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589
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From The Rockefeller Institute and the Department of Zoology, University College of the West
Indies, Jamaica, British West Indies. Dr. Locke's present address is The Biological Laboratory,
Western Reserve University, Cleveland
ABSTRACT
The fine structure and the distribution of an esterase have been studied in the cuticle of
Galleria larvae, Tenebrio larvae and pupae, and in the wax-secreting cuticle of the honey
bee, and compared with those in the cuticle of the caterpillar of Calpodes. In Galleria and
Tenebrio the pore canals are spaces passing through the lamellate endocuticle from the
epithelium to the epicuticle. They contain a filament from the cells which may be con-
cerned in their formation. The shape of the pore canal is probably determined by the
orientation of the fibres making up the lamellae in the endocuticle and is not a regular
helix. The pore canals also contain numerous filaments of another sort which pass on through
the epicuticle and are believed to be the origin of the surface wax. They are particularly
abundant in the pore canals of the honey bee wax-secreting cuticle and extend into the cell
in long pockets surrounded by an envelope of the plasma membrane. The esterase is prob-
ably concerned with the final stage of wax synthesis, for its distribution is similar to that of
the lipid filaments.
INTRODUCTION
This paper is one of several upon the structure of secretes the cuticulin first and then the lamellate
the insect integument. It will be limited to a endocuticle. The exocuticle is derived later by
description of structures which may be concerned quinone tanning of the outer lamellae of the endo-
in thc synthesis and transport of cuticular waxcs. cuticle. The wax layer is one of the last layers to
Only the structure of the cuticle has been studied, appear, and the wax or its precursors must trav-
not its genesis. erse the endocuticle. Later still, the wax is
The integument in insects consists of a single covered by the cement secreted by the dermal
layer of cells and the cuticle (Fig. I A) (Wiggles- glands.
The problem is how to account for the appear-
worth, 22, 29, 31, 33, Richards, 18, 20). In most
ance of wax on the outside of the cuticulin when
integuments the cuticle is perforated by pore
it is separated from the epithelium by a solid
canals running from the epithelium up to but not
hydrophilic endocuticle. It has been supposed
through the epicuticle (Denell, 5, 6, Richards and
that the pore canals might be the pathway for the
Anderson, 21, Locke, l l ) . The wax layer in the
movement of wax (Wigglesworth and Kramer,
epicuticle is of great physiological interest, for it 36), but this has been doubted as a general expla-
determines many of the surface properties and nation, for in many insects, notably in the cater-
forms the main barrier to water loss (Wiggles- pillars of Calpodes (Locke, 14, 16) and Diataraxia
worth, 30, Beament, 2). (Way 27), wax appears outside the cuticle not
In the formation of the cuticle the epithelium penetrated by pore canals.
589
FIGURE 1A
Diagram of the main features of
the integument in an insect. The
terminology is the same as that
used in the text.
FmURE 1B
Diagram of the structures be-
lieved to exist in the epicuticle.
The terminology is the same as
that used in the text. The wax
canal filaments may appear tu-
bular or filamentous.
590 THE JOURNAL OF BIOPHYSICAL .~NI) BIOCItEMICAL CYTOLOGY • VOLUME 10~ 1961
FIGURE
D i a g r a m of the structure of the abdominal cuticle in Galleria larvae. T h e inset shows the regions
which can be distinguished in the epicuticle. I n addition to the regions shown a thick lipid layer is
known to be present at the surface.
592 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CrrOLOGY • VOLUME 10, 1961
Tenebrionidae), the meal worm. Tenebriowas selected The Pore Canals and Their Contents: The pore
to represent a commonly occurring hard type of canals are nearly always cut obliquely, appearing
cuticle. (3) The wax-secreting cuticle from the ab- as clear crescentic areas. They are found through-
dominal sternites of the honey bee, Apis mellifica out the cuticle between the epithelial cell layer
(tlymenoptera, Apidae). This last cuticle was chosen
and the epicuticle, and their typical arrangement
because any structures within it concerned with wax
is shown in Fig. 4. They terminate below the thin
secretion would be expected to be hypertrophied.
