Download Electron microscopical reconstruction of the anterior sensory

Electron Microscopical Reconstruction of the Anterior
Sensory Anatomy of the Nematode
Caenorhabditis elegans
SAMUEL WARD,' NICHOL THOMSON, J O H N G. W H I T E AND
SYDNEY B R E N N E R
M R C Ltrborntory of Molecular Biology, H l l l s Rottd, Crrmbrzdge C B 2 2QH
D i g l n n d nnd 1 D e p n r t m e n t of Biologzcrrl C h e m i s t r y , Hnrucird
M t d i c t r l School, Boston, Mrrsscichttsetts 0 2 1 75 U S.A
ABSTRACT
The complete structure of the anterior sensory nervous system of
the small nematode C . elegans h a s been determined by reconstruction from serial
section electronmicrographs. There are 58 neurons in the tip of the head. Fiftytwo of these are arranged in sensilla. These include six inner labial sensilla, six
outer labial sensilla, four cephalic sensilla and two amphids. Each sensillum
consists of ciliated sensory neurons ending in a channel enclosed by two nonneuronal cells, the sheath and socket cells. The amphidial channel opens to the
outside a s does that of the inner labial sensilla so that these probably contain
chemoreceptive neurons. The endings of the other sensilla are embedded in the
cuticle and may be mechanoreceptive. The cell bodies of all the neurons lie near
the nerve ring and their axons project into the ring or into ventral ganglia. One
of the ciliated sensory neurons in each of the six inner labial sensilla makes
direct chemical synapses onto a muscle making these sensory-motor neurons.
The anatomy of four isogenic animals was compared in detail and found to be
largely invariant. The anatomy of juveniles is nearly identical to that of the adult,
but males have four additional neuron processes.
Nematodes have simple nervous systems
composed of only a few hundred neurons.
Seventy years ago, R. Goldschmidt made a
detailed study of the nervous system of
Ascaris by reconstruction from serial sections using optical microscopy. (Goldschmidt, '03, '08, '09). Among his elegant
anatomical drawings he produced a set of
enigmatic wiring diagrams which have never been interpreted. It is likely that some of
the bizarre connections shown in these diagrams were simply the outcome of the low
resolution of the method Goldschmidt used.
A smaller nematode would offer the possibility of determining the complete structure of a nervous system by electron microscopy allowing reliable identification of
connections. This would open the way to
understanding how the nematode's behavior could be generated by the nervous
structure.
We have chosen the small soil nematode
Caenorhabditis elegans for electron microscopical reconstruction of its nervous system. C. elegans is a favorable organism for
studying the genetics of complex processes
J . COMP. NEUR., 160: 313-338.
and many behavioral mutants have been
isolated (Brenner, '73; Ward, '73; Brenner,
'74). Reconstruction of the wild-type nervous sytem will make i t possible to recognize anatomical lesions in such mutants.
Such lesions should aid the task of understanding nervous system function by allowing correlation of behavioral and anatomical defects. In addition, the study of
genetically induced lesions may help to
understand how nervous system structure
is specified genetically.
This paper is the first of a series which
will describe the structure of the nervous
system of C. elegans. It deals with the anterior sensory nervous system. Although, in
volume, this is only a small part of the
nematode it accounts for 58 of the 300 or
so neurons found in the animal. The neurons are relatively simple and the reconstruction, though laborious, has not posed
any special problems.
MATERIALS AND METHODS
Organism
The nematode used in this work was
313
314
S . WARD, N. THOMSON, J . G. W H I T E AND S. BRENNER
Caenorhabditis elegans (var. Bristol). It was
grown at room temperature on agar petri
plates seeded with E. coli as described by
Brenner ('74).
Fixation and embedding
Worms were rinsed off a fresh petri plate
and fixed in 0.5% Os04 in 0.1 M sodium
phosphate, pH 7.4, for one hour at room
temperature. Some specimens were prefixed in glutaraldehyde by rinsing them off
plates with ice-cold 3% glutaraldehyde in
0.1 M sodium phosphate, pH 7.4, and then
cutting them in half to allow penetration of
the fixative. After two hours on ice, worms
were removed from glutaraldehyde by centrifugation, rinsed and then post-fixed in
Os04 as above.
After fixation, the worms were spread on
a thin layer of 1% agar and any uncut
worms were cut in half or cut nearer the
head if only the tip of the head were going
to be sectioned. The cut worms were covered with a drop of 1 % agar, and blocks
of agar containing a single specimen were
removed, dehydrated through a graded series of alcohols to propylene oxide, then
propylene oxide and araldite (CY 212 resin, CIBA, Ltd.) and then into araldite at
room temperature overnight. The following
day they were transferred to fresh araldite
and polymerized in gelatin capsules overnight at 60°C.
Sectioning, staining and electron
microscopy
Serial sections of approximately 500
were cut with diamond knives on a n LKB
Ultratome 111. Most of the series were cut
transversely to the long axis of the nematode but several series were cut longitudinally. Ribbons of sections were routinely
picked u p on formvarcoated 75-mesh copper grids taking care not to align a ribbon
with a grid bar. When every section was
needed ribbons were collected on formvarcoated slot grids. For analysis of the sensory
endings mesh grids were satisfactory. Grids
were stained with 5% aqueous uranyl acetate for ten minutes a t 60°C and then with
lead citrate for five minutes a t room temperature according to the procedure of
Reynolds. Each section was photographed
onto a single plate with a n AEI EM 6B or
AEI EM 802 electron microscope, except
for the region of the nerve ring which was
A
photographed in four parts and the prints
assembled into a montage.
Reconstruction
The anterior tip of the head of the nematode contains the specialized endings of
sensory neurons and processes of a number
of associated cells. In this region the processes have distinctive morphological features which can be reconstructed from
about 200 serial sections. Four such series
have been analyzed completely, and many
of the features have been confirmed in less
complete series from about 25 additional
animals. Each series of sections was enlarged and printed in rough alignment. All
individual cell processes were followed from
print to print and labelled with colored
pens on successive photographs. Small
groups of processes were followed together
to make identification easier and to prevent
accidental jumps.
The ciliated endings of many of the processes in the head suggested that these were
dendrites of sensory neurons. To confirm
this and to determine the axonal projections
of these neurons, every process in the head
was followed in its entirety. This involved
complete reconstruction of a series of 1,600
sections which was carried out as follows.
The processes were traced back from the
tip of the head for 850 sections. They form
six nerve cords each of which was traced
independently. At the same time reconstruction was begun from the other end of
the same series, identifying cell bodies and
their axonal projections. When the two sets
of assignments were joined, symmetrical
cells were compared. Complete concordance
was found. Thus the classification of cells
by their positions and axonal projections
matched the classification of processes by
the similarity of their neuron endings. The
central structures have been confirmed on
a n independent series but tracing the nerve
cords has only been done once. Nevertheless, the concordance found gives us confidence in the assignments made.
The diagrams showing the structures of
the various sensilla and the positions of
cell bodies (figs. 4, 14, 18, 40) were prepared by tracing the outlines of cells in a
sensillurn from equally spaced transverse
sections. The longitudinal projection of the
structure was then drawn by hand. Longitudinal series of sections confirmed that
315
NEMATODE SENSORY ANATOMY
interpretations based on transverse sections
were correct.
