riad less than 1/4 µm, and then bends sharply medially to aposition close to the stoma, as shown in both Figures 2 and3. Posterior to its sensory ending, the outer labial neuron(OLL) lies in a position ventral to the amphidial channel.

The paired dorsal and ventral inner labial neurons IL1(dark blue) and IL2 (light blue) extend into sensilla in thedorsal and ventral lobes of the lips where IL1 extends tothe cuticle, whereas IL2 ends approximately 1.5 µm far-ther back (Fig. 3b,c). The dendritic processes of the dorsaland ventral outer labial quadrant neurons (OLQ) in laven-

Fig. 4. Stereo image of a single thin (,100 nm) section showing thesensory endings of the inner lateral labial (ILL) and outer laterallabial (OLL) dendrites. ILL forms a flattened foot-like process underthe cuticle (Cu), with OLL dorsal to it. There are no specialized

structures in the cuticle itself; it is very thin over the sensory end ofILL. Granular material and microtubules are found within the processof ILL, while rod-like inclusions are evident in OLL. Magnificationx32,000.


der are closely associated throughout their lengths withthe cephalic dendritic processes (CEP, yellow). The den-dritic processes of the cephalic neurons are flattened andwider than the others.

Ultrastructure of the neurons

The dendritic processes of both the lateral outer andinner labial neurons (OLL and ILL) extend into theprominent lateral lobes of the lips of the third stage larva,their sensory endings terminating just under the cuticle.The structural details are best understood in longitudinalsections. Figure 4 is a stereo image (for clarity) of a singlethin section showing the sensilla innervated by theseneurons. The inner lateral labial dendrite (ILL) ends in aflattened foot-like process just under the cuticle, with theknob-like end of the outer labial dendrite (OLL) adjacentto it. The cuticle over the ends of these dendrites is thin butwithout other specialized modifications. Microtubules inthe inner labial dendrite (ILL) terminate within the foot-like sensory ending, which also contains some granularmaterial (Fig. 4). The outer labial dendrite (OLL) contains7–8 microtubules (Fig. 2) as well as granular materialwhich is in the form of rod-like structures (Fig. 4).

The dorsal and ventral inner labial sensilla (Fig. 5a–d)are each innervated by two neurons (IL1, and IL2), ofthese, IL2 is approximately 1.5 µm shorter than IL1. OnlyIL1 extends to the cuticle, where it ends in a slightlyexpanded tip (not shown). There are no specialized overly-ing cuticular structures. The dendritic process of IL1,surrounded by its socket and sheath cells, is shown inFigure 5a, approximately 0.2 µm posterior to its tip.Approximately 1.4 µm posterior to this section the tip ofIL2 is found in close proximity to the process of IL1(Fig.5b). In the next half micron (Fig. 5c,d), IL2 shows adense circular structure, the level at which the axoneme orciliary necklace is seen in IL1. Posterior to these sections,the processes end abruptly and the dendrites become verysmall in cross section, and they contain a few neuralfilaments (Fig. 8).

The expanded tips of the dendritic processes of thecephalic (CEP) and outer labial quadrant (OLQ) neuronsterminate just under the cuticle in the subdorsal andsubventral labial lobes and contain amorphous darklystaining granular material (Fig. 5a). The former end aslarge, bent, flattened structures just under an area of thecuticle; the cuticle, itself does not have any specializedstructures associated with these dendrites. In sectionsfrom another worm (Fig. 6a), following those in Figure 5,the CEP dendrite (thick arrow) is filled with microtubulesinterspersed with amorphous ‘‘tubule-associated material’’(Perkins et al., 1986). The outermost microtubules appearto have attachments to the membrane surrounding themicrotubular bundle. Additional granular material liesoutside this membrane in the dorso-medially bent part ofthe dendrite. When thin longitudinal sections (Fig. 7) areviewed in stereo, the tubule-associated material (smallarrow) is observed to form rod-like structures running