epicuticle between the rugosities, but where the
epicuticle is much thicker over the top it is pene-
RESULTS
trated by a number of finger-like spaces more or
The Cuticle in Galleria Larvae (Fig. 2) less circular in cross-section and continuous with
The main features are seen in Fig. 5. The cuticle the pore canals in the endocuticle.
is rugose over most of the larva. Each rugosity has Within each pore canal is a filament about 150
a deep root of dense material extending into the to 200 A in diameter. It originates beneath the
endocuticle, and is capped by a diffuse mass of plasma membrane of an epithelial cell and may
melanin above which the epicuticle is much extend to the peripheral end of the pore canal.
thickened. In insects in which the integument does Near the surface the pore canals contain numer-
not stretch between instars the exocuticle is a con- ous filamentous structures of a different sort about
tinuous layer, but in Galleria the cuticle stretches 80 A in diameter. These filaments emerge from
during growth and the endocuticle is only rein- the pore canals and pass through nearly all of the
forced to become exocuticle in the bases of the epicuticle (Fig. 8). They do not appear to be con-
rugosities. tinuous with the pore canal filaments. They
The endocuticle is made up of 10 to 30 lamellae resemble the structures termed wax canals and
about 0.5 to 1 # apart, the larger number being their contents, the wax canal filaments, which
found later in the instar. The lamellate appearance pass through the epicuticle in Calpodes. In Calpodes
is due to the arrangement of fibrils. These have the wax canals were inferred to be the route by
not been satisfactorily resolved but they give the which wax reaches the surface. Their nature is
impression of an arrangement as in Fig. 3. The discussed below.
endocuticle next to the epithelium does not have The EpicutMe: Six regions can be distinguished.
this ordered fibrillar structure. The fibrillar On the very outside is an irregular "fluffy" layer.
structure has already been briefly reported for This might be contamination from the food of the
Calpodes larvae (Locke, 16). larva but it will be referred to as the " c e m e n t "
Pore canals extend through the endocuticle. (Fig. 2, I) although only future work can tell
Some also pass through the dense roots into the whether or not it is the secretion of the dermal
thickened epicuticle above. (Verson's) glands. It resembles the cement in
The Epithelium and the Inner Surface of the Endo- Rhodnius (Locke, 11) and Calpodes (Locke, 16)
cuticle: Schmidt (22) has described a layer stain- both in position and texture. In carbon replicas
ing intensely with aldehyde fuchsin between the prepared from the first collodion stripping, similar
epithelium and the endocuticle in a number of " d i r t " is always present. Below this is a dense,
insects. This corresponds to the layer of cuticle well defined layer of very uniform thickness
without fibrils which separates the lamellate (about 60 A) (Fig. 2, H). There is good physiologi-
endocuticle from the epithelium. The fibrils from cal evidence that one lipid layer at the surface of
the innermost lamella can be seen to end in this the epicuticle is a well ordered monolayer or
amorphous layer. Closer to the epithelium the bimolecular layer (Beament, 3). Such a layer, if
texture changes and becomes granular (Fig. 1 I). fixable by OsO4, would show as a thin dark line,
The epithelial edge is covered with rather short and this layer (Fig. 2, H) is, therefore, tentatively
finger-like processes, some of which are longer homologised with the oriented lipid layer inferred
and contain the pore canal filament penetrating from physiology. Favouring this hypothesis,
the endocuticle (Fig. 10). In all insects examined, layer II and the cement are the only layers of the
the plasma membranes of the epithelium below epicuticle not penetrated by the wax canals. Also
the cuticle have been found to have desmosomes supporting this interpretation is the uniform clear
similar to those described in Hydra (Wood, 37). layer (about 80 A thick) (Fig. 2, III) below it.
FIGURE 5
Electron micrograph of a thick transverse section to show the m a i n features.
Ep, epicuticlc; End, endocuticlc; Epith, epithelium.
( O s m i u m tctroxide, Araldite). X 3,200.
FIGURE 6
Photomicrograph of 2 ~ section after incubation in 5-bromoindoxyl acetate to demon-
strate esterase. Nearly all the black part in this picture is due to the indigo produced.