To reconstruct the shapes of the amphidial cells the transverse outlines of each
cell were traced into a computer directly
from the photographs using a coordinate
digitizer to draw the cell outlines. A computer graphics terminal then displayed
longitudinal projections which could be
rotated to aid visualization. The drawings
of these cells shown in figures 19, 20 and
21 were prepared from print-outs of the
computer display. The details of the system
will be described elsewhere.
Terminology
We call all the sense organs in the tip
of the nematode’s head “sensilla” which
cornforms with the definition given by Bul. simlock and Horridge (’65, p. 1608)
ple types of sense organs involving only a
few neurons.” In much of the earlier nematode literature, sensilla has been used only
for the amphids; other sense organs are
designated as “papillae, setae, etc.” We
also adopt two new terms “sheath” and
“socket” cells for the two non-neuronal
cells invariably associated with each sensillum. The reasons for this are presented
I ‘ . .
in the discussion. We have followed the
nomenclature of DeConinck (’65) in naming
the individual sensilla.
RESULTS
T h e arrangement of sensilla
The tip of the head of C. elegans is shown
e n f a c e in the scanning electron micrograph, figure 1. There are six symmetrical
lips surrounding the triangular opening
of the mouth. Each lip is surmounted by a
papilla which is the ending of the inner
labial sensillum. The lateral lips contain an
inpocketing of the cuticle which is the opening of the amphid. Each sub-dorsal and
sub-ventral lip has a pair of bumps and the
lateral lip has a single bump visible. These
are the endings of the cephalic and outer
labial sensilla.
All of the cells found in the anterior 10 p
of C. elegans’ head are summarized in table
1. The non-neuronal cells were identified
easily by their positions and morphology.
The neurons were identified by synaptic
connections in the central nervous system.
Most of the neurons can also be recognized
just by the fine structure of their anterior
terminals.
The types of neurons in the tip of the
head are summarized in table 2. (The labels
given in this table will be used throughout
this paper.) There are a total of 58 neurons
arranged in bilateral, or apparent four and
six-fold symmetries. This symmetry reduces
the types of neurons to 21. With two posTABLE 1
Cells tn the ciizterior 10 y of C. elegans
Cell type
Muscle
Hypodermal
Labial
Epidermal
Arcade
Sheath
Socket
Neuron
Total cells
Fig. 1 En face scanning electron micrograph.
The six papillae surmounting the lips are the endings of the inner labial sensilla. Other sensilla endings are the small bumps and the inpocketing of
the amphids which are labeled a s indicated in
table 2. This picture was taken by Mr. Len Christianson and Dr. Peter Andrews and kindly provided
by Dr. David Hirsh. Magnification, X 8,000.
Number
8
8
6
9
18
18
58
125
The hypodermal cells ring the circumference of the
nematode and secrete the cuticle. The muscles lie longitudinally just under the hypodermis. The labial epidermal cells form the divisions of the six lips. The arcade
cells line the esophagus: three syncytial anterior cells
overlap six syncytial posterior cells. The nuclei of all the
above cells lie 50-100 y from the tip of the head. The
sheath, socket cells and the neurons make up the sensilla.
316
S . WARD, N . THOMSON, J G WHITE AND S BRENNER
TABLE 2
A?]tcprior
Sensillum
Neuron
labels
itt’ii
ro i t s
N u m b e r of
n e u r o n types
Total
neurons
s1x
two (1)
two (v)
4
-
2
2
1
Outer Labial ( 0 )
0
1
six
6
Cephalic ( C )
C
1
four (d,v)
4
Amphid (A)
a-1
12
lnner Labial (1)
Not in sensilla
1.2
m,n
Syinmetry
X
1
Y
1
Total
types 21
two (1)
two (d)
four (d,v)
Total
neurons
12
2
24
2
4
58
N e u r o n s all m a k e synaptic c o n t a c t to o t h e r cells i n t h e c e n t r a l nervous system. T h e letters in p a r e i i ~
t h e s e s after t h e n a m e of e a c h s e n s i l l u m and t h e n e u r o n l a b e l s will be used to label t h e sensilla and t h e
n e u r o n s throughout t h i s paper. E a c h n e u r o n type reflects a s y m m e t r i c a l grouping of n e u r o n s with ne‘vly
idcntical f i n e s t s u c t u r e at their e n d i n g s . T h e s y n i m e t r ~of t h e w ~ r o u p i i i g IsS indicated (1. l a t e r a l . v. suhventral: d. sub-dorsal,. T h e r e :we two additional antcrior n e u r o n s which i n n e r v a t e t h e p h a r y n x xbout 12 p.
from t h e tip of t h e head. T h e m a l e h a s four a d d i t i o n d c e p h a l i c n c u r o n processes.
sible exceptions (see below) all of the neurons appear to be sensory.
Figure 2 is a micrograph of a transverse
section cut 1 p from the tip of the head
which shows the arrangement of the sensilla. Each of the six lips contains one inner
labial and one outer labial sensillum. The
sub-dorsal and sub-ventral lips each contain a cephalic sensillum. The lateral lips
each contain a n amphid. This arrangement
of sensilla is summarized by figure 3 which
shows diagrammatically all the sensilla and
all the neurons in the head. The structure
of each of these sensilla will be presented
in the following sections.
T h e inner labial sensilla
The structure of a n inner labial sensillum is diagrammed in figure 4. Electron
micrographs of transverse sections of this
sensillum are shown in figures 5-13, and
a longitudinal section in figure 15. Two
sensory neurons innervate each sensillum.
The ending of inner labial neuron 2 penetrates through a small opening in the
cuticle at the tip of a papilla (figs. 4, 5, 6).
The other neuron ends embedded in the
cuticle approximately 0.5 p below this opening (figs. 4, 7). Both neuron processes
then pass back through a channel in the
cuticle and sub-cuticle. This channel is
lined by extra-cellular material and bends
inward before being encased by a special-
ized non-neuronal cell, the socket cell. The
socket cell wraps around the channel approximately 1 p from the ending and seals
to itself with a tight junction (figs. 28-30
for similar junctions). The extracellular
material lining the channel ends where
the socket cell forms a tight junction to another specialized non-neuronal cell, the
sheath cell.
The sheath cell is goblet-shaped and
surrounds the nerve channel. The neurons
penetrate through the base of the sheath
cell to reach the inside of the channel with
tight junctions sealing the channel from
the pseudo-coelom. The sheath cell membrane (the inner surface of the goblet) is
deeply invaginated into lamellae below the
region where the neurons penetrate the
sheath cell. The spaces between lamellae
Fig. 2 Transverse section 1 p from the tip of
the head. Sensilla and neurons are labeled as in
table 2. The right sub-ventral side is more anterior
than the left sub-dorsal. The specimen was prefixed in glutaraldchyde. Magnification. X 13.000.