Fig. 5. Transverse sections at several levels just posterior to thenose of a third-stage larva, showing the dorsal inner labial sensillumand the tips of the IL1 and IL2 dendrites. The very tips of the OLQ andCEP dendrites are also shown. a: Section approximately 0.2 µm fromthe anterior tip of the dendrite IL1, its socket (So) and sheath (Sh) cellssurround it. The tips of OLQ and CEP are evident, as well. Thesecontain mostly fine granular material. b: Section approximately 1.4µm posterior to the tip of IL1. The densely stained tip of IL2 is seenadjacent to IL1. c: Section approximately 1/4 µm posterior to b. Theaxoneme is visible in IL1, while IL2 shows an electron-dense ring. d:Section approximately 1/4 µm posterior to c. The center of the axonemein IL1 is densely stained. The electron dense ring in IL2 continues inthis section. Scale bar 5 0.5 µm.


parallel to the microtubules (which are not clearly seen inthis image). Posterior to the flattened sensory ending (Fig.6a), the dendrite contains nine microtubules, some ofwhich are singlets, while others have partially formedsecondary fibrils (Fig. 6b). In this section the socket cells(So) associated with these neurons are clearly seen. Theself junctions of each socket cell (Perkins et al., 1986), aswell as tight junctions between the two cells are evident. Insubsequent sections (Fig. 6c–f) the nine microtubulesbecome doublets surrounding two eccentric small singlets.There are filamentous connections to the membrane of thedendrite. At the posterior end of the ciliated dendriticprocess, the nine microtubules are all large doublets,connected to the membrane by ‘‘Y’’-shaped elements.

The OLQ dendrite (thin arrow) contains four dense fila-ments (doublet microtubules in C. elegans hermaphroditeaccording to Perkins et al., 1986) joined into a square by cross

bridges (Fig. 6a). In a configuration very similar to thatseen in C. elegans hermaphrodites, an ‘‘X’’-like structure isformed by fine radial filaments connected to a central element.In subsequent sections the outer cross-bridges disappear,while the radial arms become more prominent (Fig. 6b,c).In the following sections a fifth filament appears, (Fig. 6d),subsequently the filaments form three dumb-bell likestructures, with thin attachments to the membrane of thecilium (Fig. 6e). The radial arms are no longer present,although the central element is still visible. In the nextsections nine doublet microtubules, with ‘‘Y’’-shaped attach-ments to the membrane are apparent (Fig. 6f).

Cell bodies of the labial and cephalic neurons

Posterior to the sensilla they innervate, the labial andcephalic neurons merge with other neurons to form three

Fig. 6. Sections through the subdorsal lobe of the lip of a thirdstage larva, showing OLQ (small arrow) and CEP (large arrow). Thesesections are tilted to high angles in order to obtain transverse imagesof the microtubules. a: Section tilted approximately 65°. The OLQdendritic process has four doublet microtubules joined to each otherand to a central element by bridges. The CEP dendritic process is filledwith an array of large microtubules interspersed with tubule associ-ated material (TAM). b: Section tilted approximately 55°. The OLQprocess shows the four putatively doublet microtubules (Perkins et al.,1986) connected to the central element, but the side bridges arereduced. The CEP process shows nine microtubules, some of which aredoublets, while some have incomplete secondary fibers. The twodendritic processes are surrounded by their socket cells (SO), each of

which shows the typical self-junction (SJ). c: Section tilted approxi-mately 40°. The OLQ process shows the four dense doublet microtu-bules. The CEP dendrite has nine doublet microtubules surroundingtwo small eccentric central singlets. d: Section tilted approximately30°. There are five densely stained doublets in the OLQ process. In theCEP process, the nine doublets are attached to an apical ring,surrounding the two central elements. There are ‘‘Y’’-shaped bridgesattaching each doublet to the membrane in this and the two followingsections. e: Section tilted approximately 20°. There are three dumbbell-shaped structures, attached to the membrane by thin bridges, in theOLQ process. f: The OLQ process shows nine doublet microtubulesattached to an apical ring. There is an eccentric central element. Scalebar 5 0.5 µm.


distinct bundles. A typical section is shown in Figure 8.The neurons IL1D, IL2D, OLQD, and CEPD merge withothers to form a subdorsal bundle. OLL, ILL, and (curi-ously) IL1V merge into a larger lateral bundle which alsoincludes the amphidial neurons, while IL2V, OLQV, andCEPV form part of a subventral bundle.