(cf. Fig. 36.)
Phase contrast. X 1,500.
FIGURE 7
Surface view of whole m o u n t of the epicuticle treated as in Fig. 6 and showing the
esterase localised most densely in the pore canals.
Phase contrast. X 1,500.
594 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
M. LOCKE Pore Canals and Related Structures 595
FIGURE 8
Electron micrograph of a transvcrsc sectlon through the cpicuticle bctwccn two rugosltics.
m, melanin; d, dcnsc layer; c, cuticulin; era, ccmcnt; pc, pore canal containing wax canal filamer
ol, oriented lipid layer.
(Osmium tctroxidc, Araldltc). X 50,000.
FIGURE 9
Diagram of the structure of the tergal
cuticle in Tenebrio larvae. In some pore
canals the porc canal filament extends
almost to the epicuticle.
These layers ( H and III) m a y well be p a r t of a Tenebrio Pupal Cuticle (Thoracic and Abdominal
lipid protein complex of the sort studied by Tergites 48 Ilours after Moulting)
Stoeckenius (24), but for the m o m e n t they will be
referred to as the oriented lipid layer. Below M a n y of the features described in Galleria can be
layer III is a thicker, less regular, dense layer seen in the survey picture (Fig. 13): epithelium
with finger-like processes, Schmidt's layer, a wide
(Fig. 2, IV) which m a y have been secondarily
derived from the thick (0.2/~) homogeneous dense lamellate endocuticle with pore canals cut
layer below (V) which makes u p the bulk of the obliquely, b u t with a smoother a n d more uniform
epicuticle. Layer IV is p r o b a b l y the homologue of epicuticle.
T h e texture of Schmidt's layer distinguishes it
the cuticulin layer in Calpodes. Layer V is greatly
very sharply from the lamellate endocuticle
thickened to form the caps to the rugosities. T h e
(Fig. 12). T h e granularity has an orientation
n o n - c o m m i t t a l term dense layer was coined for it
similar to t h a t in the lamellae a n d usually the
in Calpodes. W i t h i n it there is a region (VI) with a
pore canals already have the crescentic cross sec-
looser texture containing islands of layer V where
tion characteristic of the endocuticle. Sometimes
the pore canals end a n d the wax canal filaments
the pore canal filament traverses this layer without
ramify.
any space a c c o m p a n y i n g it. In Calpodes the p u p a l
The Distribution of Esterase: Figs. 6 a n d 7 show cuticle is very like t h a t in Tenebrio, a n d Fig. 15 of
the distribution of esterase. T h e pore canals show
Calpodes shows two well defined pore canal fila-
u p clearly close to the epicuticle a n d a r o u n d and ments which are not surrounded by a discrete pore
within the roots. T h e epicuticle is also strongly canal until they reach the endocuticle.
coloured. T h e failure of the inner ends of the pore T h e pore canals contain wax canal filaments
canals to colour in this sort of cuticle is p r o b a b l y over a m u c h greater p a r t of their length t h a n in
not an artefact. In the h a r d brown sclerite at the Galleria. T h e epicuticle is exceedingly h a r d a n d
base of the leg the cuticle is different a n d the pore all the detail seen in Galleria could not be resolved
canals contain a n esterase t h r o u g h o u t their in the sections obtained, b u t the general p a t t e r n
length. These results are summarised in Fig. 36. is similar. T h e pore canals t e r m i n a t e below a n d
FIGURE 11
A thicker section from the region between the cells a n d the endocuticle showing how
the oriented fibrcs of thc cndocuticle merge into the poorly oriented g r a n u l a r layer
above the finger-like projections from the cells. T h e endocuticle has very little con-
trast, and fibre orientations arc difficult to make out on thin sections.
( O s m i u m tctroxide, Araldite). X 50,000.