Figs. 5 , 6 Tip of the inner labial sensillum. The
very tip of the papilla is shown in transverse section, revealing that the inner labial neuron 2 opens
to the outside through a channel in the cuticle. Figure 5 is a right sub-dorsal sensillum and figure 6 is
a right sub-ventral sensillum from different animals. Magnification, X 21,000.
Fig. 7 Transverse section 0.5 p from the tip
of a n inner labial sensillum. This electronmicrograph shows the ending of the inner labial neuron
1. Magnification, X 21,000.
NEMATODE SENSORY ANATOMY
317
318
S. WARD, N . THOMSON, J. G . W H I T E AND S . BRENNER
Fig. 3 Summary of the arrangement of neurons and sensilla in the head. All of the sensilla
are shown as neurons surrounded by sheath cells (lightly shaded) with socket cells (darkly
shaded) adjacent. Ciliated neurons are black, unciliated neurons are unshaded. Labeling is as
in table2.
are continuous with the sensory neuron
channel, as drawn in figure 4 and shown
in figures 10, 11 and 12.
The endings of the two inner labial sensory neurons have characteristic fine structure. Sensory neuron 1 has a basal body
with seven doublet microtubles. These extend various distances up the nerve in a
ciliary ring, some reaching the tip and
fusing with an electron dense material.
This neuron also has a striated ciliary
rootlet extending posteriorly from its basal
body for about 7 p as shown in figure 15.
There are one to four vesicles in this nerve
just below its basal body. In glutaraldehyde
fixed preparations the pesicles range in
diameter from 250-900 A and do not contain electron dense material.
Inner labial neuron 2 is exposed to the
outside at its tip. It has a basal body with
NEMATODE SENSORY ANATOMY
five to seven doublet-microtubules about
2.5 p from its tip. These doublet tubules
become singlets as they extend toward the
tip, and two of them extend to the very end
of the neuron. There is no ciliary rootlet.
Figure 4 shows a n unciliated accessory
neuron ending adjacent to a socket cell. A
cell of this type is found invariably associated with the sub-ventral sensilla. In some
animals this cell has a branch to the lateral
sensilla a s well. These accessory neurons
319
sometimes end embedded in the socket cell
but do not have specialized junctions at
their endings. The dorsal sensilla have a
similar neuron associated with them but
this dorsal neuron is a branch of the lateral accessory neuron m discussed below.
The lateral inner labial sensillum has
two additional ciliated accessory neuron
labeled m and n . These neurons wrap
around a branch of the lateral inner labial
socket cell just below the region where the
Fig. 4 Inner labial sensillum. A right sub-ventral sensillum is shown diagrammatically in longitudinal and transverse section. T h e six inner labial sensilla have identical structure except for
t h e accessory neuron. T h e outlines of t h e cells were simplified slightly from actual tracings and
t h e n u m b e r of m e m b r a n e invagination in the sheath cell w a s reduced for clarity. T h e blackened
junctions between cells represent tight junctions. Vesicles are shown n e a r t h e basal body of neuron 1. T h e socket cell and t h e anterior part of the sheath cell are surrounded by a labial epidermal cell to which the socket cells make additional tight junctions. T h i s cell is visible in figures
8 a n d 9.
320
S . WARD, N. THOMSON, J. G. WHITE AND S. BRENNER
basal body of the sub-dorsal and sub-ventral
neurons. Both the cephalic and outer labial
neurons have a small electron dense branch
which extends to a bump in the cuticle
without penetrating through the cuticle.
This bump is visible in the scanning elecT h e outer labial and cephalic
tron micrograph, figure 1.
sensilla
The lateral outer labial sensilla have a
The six outer labial and four cephalic neuron, sheath and socket cell arrangesensilla end beneath the cuticle and have ment similar to the sub-dorsal and subsimilar structures. The lateral pair of out- ventral sensilla, but the fine structure is
er labial sensilla differ somewhat from the different. The neurons end about 0.5 p less
sub-dorsal and sub-ventral pairs. The sub- anteriorly than the other outer labial senventral cephalic and outer labial sensilla silla. Their endings are shorter and smaller
are shown diagrammatically in figure 14. and are electron dense. These endings reFigures 8-13 are electron micrographs of semble the small electron-dense bump of
the other outer labial neurons without the
transverse sections through these sensilla.
In each sensillum a single neuron passes anterior extension. Their basal body is withthrough a close-fitting channel about 1.5 in 1 p of the endings and does not have a
microns long through the cuticle and sub- striated rootlet connecting to it. Because of
cuticular matrix and ends embedded in the these differences in fine structure, the descuticle. The neuron channel widens slightly ignation of these lateral sensilla as outer
labial is based on other similarities to the
where the socket cell wraps around it and
seals to itself. Like the inner labial socket sub-dorsal and sub-ventral sensilla: the
cells, the socket cell abuts onto a sheath positions of their cell bodies; the similarity
cell with a tight junction. The sheath cell of their arrangement in the nerve cords;
is again goblet-shaped with the neuron pen- and the position and morphology of their
sheath cells.
etrating into the channel through its base.
The channel is sealed from the pseudoA m p hid s
coelom with tight junctions between the
neuron and the sheath cell. Both the outer General structure
labial and the cephalic sheath cells have
The amphids are two large lateral sendeeply invaginated membrane lamellae sep- silla each containing 12 sensory neurons
arated by spaces which are continuous with and a sheath and socket cell. The amphid
the channel surrounding the neuron.
structure is shown diagrammatically in figThe cephalic and outer labial neurons ure 18. Various aspects of the structure can
differ from each other in size and in the be seen in figures 1 G 2 7 and 31 and 32.
fine structure of their tips. Each cephalic
The general arrangement of neurons,
neuron has a basal body with six to eight sheath and socket cells shown in figure 18
doublet microtubules which extend toward is similar to that of the other sensilla. Eight
the tip for about a micron in a ciliary ring. of the neurons end in a channel which
The neuron widens just above where i t opens to the outside through the cuticle.
enters the subcuticular matrix, and its This opening is formed by a n infolding of
microtubules are joined by other tubules the cuticle. The socket cell wraps around
organized in groups around an amorphous the channel and seals to itself a s do the
electron dense core. These tubules extend socket cells of the other sensilla. The socket
to the end of the neuron. The neuron has cell is shown clearly in the longitudinal
no ciliary rootlet.
Figs. 8-13 Seriesoftransverse sections through
The sub-dorsal and sub-ventral outer
the inner labial, outer labial and cephalic sensilla.
labial neurons have basal bodies with five The
series is through a right sub-dorsal lip of a sinto seven doublet microtubules. Apparent gle animal fixed only with osmium. Labeling is as
singlet tubules extend anteriorly from in table 2 and in addition, S o , socket; Sh, sheath;
the basal body in a characteristic joined Cu, cuticle. The sections are approximately 0.5 p
diamond pattern, which reaches almost to apart and correspond roughly to the transverse
sections diagrammed in figures 4 and 14. Unlabeled
the end of the neuron (fig. 10). A striated processes
are branches of non-neuronal cells or of
rootlet 3 p long extends posteriorly from the the right amphid. Magnification, x 26,000.
socket cell surrounds the neurons (fig. 2).