The section in Figure 8, which is approximately 38 µmfrom the nose of the worm, shows the beginning of the cellbody of IL1D. The next cell body found is that of IL1V, inthe lateral bundle, approximately 64 µm from the nose ofthe worm (Fig. 9a). The dendrite of IL1V is found in thelateral bundle at the level of Figure 8, but moves into thesubventral bundle for several microns, and then bendsback into the lateral bundle where it joins its cell body. Thearrangement of the cell bodies of the labial and cephalicneurons on one side of the body is shown diagrammaticallyin Figure 9a, and in a foreshortened three-dimensionaldrawing in Figure 9b. Seven more cell bodies are locatedjust anterior to the nerve ring 85 to 90 µm from the worm’snose. The dendrite of CEPD follows a tortuous path aroundthe nerve ring to its cell body, the only one found posteriorto the nerve ring.

DISCUSSION

The anterior sensory ultrastructure has been describedin detail and in its entirety in only one developmentallyarrested larval nematode, the dauer larva of Caenorhabdi-tis elegans (Albert and Riddle, 1983). In our previous paperof this series (Ashton et al., 1995), we described theamphidial sensilla, dendrites and associated cell bodies ofthe infective larva of S. stercoralis, essentially a dauerlarva, making this the second such larva in which theseimportant chemosensory structures are fully described.Because the amphids are the only anteriorly situatedsensilla open to the external environment in the S. sterco-ralis infective larva, the amphidial neurons must play aprimary role in the host-finding and infective processes. Inthis paper, we complete the description of the anteriorsensory neuroanatomy of this parasite with the character-

ization of the labial and cephalic sensilla. These sensilla,presumed to be mechanoreceptors, must be important inhost-contact and skin-penetration as well as in subsequenttissue invasion, processes in which thigmotaxis is probablyinvolved.

The S. stercoralis infective larva shows striking similar-ity to the dauer larva of C. elegans in both form andfunction. Indeed, parasitologists and nematologists areincreasingly recognizing the free-living, soil-dwelling infec-tive larvae of parasitic nematodes as dauer larvae (Hotezet al., 1993). It is appropriate, therefore, to use the C.elegans dauer larva as a model for these. Here we comparethe labial and cephalic sensilla of the S. stercoralis larva,and also their counterparts in the incompletely studiedinfective larva of Ancylostoma duodenale, a hookworm ofhumans (El Naggar, 1987), with their homologs in the C.elegans dauer larva. The basic classes of sensilla arepresent, and occur in the same number, in these threespecies, but they vary between species in the number ofneurons per sensillum, and in the fine structure of thesensory nerve endings.

The larvae being compared, while all resting and dis-persal stages, have other functions as well. It is notsurprising, therefore, that their sensory nervous systemsshow both similarities and differences. Like C. elegans,both S. stercoralis and A. duodenale have six inner labialsensilla surrounding the mouth. In the C. elegans dauerlarva, all six of these sensilla are similar in structure. Theyoccur one per lip, and are shifted from the anterior labialsurface, where they occur in active life stages, to the innerlabial margin, where each opens to the exterior via a smallcanal (Albert and Riddle, 1983). Each sensillum containstwo dendritic processes of unequal length.

S. stercoralis has two lateral trilobed lips with one innerlabial sensillum per lobe. In sharp contrast to C. elegans,the inner labial sensilla of the S. stercoralis L3 do not opento the outside. Rather, they end under the cuticle and,furthermore, the two lateral members of this class ofsensilla differ structurally from their four remaining coun-terparts. Only the latter resemble those of C. elegans in

Fig. 7. Stereo image of a single thin longitudinal section through the tip of the CEP dendritic process(large arrow). The tubule associated material (small arrow) is seen to be in the form of rods runningbetween the microtubules themselves. (The microtubules are not easily seen in longitudinal view.)Magnification x32,000.