598 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
M. LOCKE Pore Canals and Related Structures 599
Electron Micrographs of Tenebrio Larval and Pupal Cuticle
FIGURE 1~
Slightly oblique transverse section of Tenebrio p u p a l cuticle in the region between the
cell a n d the endocutiele. Note the crescent s h a p e of t h e pore canal space even in t h e
poorly oriented g r a n u l a r layer above the finger-like projections from the cells, a n d the
contrast in textures between endocuticle a n d the g r a n u l a r layer.
pcf, pore canal filament; pc, pore canal; end, endocuticle; g, g r a n u l a r l a y e r - - e n d o -
cuticle in the process of formation; d, desmosome.
( O s m i u m tetroxide, Araldite). M 50,000.
FIGURE 13
T r a n s v e r s e section of p u p a l cuticle to show the m a i n features, cf larval cuticle in
Fig. 14.
ep, cpicuticle; end, cndocuticle; pc, pore canals; g, g r a n u l a r layer cndocuticle in the
process of formation.
( O s m i u m tetroxide, Epon). X 2,500.
FIGURE 14
Transverse section of larval cuticle to show t h e m a i n features. T h e pore canals are
smaller t h a n in t h e p u p a ; there is a n exocuticle, a n d the region with ramifying w a x
canals is m u c h wider.
ep, epicuticle; ex, exocuticle; end, endocuticle.
( O s m i u m tetroxide, Epon). X 2,500.
FIGURE 16
Slightly oblique tangential section of Tenebrio larval cuticle showing the finger-like
proccsscs of the cells on t h e right a n d the innermost, poorly ordered, g r a n u l a r layer of
the cuticle on the left. Some of the cellular processes extend into this layer carrying the
pore canal space with t h c m .
pc, incipient pore canal space.
( O s m i u m tetroxide, Epon). X 42,000.
FIGURE 17
T a n g e n t i a l section just above t h a t of Fig. 16 showing well defined pore canals with
axial filaments a n d g r a n u l a r contents. C o m p a r e the dense h o m o g e n e o u s texture of this
fully f o r m e d endocuticle with the g r a n u l a r precursor in Fig. 16.
pcf, pore c a n a l filament.
( O s m i u m tetroxidc, Epon). X 32,000.
602 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
Tenebrio Larval Cuticle
FIGURE 18
Electron micrograph of a tangential section about a/4 of the way through the cuticle
from the epithelium; i.e. well above that of Fig. 17. I n this example no pore canal fila-
ments are present (cf. Fig. 26) but the pore canals contain n u m e r o u s wax canal fila-
ments. T h e pattern made by the pore canals and their shape in transverse section
suggest that the lamellae of the endocuticle are made up as in Fig. 3, but the cuticle
never appears fibrous as in Galleria (cf. Fig. 4).
( O s m i u m tetroxide, Epon). X 35,000.
FIGURE 19
Photomicrograph of 1 ~ tangential section rather below that of Fig. 19, incubated in
5-bromoindoxyl acetate. T h e pore canals react intensely for esterase and all the density
in this picture is due to the deposit of indigo.
Phase contrast. X 2,600.
FIGURE ~0
Electron micrograph. One pore canal from Fig. 18 enlarged to show the wax canal
filaments. X 100,000.
FIGURE ~1
Photomicrograph of 1/z transverse section of cuticle from an intersegmental m e m b r a n e
incubated in 5-bromoindoxyl acetate. Nearly all the density in this picture is due to
esterase. It is most marked in the cells, pore canals near the epicuticle, and at the base
of the wedges of exocuticle.
Phase contrast. X 1,500.
FIGURE ~
Photomicrograph of 1 ~ transverse section of tcrgal cuticle treated as in Fig. 21 to
show the presence of esterase. The reaction is most m a r k e d in the cells and throughout
the length of the pore canals.
Phase contrast. X 1,500.
FIGURE ~5
T a n g e n t i a l section j u s t below t h a t in Fig. 24 showing the j u n c t i o n between the lamel-
late cuticle with pore canals on the left a n d the i n n e r m o s t epicuticle with r a m i f y i n g
w a x canals on the right. J u s t below the epicuticle the w a x canal filaments are ar-
r a n g e d in a ring a r o u n d the periphery of each pore canal.