Each contains a basal body near its ending
containing nine doublet microtubules which
extend anteriorly for about 1 F . The neuron
m also has a short ciliary rootlet.
NEMATODE SENSORY ANATOMY
32 1
S. WARD, N. THOMSON, J. G. W H I T E AND S. BRENNER
322
Fig. 14 Outer labial and cephalic sensilla. The left sub-ventral outer labial and cephalic sensilla are shown in longitudinal and transverse section in a manner similar to figure 4.
section, figure 17. The socket cell surrounds a sheath cell and joins to it with a
tight junction (fig. 27). The neuron cham
nel formed by the sheath cell is lined for
part of its length by a densely staining
extracellular material.
Sheath cell
The ending of the amphidial sheath cell
is a large bilobed structure as shown in
figure 19. The longitudinal section shown
in figure 18 is drawn as if cut along the
radial axis of the nematode parallel to the
line of view of the sheath cell shown in figure 19. Figure 18 shows that the neurons
enter the sheath cell at the base of its channel with tight junctions sealing their entry.
Unlike the sheath cells of the other sensilla
the amphidial sheath cell does not have
deeply invaginated membrane lamellae
opening into the neuron channel. Instead,
it has a large golgi apparatus giving rise
to many large vesicles along its concave
face facing the channel (figs. 17, 18, 25).
NEMATODE SENSORY ANATOMY
323
Fig. 15 Longitudinal section of an inner labial sensillum. The section is cut longitudinally tangential to the cuticle. The electron-dense tip of the papilla is shown. Below that, the section crosses the
subcuticular endings of the outer labial and cephalic sensilla. The junction between the socket and
sheath cells is indicated by arrows. The striated ciliary rootlet in neuron 1 shows clearly. The average
spacing of striations is 750 A. Magnification, x 14,000.
Fig. 16 Tangential section through an amphid. This section shows the seam of the socket cell particularly clearly, as well as clustering of the neuron endings (N) as they pass into the socket cell channel. Vesicles (v) inside the sheath cell adjacent to the channel boundary are also evident. Magnifkation, X 14,000.
Fig. 17 Radial section through an amphid. The opening of the amphid through the cuticle is shown
with the neurons passing up the amphidial channel. The large white-ish vesicles (v) on the right are
inside the amphidial sheath cell. Magnification, X 14,000.
324
S . WARD, N. THOMSON, J. G. WHITE AND S. BRENNER
Neurons
Reconstructions of the endings of 12 sensory neurons which innervate the amphid
are shown in figures 20 and 21. They all
contain basal bodies with nine outer doublet
microtubules located in the relative positions shown. They also contain 2-6 central
singlet microtubules. In the endings e-1,
the doublet tubules extend anteriorly in a
ciliary ring nearly to the tip of each neuron
and become singlet tubules near their endings (figs. 26, 27). Although the neuron
endings e and g-k appear similar in morphology, each can be identified uniquely by
the relative position of its basal body, its
point of entry into the sheath cell, and,
most importantly, its relative position in
the sheath cell channel. This positioning
will be described below. The eight cells
e-1 all pass up the amphidial channel near-
Cuticle -
Socket ___
Sheath ___
Neurons
-
Fig. 18 Amphid. A right amphid is shown in radial and transverse section in the manner
of figures 4 and 14.The shapes of the cells were simplified slightly for clarity. Dense vesicles are
shown near the basal bodies of the neurons. A large golgi apparatus and mitochondria are
shown in the base of the sheath cell. The scale of the transverse sections i s one half that shown
for the longitudinal sections.
325
NEMATODE SENSORY ANATOMY
;
I
/;?
Fig. 20 Amphidial neurons a d . The neurons
are shown viewed from the side a s for figure 19.
They are based o n computer reconstruction of
transverse cell outlines. The rings of doublets indicate the position of the basal bodies.
Fig. 19 Amphidial sheath cell. This drawing
views a right amphidial sheath cell from the outside of a verticaI nematode. T h e drawing was based
on a computer reconstruction of cell outlines from
series of transverse sections.
ly to its opening, as indicated in figure 18
and shown in figure 17. Because cells f and
1 are branched there are a total of ten ciliated processes passing up the amphidial
channel .
The unusually shaped neuron endings
a 4 do not run all the way up the amphidial
channel. Cells a-i: run in the sheath cell
channel up to their basal bodies but above
this they poke their way into the sheath
cell itself, like hands into a balloon, ending
surrounded by the sheath cell. Cell d also
pokes into the sheath cell but does so from
outside of the sheath channel. It sends into
the channel a small branch which is sealed
off at its base by tight junctions to the
sheath cell. None of these four neurons
makes gap junction synapses to the sheath
cell membrane; nevertheless, their shape
creates an enormous surface area of membrane adjacent to the sheath celi membrane. There is always a 75-150 A space
between the two membranes. For cells a-c
this space is continuous with the sheath
cell channel. For cell d it is separated from
the channel and the pseudo-coelom by tight
junctions.
The arrangement of the four neurons
( a d ) in the sheath cell is indicated in fig-
Fig. 21 Amphidial neurons el.T h e neurons
are shown a s in figure 20.
ures 18, 31 and 32. The cell with fingerlike projections, d, is always dorsal; the
branched ceI1 a is dorso-lateral (it is the
cell shown poking into the sheath in fig.
18); cell c has its wings extending both
dorsally and ventrally; cell b has its wing
ventral and the small branch dorsal in the
channel.
Tracings of the cell positions at three
different levels in the amphids are shown
in figure 31; in figure 32 the arrangement
326
S . W A R D , N. THOMSON, J. G . WHITE AND S . BRENNER
Figs. 22-25 Transverse serial sections through a n amphid. The series is through a right
amphid of a n animal fixed only with osmium. The neurons can be identified by comparison
with figure 31. T h e sections are at approximately 1 p intervals. Magnification, X 18,000.
32 7
NEMATODE SENiSORY ANATOMY
of neurons in the left amphid of different
adult animals is compared. The arrangement is identical in these four animals and
is the same in males and juveniles. This invariant arrangement makes it possible to
identify each amphidial neuron by its position in a single transverse section.
Other neurons
There are six neurons in the tip of the
head which are not intimately associated
with sensilla. Two of these neurons end in
unciliated large bulbs, located between the
sub-dorsal cephalic sheath cell and the
amphidial sheath cell. These are labeled x
in figures 3 , 12 and 13. Four other unciliated neurons end in thin sheet-like structures. The ventral pair extends on the ventral side of the sub-ventral inner and outer
labial sheath cells just below their anterior
endings. The dorsal pair extends similarly
between the dorsal inner and outer labial
sheath cells. They are labeled y in figures
c
Fig. 31 Arrangement of amphidial neurons.
Tracings of transverse sections of the left and right
amphids of a single animal are shown. The neurons
are labeled as in figures 20 and 21.
Fig. 32 Comparison of the arrangements of
amphidial neurons in different animals. The arrangement of neurons identified by their fine structure, shape and position is shown for three different animals. The left amphids are shown as in
figure 31 at a level corresponding to the middle
tracing in figure 31.