that each contains two simple dendritic processes of un-equal length, one extending further toward the distal endof the sensillum’s channel than the other. In S. stercoralisthe two neurons are indistinguishable, except by length,since a ciliary rootlet is absent in both dendrites, itspresence or absence distinguishing them in C. elegans(Ward et al., 1975; Albert and Riddle, 1983). Each of thetwo neurons constituting the remaining, laterally- situ-ated pair of inner labials, enters the medial lobe of acorresponding lip. Each extends through sensillar support-ing cells and terminates adjacent to the cuticle in afoot-shaped distal end, unlike the other inner labial neu-rons of S. stercoralis and all inner labials of C. elegans, allof which have simple tips. The foot-shaped sensory end-ings are complex, containing many parallel microtubulesand electron dense granular material characteristic of

mechanoreceptors (Chalfie, 1993; Bargmann, 1994; Garcia-Anoveros and Corey, 1996). The overlying cuticle is thin,but otherwise unmodified.

In the skin penetrating infective larvae of hookworms,the six inner labial sensilla are again closed. The dendriticprocesses end in discoid tips that are linked to the cuticu-lar cortex via a ligamentous intracuticular band (ElNaggar, 1987), a structural arrangement considered byWright, (1976, 1980) to characterize a stretch receptor.It is interesting that in both parasitic species these mostanteriorly situated sensilla (i.e., the inner labial sensilla)apparently have no chemosensory function, as they arethought to have in C. elegans, there being no communica-tion to the exterior. Instead, they end beneath the cuticle,suggesting that they have been converted to mechanosen-sory sensilla.

Fig. 8. Transverse section approximately 38 µm from the anterior end of the third stage larva, showingthe location of the dendrites of the labial and cephalic neurons in three bundles. The dauer-specific cuticle(Cu) is clearly seen in this section. This section is just anterior to the cell body of IL1D; this dendrite’sprofile is much larger than those of the other dendrites. Scale bar 5 1 µm.


The six outer labial and four cephalic neurons in allthree species end under the cuticle. In S. stercoralis thesensory endings of the cephalic neurons and their closelyassociated sublateral outer labials occur in the relativelysmall, submedial lobes of the lips. This places them in aslightly less prominent position than lateral inner labialswith respect to direct frontal engagement with the environ-ment, but, perhaps in a more exposed position withreference to antero-radial interactions. In C. elegans and

in A. duodenale these same neurons, similarly associated,end under slightly elevated cuticular domes sublaterallysituated at the bases of the lips. In C. elegans the fourcephalic neurons end in digitiform swollen tips that bendas they exit their socket cells and then run parallel to theadjacent cuticle, into which each is anchored by a smallnubbin. These sensory tips contain singlet 13-protofila-ment microtubules interspersed with cores of electrondense material (Perkins et al., 1986). This is a well-studied

Fig. 9. a: Drawing approximately to scale made from a reconstruction, made with the surface-rendering program SYNU (SYNthetic Universe; a suite of programs for manipulating three-dimensionaldata sets), to show the positions of the labial and cephalic cell bodies in relation to the other parts of theanterior of the third-stage larva. b: Foreshortened drawing made from the SYNU reconstruction to showthe three-dimensional relationships of the cell bodies to each other. For abbreviations, see list.


mechanosensory ending of the kind found in touch recep-tors that mediate avoidance responses by this species(Bargmann, 1994; Driscoll and Kaplan, 1997). Four of sixouter labial sensilla are very closely associated with thefour cephalics; Wright (1980) shows both neuronal classesto have the same general terminal structure. Consideringtheir close apposition at their sensory endings in a varietyof species, one is led to speculate that they may acttogether to recognize deformations in the overlying cuticlethat indicate contact.

Nothing is known of the neuronal cell bodies in A.duodenale. Turning to the cell bodies of the labial andcephalic neurons of C. elegans (as known from the hermaph-rodite (White et al., 1985)) and S. stercoralis, their posi-tions in the two species are remarkably similar. The onlydifferences involve two types of inner labial neurons. Oneof the two neurons (IL1D) in each dorsal pair of innerlabials is very short, its cell body occurring only 38 µmfrom the worm’s nose. Additionally, the cell body of one ofthe paired ventral inner labial neurons, IL1V, thoughfurther back at 64 µm from the nose, is still far anterior tothe nerve ring. In C. elegans, all the relevant cell bodies liein a ganglion situated just anterior to the nerve ring,except the two dorsal cephalic neurons, whose cell bodieslie just posterior to it (White et al., 1986), as do theirapparent homologues in S. stercoralis.