3 , 12 and 13. These cells may be homologous to Goldschmidt’s (’03) four “Faserzellen” in Ascaris.
Males
Series of transverse serial sections through
the tip of the head of three adult male
worms have been examined. The arrangement of sensory cells is identical with that
of the hermaphrodite except that there is
an additional sensory process in the channel of each of the cephalic sensilla. This
process is a modified cilium but is smaller
than the cephalic nerve ending. It penetrates the cuticle of the male, opening to
the outside through a small papilla as
shown in figure 33. This opening is continuous with the cephalic nerve channel so
that in the male the entire channel appears
to open to the outside.
These four extra processes exit posteriorly from the sheath cell channel with
tight junctions to the sheath cell, as do the
cephalic neurons. I t is not yet known whether they represent four additional neurons
or are extra branches of neurons also
found in the hermaphrodite.
Deirids
The deirids are a pair of lateral, cervical,
sub-cuticular sensilla located about 70 p
from the tip of the head. The ending of one
of them is shown in figure 34. Like the other sensilla they have a ciliated sensory neuron in a channel surrounded by a socket
cell and an invaginated sheath cell. The
neuron channel does not penetrate the
cuticle but ends longitudinally in the sub-
328
S. WARD, N . THOMSON, J. G . WHITE AND S. BRENNER
NEMATODE SENSORY ANATOMY
cuticle, dorso-laterally. The fine structure
of the ending closely resembles the endings
of the cephalic sensilla showing many microtubules clustered around electron-dense
material. Fluorescence microscopy of formaldehyde-treated worms shows that both
the deirid and cephalic cell bodies and
processes contain catecholamines and these
six cells are the only anterior cells which do
so (J. Sulston, personal communication).
Juveniles
Figure 35 shows a complete section of a
first or second stage juvenile. All of the sensilla are present and are identical with
those in the adult hermaphrodite except
that the sheath cells are not as extensive
and none of the non-ciliated accessory neurons are branched.
Positions of cell bodies
The cell bodies for all cells in the anterior tip of C. elegans lie further back in the
head. The neurons, sheath and socket cells
connect to their cell bodies by long, thin
processes that run down the head in six
cords reflecting the arrangement of sensilla in the lips. Sections of the nerve cords
are shown in figures 38 and 39. Although
the sheath and socket cells run as nervelike processes down the cords to their cell
bodies, they do not give or receive synapses
to any other cell and are, therefore, not
neurons. In addition to the processes from
cells in the tip of the head, 14 other processes are found in the anterior nerve cords.
Figs. 26, 2 7 Fine structure of the amphidial
neurons. High magnification electronmicrographs
of transverse sections of the amphidial neurons of
a glutaraldehyde-fixed specimen. (26) 3 p from
the amphidial opening. Magnification, x 31,000.
(27) 1 ,u from the opening. Magnification, X
45,000.
Figs. 2 8 , 2 9 , 3 0 Cell junctions. High magnification pictures of the junctions between cells of a
glutaraldehyde-fixed specimen. (30) A socket cell
junction to itself. (29) A sheath-socket cell junction.
(28) A sheath-epidermal cell junction. E, epidermal
cell. Magnification, X 45,000.
Fig. 33 Male. The additional process which is
in the male cephalic sensilla is shown ending exposed to the environment in the channel of a n extra
male papilla. This is a right sub-dorsal lip with the
endings of the cephalic a n d outer labial neurons
shown. Magnification, X 32,000.
Fig. 34 Deirid. The ending of the right deirid
is shown. This transverse section was cut about
80 ,u from the tip of t h e head. T h e projections of
the cuticle a r e the lateral “treads”. D, deirid neuron. Magnification, X 27,000.
329
These terminate about 15 p from the tip
of the head. Two are interneurons which
innervate the pharynx, and these are described in a subsequent section. Six others
(one in each cord) are tiny processes from
glial cells which line the inside of the nerve
ring. Another four (2 sub-dorsal, 2 subventral) are tiny processes from neurons
which synapse onto muscles in the ring.
Two more are tiny lateral processes from
other neurons in the ring.
The positions of the cell bodies for all
the processes in the anterior are shown in
figure 40. Note that the positions of sheath
and socket cell nuclei are not perfectly bilaterally symmetrical. The significant alterations are in the position relative to the
first bulb of the pharynx. In the left dorsal
cord, the outer labial socket cell nucleus is
adjacent to the inner labial, anterior to the
bulb, whereas in the right dorsal cord this
outer labial socket cell nucleus is displaced
posteriorly. A similar displacement is observed for the inner labial sheath cell in
the two lateral cords. However, in all cases
the sequence of cell bodies within each
cord and the dorso-ventral positions of the
cell bodies relative to the cords are the
same. Two identical dorsal motor neuron
cell bodies are also not bilaterally symmetrical: they are in reverse order on the two
sides. The above variations in symmetry
may be trivial due only to mechanical displacement by the pharynx so that the cell
body positions actually reflect the bilateral
symmetry of the sensilla in the head exactly.
The additional four- and six-fold arrangements of sensilla in the head are reflected
in cell body position also but not as precisely. For example, if one compares the dorsal
to the ventral cords one finds the same anterior-posterior sequence of neuron cell bodies (inner labial two, inner labial one, outter labial, cephalic) but the spacings and
dorso-ventral displacements of the nuclei
differ. The dorsal cephalic cell bodies are,
in fact, posterior to the nerve ring itself.
The positions of the lateral outer labial
neuron cell bodies are similar to those of
the outer labials in the dorsal and ventral
cords and are not as posterior as the cephalic cell bodies. This is evidence supporting the designation of the lateral sensilla as part of the outer labial symmetry
group.
330
S . WARD, N. THOMSON, J. G. W H I T E AND S. BRENNER
Pharyngeal inneruation
Two neurons found in the lateral cord
enter the anterior end of the pharynx. This
connection was described in Ascaris by
Goldschmidt (Bullock, 'SS), and our attention was drawn to it in C. elegans by R. L.
Russell (personal communication). Figures
36 and 37 show the extension of this neu-
331
NEMATODE SENSORY ANATOMY
ron into the pharynx. The neuron ends in
an electron-dense specialization adjacent
to two pharyngeal neurons with a possible
gap junction between them.
The two neurons entering the pharynx
are interneurons which receive synaptic
input in the ring and are afferent to the
pharynx (White, Thomson and Brenner,
unpublished). They are the only two neurons which connect the somatic nervous
system to the pharynx.
labial neuron a direct sensory-motor neuron. However, these neurons also synapse
onto interneurons, including the neuron
which connects to the pharynx, and they
have a peculiar synapse-like association
with hypodermal cells. The details of these
connections and the synapses of the other
neurons will be described elsewhere.
DISCUSSION
Function
C . elegans has at least three sensory reAxonal projections
sponses: chemical mechanical and therFor clarity the axonal projections of the mal (Ward, '73; Dusenberry, '73; Ward,
sensory neurons are not shown in figure 40. unpublished). The chemical sense includes
The axons of the sensory neurons, except detection of at least four classes of attractfor 11 of the amphidial neurons and neu- ants: cyclic nucleotides, anions, cations
ron m, enter the circumpharyngeal nerve and hydroxyl ions (Ward, '73). The nemaring. The axons first pass posteriorly out- tode is also repelled by acid, some form of
side the nerve ring remaining grouped in carbonate ions, and aromatic compounds
symmetrical cords. They then turn back (Dusenberry, '74; Brenner and Ward, unto form the neuropil of the ring making published). From the altered chemotaxis of
both gap junctions and chemical synapses head-defective mutants, Ward ('73) cone n passant as they course anteriorly. Eleven cluded that the receptors detecting the atamphidial neuron axons enter the ventral tractants must be located on the head.
ganglia through commissures passing venThe structures of the sensilla on the head
trally between the muscles and hypodermis. suggest which of them could detect these
The synaptic connections of all of the attractants. Chemoreceptive neurons must
neurons from the head have been deter- either directly contact the environment surmined. With the exception of the pharyn- rounding the nematode or contact another
geal neurons and the neurons x, all the specialized cell which then contacts the
neurons give synapses in the ring, consist- surround. All of the neurons in the amphid
ent with their designation as sensory. The meet these criteria, as does the inner labial
axonal processes from all six inner labial neuron 2. Eight of the amphidial neurons,
neurons 1 make direct chemical synapses 4, reach nearly to the opening in the
to muscle arms from the thirty-two most cuticle of the amphidial channel. The three
anterior body muscle cells. One such syn- neurons a-c also contact the open channel
apse is shown in figure 41. Each neuron further down. The neuron d has only a
makes several such synapses. This synaptic small branch reaching the open channel;
connection to a muscle makes the inner however, it has a large surface area adjacent to the sheath cell which bounds the
Fig. 35 Juvenile. A complete eansverse sec- channel, and therefore it might be a section about 0.5 p from the tip of the head of a j u v e
ondary sensory neuron such as is found in
nile is shown. Compare this figure to figure 2 and
notice that the non-neuronal cells are much less the mammalian taste bud (e.g., Murray and
extensive and that the sheath cells are smaller and
Murray, '70).
less invaginated. Magnification, X 17,500,
Amphids in other nematodes have been
Figs. 36, 37 Pharyngeal neuron. Figure 36
assumed to be chemoreceptors because they
shows the pharyngeal neuron (P) extending an el=open to the outside, but no more direct evitron-dense process into the pharynx. Figure 37
shows this process two sections later ending as a
dence of their function has been presented
densely staining process with a possible gap junc(deconinck, '65; Bird, '71 ; Croll, '70). Howtion to neuron (3) within the pharynx. The two
ever, the recent isolation of non-chemotacneurons labeled 3 and 4 both may be associated
tic C. elegans mutants which are defective
with the ending of the pharyngeal neuron. They
appear to be fused in figure 37, but successive s e e
in their amphidial neurons supports the
tions show that this is not so; the membrane bouninterpretation that the amphid is a chemodaries are not perpendicular to the plane of section
receptor (J. Lewis, personal communicafigure 37. Both neurons 3 and 4 have cell bodies
tion).
with the pharynx. Magnification, X 30,000.
332
S. WARD, N. THOMSON, J. G W H I T E AND S . BRENNER
NEMATODE SENSORY ANATOMY
The function of the amphidial sheath
cell remains unknown. The large golgi apparatus with its forming face facing the
amphidial channel suggests that it secretes
material into the channel (Revel, '71). Presumably this material is stored in the large
sheath cell vesicles and released into the
channel. The sheath cell has been identified
as a gland cell in other nematodes and
esterase activities have been detected in
the amphidial channel (Bird, '71 ; McLaren,
'72). Its secretions may include mucus to
protect the exposed neuron terminals or
secretions which might be involved directly
in neuron specificity or function.
The sheath cells surrounding all of the
sensilla except the amphids have striking
lamellar membrane invaginations which
increase enormously the surface area of
sheath cell membrane in contact with the
neuron channels. Such membrane specialization might be for ion uptake and secretion so that these cells could regulate the
ionic environment surrounding the sensory
neurons thus affecting their sensitivity.
The role of the socket cells appears to be
support, but they may secrete the extracellular material lining the neuron channels.
The four additional sensory processes in
the male cephalic sensilla are likely to be
chemoreceptive because they contact the
Fig. 38 Sub-dorsal and sub-lateral anterior
nerve cords. A transverse section approximately
30 p from the tip of the head. The sub-dorsal cord
is the cluster of small processes at the top left. T h e
amphidial neurons are the larger processes below
the amphidial socket cell body; the small processes
are from the other lateral sensilla and from other
lateral cells (see text). Magnification, x 13,000.
Fig. 39 Neuron cell bodies. A transverse section approximately 50 p from the tip of the head
showing some of the neuron cell bodies on the right
sub-dorsal side. mn, motor neuron. Magnification,
X 15,000.
Fig. 41 Nerve ring. A transverse section of the
right lateral and ventral regions of the nerve ring.
The small processes labeled 1 and 2 to the lower
left of center are the sub-ventral inner labial neurons as they pass posteriorly. After turning around
they become larger and are the processes with vesicles labeled 1-1 and 1-2 which are passing anteriorly. The lateral inner labial neuron 1 is also
labeled at the top of the section. The arrow shows
a synapse to a muscle process (M).T h e muscles in
nematodes receive innervation by sending processes to the nerves; the process labeled M is from the
most anterior head muscle. The synapse is recognized by its characteristic electron dense p r e
synaptic specialization. I t extends for several sections and this neuron makes four such synapses to
the muscle. Magnification, X 17,000.
333
outside directly. A likely function for such
a neuron would be to detect a sexual attractant released by a hermaphrodite. Sexual attractants have been described for
several dioecious nematode species (Greet,
'64; Green, '66; Cheng and Samoiloff, '71)
but no evidence for a sexual attractant in
C. elegans has been reported yet.
The mechanical and temperature sensitivity of C. elegans has not been studied
extensively, but one can observe easily that
a worm touched on the head backs up.
Since we do not know how mechanical deformations are propagated along the cuticle we cannot predict which sensilla mediate this response. The inner labial, outer
labial and cephalic sensilla are all candidates for mechanoreceptors because they
have neurons ending embedded in the cuticle. They might also function a s proprioreceptors since the fine structure of the
cephalic sensilla resembles that of the proprioreceptive campaniform sensilla in insects (Moran et al., '72). Isolation of mutants altered in mechanosensitivity and
identification of their anatomical defects
might help to specify the function of these
sensilla.
Comparison to nematodes and other
invertebrates
The arrangement of nematode sensilla
has been studied extensively with the light
microscope because of its taxonomic importance (Chitwood and Wehr, '34; Chitwood and Chitwood, '38; DeConinck, '65).
Chitwood and Wehr proposed that the primitive ancestral pattern of labial sense organs should consist of three sensilla per
lip plus the amphids. They could arrange
the phylogeny of various species so that it
reflected modifications of this basic plan.