It is interesting to relate these neuroanatomical observa-tions to the roles these larvae play in the life of each ofthese species. All of the larvae are environmentally resis-tant, non-feeding, resting stages that will resume develop-ment when conditions turn favorable: when a host arrivesfor the parasite larvae or food becomes adequate relative toworm numbers for C. elegans. In the adult hermaphroditeof this species, several different sets of neurons detectvarious mechanical stimuli. In addition to those alreadymentioned, the amphidial neuron ASH, besides detectingosmotic stimuli, mediates nose-touch avoidance. Head-oncollisions are detected primarily by this neuron, with asmall fraction of the response mediated by OLQ and by theaccessory neurons FLP (Kaplan and Horvitz, 1993). Thelatter are apparently absent in the two parasites. Themicrotubule touch cells (Chalfie and Nichols, 1982; Chalfie,1993), which extend along the body of C. elegans, mediatelight body touch as well as diffuse mechanical stimuli(such as taps on the dish containing the worms (Kaplan,1996; Driscoll and Kaplan, 1997)). Foraging behavior(continuous, exploratory, head movements) and the head-withdrawal reflex are mediated by OLQ and IL1 (Hart etal., 1995).

On the other hand, the dauer larva of C. elegans, anon-feeding stage showing no foraging behavior, waitsuntil ecological conditions change (Riddle, 1988), and isinactive until stimulated, as by touch, for example. A touchon the nose with a hair will cause the larva to back away(Kaplan and Horvitz, 1993). In sharp contrast, when a hostis near, the infective larva of S. stercoralis engages activelyin host-finding behavior: it must attach to the host andpenetrate its skin before it can resume development. Aspart of this behavior, it explores its surroundings by meansof head motions remarkably similar to the foraging behav-ior in C. elegans, although the S. stercoralis infective larvadoes not feed. Additionally, it extends itself from particu-lates on the substratum, and if touched by a hair, it willfrequently respond by attaching. It seems highly probablethat this host-contacting/attaching behavior is mediated,

at least in part, by homologues of the mechanosensoryneurons IL1 and OLQ of C. elegans. The conserved,specialized structure of several of the anterior mechanosen-sory sensilla is consistent with a responsiveness to ante-rior touch, although the induced reaction is apparentlyreversed in the parasite larvae. As already described, theinner labial sensilla are modified from putatively dual-function chemosensory and mechanosensory organs suchas occur in C. elegans, to apparently unifunctional, mecha-nosensory sensilla, with especially elaborate developmentof the lateral pair in S. stercoralis. This increased special-ization is precisely what might be expected in a parasiticnematode larva that, having found a host, needs to attach,find an epidermal irregularity or a hair follicle and initiatepenetration, migrate through the skin and eventually borethrough other tissues to reach a final, distant destinationin the host. Sensitive touch and stretch receptors, comple-menting a complex of chemoreceptors, would seem essen-tial to fufill these functions.

ACKNOWLEDGMENTS

This work was supported by NIH grant RR 02483 to theIVEM and Biomedical Image Processing Resource, Univer-sity of Pennsylvania (Dr. L.D. Peachey, Dir.), NIHgrantRO1 A1 22662 (to G.A.S.), The Research Foundation of theUniversity of Pennsylvania (to G.A.S.), and The UpjohnCorporation (to G.A.S.).

We thank Xueqin Wang of the Physics Department forpreparing the scanning electron micrograph, Susan Tram-mell for drawings of the neuronal cell bodies, CatherineNghiem for her work on the stereo images, and Dr. MartinChalfie for his comments on the manuscript.

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sensory neuroanatomy of a skin-penetrating nematode parasitestrongyloides stercoralis. ii. labial...

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