They noted with chagrin, however (and
this is confirmed by DeConinck ('65), that
no nematode has been discovered with three
sensilla (excluding the amphids) on its lateral lips. Because the dorso-lateral sensilla
is invariably absent, DeConinck proposed
a different symmetry arrangement for the
ancestral nematode: an inner ring of six;
one outer ring of six; and a n outer ring of
four sensilla plus the two amphids. DeConinck proposed that the outer ring of
four reflected the bilateral symmetry of the
body rather than the hexaradiate symmetry
of the head.
334
S. WARD, N . THOMSON, J. G. W H I T E AND S . BRENNER
\
-1
c
8
c
Qp
Q
6
h
Figure 40
NEMATODE SENSORY AXATOMY
The arrangement of cephalic sensilla in
the head of C. elegans conforms to the general nematode plan with no dorso-lateral
sensillum. However, the cervical deirid is
located dorso-laterally; i t is similar in fine
structure to the cephalic sensilla and like
the cephalic neurons the deirid neurons
contain catecholamines. Therefore, the
deirid could be regarded as homologous to
the cephalic sensilla. Thus, these sensilla
would have a hexaradiate arrangement,
with the lateral member displaced, supporting the model proposed by Chitwood
and Wehr.
The structure of individual sensilla in
C. elegans resembles sensilla in other nematodes. Goldschmidt (’03) first described
that two accessory cells were associated
with most of the sensilla. Goldschmidt
found only one associated with the amphid
but this was later corrected by Hoepli (‘25).
The resolution of the electron microscope
has allowed us to describe the topology of
these cells more accurately and we have
renamed the cell which corresponds to
Goldschmidt’s Geleitezelle (accompanying
or escort cell) the socket cell, and the cell
corresponding to the Stiitzelle (support cell)
the sheath cell. One reason for introducing
this new terminology is that Goldschmidt’s
missing the Geleitezelle for the amphid has
caused a subsequent confusion in terminology for the two cells found associated with
amphids in other nematodes. In addition
the terms “accompanying” and “support”
cells have been used differently by different authors and some reviewers have confused them with labial cells.
Ciliated endings of nematode sensory
neurons have been described for several
species (for references, see McLaren, ’72)
and the complete structure of amphids from
electron micrographs has been described
recently by McLaren (’70) for Dipetalo~~
-~
~
Fig. 40 Anterior nerve cords and sensilla cell
bodies. The nerve cords and cell bodies of all cells
which have processes in the anterior nerve cords
are shown. They are drawn as if projected radially
onto a cylinder through the length of the worm and
the cylinder were cut along the rnid~dorsalline and
unrolled. The positions of the first bulb of the pharynx and the nerve ring are noted on the side. In
addition to previous labels, cells are labeled as follows: G, glia; mn, motor neuron; in, inter-neuron;
P, pharyngeal neuron. Non-neuronal cells are
shaded. Not all the cell bodies in this region are
shown. The neurons changing cords in front of t h e
pharynx bulb are m and I Ac.
335
n e m a viteae, and by Storch and Riemann
(’73) for Tobrilus aberaans. The amphid
in D. viteae is similar in structure to that
of C. elegans and has accessory cells corresponding to the sheath and socket cells.
The accessory cells appear to be present
in T . aberrans as well from the electronmicrographs shown, but they are not described as part of the amphid. In both cases,
neurons with modified cilia pass out through
a channel open to the outside. Other neurons ending in the sheath cell were not
described but are found in other nematodes
(Burr and Webster, ’71).
A s has been noted before (Lee, ’SS), the
sensilla in nematodes resemble those in
other invertebrates, especially arthropods
(e.g., Slifter, ’70; Hayes, ’71; Harris and
Mill, ’73). They all have ciliated sensory
neurons surrounded by specialized accessory cells. This similarity probably reflects
evolution of convergent solutions to the
problem of how to get a sensory neuron into
or through an exoskeleton. Nonetheless, the
possibility that the similarities in structure
reflect a common primitive invertebrate
ancestor with specialized sense organs
should not be automatically dismissed.
T h e sensory-motor neuron
The finding that all six of the inner labial
neurons 1 make direct chemical synapses
to muscle arms establishes that these cells
are sensory-motor neurons. Direct sensorymotor neurons have been postulated as intermediates in the evolution of nervous systems. Coggeshall (’71) described a possible
sensory-motor neuron in the sea-hare
Aplysia, and a possible sensory-secretory
neuron has been described in Trichuroid
nematodes (Wright and Chan, ’73). Interestingly, Goldschmidt (’08) proposed that
one of the anterior sensory neurons in
Ascaris might make a direct neuromuscular connection, but he could not be certain
of this connectivity using only the light
microscope. Our results suggest that Goldschmidt was correct so that sensory-motor
cells may be common in nematodes. Since
the nematode is a primitive invertebrate,
such cells may indeed represent an important stage of the evolution of complex nervous systems.
lnvariance and symmetry
The detailed comparison of the sensory
336
S. WARD, N THOMSON, J . G WHITE AND S. BHENNER
terminals of several worms reveals little
anatomical variation. The shape and p s i tion of the ciliated neuron endings and the
arrangement of sheath and socket cells
were identical in all animals examined. This
invariance is also reflected by the symmetry
of the sensilla. The animals are exactly bilaterally symmetric. The striking hexaradiate symmetry is not exact. The lateral pair
of each type of sensilla are distinct from the
sub-dorsal and sub-ventral pairs: the outer
labial have smaller terminals, the inner
labial have additional accessory neurons,
the “lateral cephalics,” the deirids, are
displaced caudally. In addition, the inner
labial sensilla do not have a medio-lateral
plane of symmetry because in all sensilla
the neuron 1 is most dorsal. These variations might indicate the influence of position on the development of sensilla specified
identically genetically.
Four of the accessory neurons (the ventral inner labial accessory and the neuron
m) vary both between and within animals
in the number and length of their terminal
branches. However, since the branches are
absent in juvenile animals the growth of
these processes may be age dependent and
the variations found to be due to slight differences in age of the animals sectioned.
There is also slight variation in the fine
structure within the terminals of some of
the neurons: the number of doublet microtubules in the neurons of the inner, outer
and cephalic sensilla is variable and the
presence of vesicles in the region of the
basal bodies varies from animal to animal.
This variation probably reflects the “noise
level” in the developmental pathways specifying these structures since the animals
were grown under identical conditions and
are likely to be isogenic (Brenner, ’74).
Although individual worms are not precise replicas of each other down to the finest
details they are remarkably exact copies. By
a combination of properties each individual
cell and its processes may be uniquely
identified in different animals. This is clearly essential for the ultimate interpretation
of the anatomical findings in terms of function, but it is also important because it allows small anatomical changes to be reliably recognized in sensory mutants without
complete reconstruction. A number of nonchemotactic mutants have already been
isolated (J. Lewis, personal communication
and S. Ward and P. St. John, unpublished)
and other sensory-defective phenotypes are
being sought. These will be useful to further probe the structure and function of
the nematode’s sensory nervous system.
ACKNOWLEDGMENTS
We thank Mrs. Eileen Southgate and Mr.
Paul St. John for their patient and pairstaking help following processes through
series of sections, and Mrs. Annette Lenton
and Mr. Lazlo Mesaly for preparation of
illustrations. Mrs. Susan Baker helped with
the electron microscopy. This research was
supported in part by a Special Fellowship
to S. W. from the National Institute of
Neurological Diseases and Stroke and by
Grant GB 36970 to S. W. from the National
Science Foundation.
LITERATURE CITED
Bird, A . F. 1971 The Structure of Nematodes.
Academic Press, Inc., New York.
Brenner, S . 1973 T h e genetics of behavior. Br.
Mfd. Bull., 29: 269-271.
1974 The genetics of C ~ ~ ~ ~ i r o r h d ~ t l r t r s
e k g r r n s . Genetics, 77: 71-94.
Bullock. T. H. 1965 The pseudocoeloinate phyla,
V : Nemntoda. In: Structure and Function in
t h e Nervous System of Invertebrates, I . T. H .
Bullock and G . A. Horridge. W. I!. Freeman
and Co., San Francisco.
Bullock, T. H., and G . A . Horridge 1965 Structure and Function in the Nervous Systems of
Invertebrates, 11. W. H . Freeman and Co., San
Francisco.
Burr, A . H., and J . M . Webster 1971 Morphology
of the Eyerpot and description of two pigment
granules in the esophageal muscle of a marine
nematode, Oricolcrimus wsicccrizrs. J. UltrastrucLure Iies., 3 6 . 621-632.
Cheng, K., and M . K. Samoiloff 1971 Sexual attraction in the free living nematode P ~ i ~ c ~ g r ~ l l i l s
silirsirrc’ (Cephalobidar). Canad. J. 2001.. 4 9 :
1443.
Chitwood, B. G., and M. B. Chitwood 1938 An
Introduction to Nematology. Monumental Print,
Baltimore.
Chitwood, B. G.. and E. E.Wehr 1934 The value
of cephalic structures as characters in nematode
classification with special reference to thc superfamily Spiruroidea. 2. Parasitenkunde. 7: 273%
335.
Coggeshall, R . E. 1971 A possible sensory motor
neuron in Aplysicr cc~lifomicu Tissue and Cell,
3 : 637-648.
Croll, N. A. 1970 The Behavior of Nematodes.
E. Arnold Ltd., London.
De Coninck, L. 1965 Classe des Nematodes.
Traite d e Zoologie I V (Nemathelminthes). I).
Grasse, ed. Masson, Paris.
Dusenberry, D. B. 1973 Countercurrent separ.1tion: A new method for studv behavior of small
aquatic organisms. Proc. N a t . Acad. SCI., 70.
1349-1352.
NEMATODE SENSORY ANATOMY
337
McLnren, D. J. 1970 Preliminitry obscrvations
1974 Analysis of cheinotzixis in the reon the sensory structures in adult filariae. Trans.
matode C[cc'?io,.htibrlitis c , / ( y ~ [ r ? iby
s CountercurRoyal Soc. of Tropical Medicine and flygicne,
rent selection. J. Exp. Zool., 188: 4 1 4 8 .
64 : 191-1 92.
Goldschiiiidt, R. 1903 Histologische Untersuch1972 Ultrastructural and cytochcmical
ungen an Nematoden, 1 Die Sinnesorganc von
on the sensory organellcTs and nervous
( 1 1 1 ~ t l l l f ~ ~ J ' ~ ( ' L.
~ l ltllld
~ ~ ~ A.
' . \ l l t i ' ~ ~ r l / f , ~ ~ ~ ~ ~ J / l [ , /studies
~ , .
system of Dipr~frrlo?ztwzcruttcsr/[r (Nernatod;i:FilCloq. Zool. Jahrh. Anat., 1 8 : 1-57.
ariordea). Parasitology, 6 5 . 507-524.
1908 Das Nervensysteln von Asc'rrrts
Moran, D. T.. K M . Chapman and K. A . Ellis 1971
Ittmhricoitlc~sund r n c ~ ~ ~ ~ t l o c c ~ pI .hZtsch.
t r l ~ c , Wiss.
The fine structure of cockroach c a m p m i f o r m
Zool., 90: 73-136.
sensilla. J . Cell. Biol., 4 8 : 155-173.
1909 Das Nervcnsystem von Asc'rrrr,\
Murray, R G . , and A. Murray 1970 The Anat1urnbridoitlc~a und rnc,~~~r2oc.c,ph[rl[i.
11. Ztsch.
omy arid Ultrastructure of Taste Endings. 1 1 1 .
Wiss. Zool., 92. 306-357.
Taste and Smell in Vertebrates. A CIBA FoundaGreen, C. D. 1966 Orientation of male kfc,t<zrotion Symposium. G . E. W. Wolstenholme and J .
dcrcr rostocicwsis Woll. and H. sch(rc2/~trs
schem.
Knight, eds. J. A. Churchill, London.
to the females. Ann. Appl. Biol., 58: 327-339.
Revel, J P. 1970 T h e role of the Golgi ;ippa'aGreet, G . N . 1964 Observations on sexual attractus of cartilage cells in the elaboration of matrix
tion and copulation in the nematode P r i i z w g i - o / c r r glycosaminoglycans. In. Chemistry and Molecm t i s rigidii.3. Nature, 204. 96-97.
ular Biology of the 1ntercellul;ir Matrix, 3 . E. A.
Harris, D. J.. and 1'. J. Mill 1973 The UltrastrucBalaza, 4. Academic Press, Inc., New York.
ture of a chemoreceptor s e n d l a in Cinifio ( A r t r Slifer, E. I I
1970 The structurv of arthropod
n c v t l t r , Artrchiiidtr). Tissue and Cell, 5 : 6 7 9 4 8 9 .
chemoreceptors. Ann. Rev. Ent.. 1 5 . 121-142.
Hoppli, K. 1925 Ubcr das Vordcrende der A s Storch, V . , and F. Riemann 1973 Z u r U l t r a cariden. Vergleichendr histologische Untersuchstruktur der Scitcmorgane (Amphidtw) des limen unter besonderen Berucksichtigung der Zellnischen Nematoden Tobriirs abcrrans ( W . Scheikonstanzf'l.nre. Z. Zellforsch. 2 : 1-68
der. 1925) (Ncmatoda, Enopida). Z Morph.
Horridge, C. A. 1968 The origins of t h e nervous
Tiere, 74 : 163-1 70.
system. I n : T h e Structure a n d Function of NerWard, S. 1973 Chcmotaxis by t h e nematode
vous Tissue. C: €%. Bourne, ed. Academic Press,
C[reiior)itrh~litisc/c*gr[u.\: identification of ;ittractInc., New York.
ants and analysis of the response by us<' of muHayes, W. F. 1971 Fine structure of the chemotants. Proc. Nat. Acad. Sci. USA. 7 0 . 817-821.
receptor sensillum in I ~ m t i l i i s .J Morph, 133.
Wright, K. A , . and J . Chan 1973 Sense receptors
205-240.
in the bacillary band of' trichuroid nematodes.
Lee, D. L. 1965 The Physiology of Nematodes.
Tissue and cell, 5 : 373-380.
Oliver and Boyd, London.