( O s m i u m tetroxide, Epon). X 24,000.
FIGURE ~6
T a n g e n t i a l section just below the epicuticle, i.e. above t h a t in Fig. 18, showing three
pore canals in transverse section with the w a x canal filaments a r r a n g e d in a ring at t h e
periphery a n d the pore canal filament in the center. T h e pore c a n a l filament is n o t
always present in this region (cf. Fig. 20).
( O s m i u m tetroxide, Epon). X 100,000.
606 THE JO~NAL OF BIOPHYSICAL _END BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
M. LOCKE Pore Canals and Related Structures 607
the wax canals pass through the epicuticle, which electron microscope study of thick sections of
has a thin, very dense outer cuticulin layer (Fig. cockroach cuticle (Richards and Anderson, 21),
2, IV) and a broader, homogeneous, dense inner and in Rhodnius the crescentic holes seen in cross
region (Fig. 2, V). section were taken to confirm their helical nature
The distribution of esterase was similar to that (Locke, 11). However in the cuticles examined in
of the Tenebrio larval cuticle described below. this paper, another shape is more probable and the
hypothesis that pore canals are usually helical may
The Tergal Cuticle in Tenebrio Larvae, 24 to 36 have to be reconsidered.
Hours after Moulting (Fig. 9) In many sections some pore canals have a
circular outline (Fig. 26). The basic shape is prob-
The main features are shown in the survey pic- ably that of a cylinder bent in a helix or in some
ture (Fig. 14). The epicuticle is wider than in the other way. If the pore canals are indeed helical,
pupa, due mainly to the thicker dense layer (V) the shape and the orientation of the holes seen in
containing the ramifying wax canals. The outer cross section vAll be influenced by two main con-
lamellate cuticle ( = exocuticle) is hard and dense, siderations: (s) the phase relations of different
although with the light microscope it is not much helices, and (2) the plane of the section. The shape
darker in colour than the endocuticle. Details of the tube and the pitch and regularity of the
are shown in the slightly oblique tangential sec- helix do not affect the following conclusion.
tions cut at different levels (Figs. 16-18, 20, 24- (1) The Phase Relations of D(fferent Helices: The
26). orientation of the crescentic holes seen in section
The epithelium has many finger-like processes is influenced by their position relative to each
probably correlated with the secretion of endo- lamella. In any one plane the outline of a pore
cuticle taking place at this time (Fig. 16). A few canal has approximately the same orientation as
of the processes taper off and pass into Schmidt's that of any other pore canal in a similar position
layer surrounded by a pore canal space. In the with respect to the lamella (Fig. 18). Whether or
lower endocuticle the pore canals have a denser not they are helices, therefore, they are in phase,
lining and contain a single pore canal filament and the pattern repeats itself in each lamella.
150 to 200 A in diameter, together with small (2) The Plane 6f the Section: If a number of
irregular granules (Fig. 17). As in Galleria the pore helical tubes are cut in transverse section, then
canal filament may extend throughout the length usually the crescentic holes observed would have a
of the pore canal. random orientation as in Fig. 23A. Even if they
In about their distal third, the pore canals con- are all in phase, the crescents will have all orienta-
tain bundles of 4 to 20 wax canal filaments 100 to tions except in the rare sections exactly in the
130 A in diameter (Figs. 18, 20). In the lamellae plane of the lamella, or, when all the pore canals
just below the epicuticle, the wax canal filaments
are arranged in a ring around the inside of the
tube (Fig. 26). The pore canal stops at the junction
with the epicuticle and the wax canals form an a ¢" ~ . • • ) " ,~ ( ) , ~-', ,~ f r ~ ~f -~ ] ~ tr
irregular feltwork (Fig. 25). Above the feltwork
the wax canals are arranged perpendicular to the (-
e" ., .,
I J
,. ,,
~
g
I --
P:
¢
r"
-- i"
.
surface before passing through the thin dense
layer of cuticulin (IV). Fig. 24 is the best evidence
obtained that the cuticulin layer (IV) is penetrated
by the wax canals. There is no loose-textured
cuticle with intertwining wax canal filaments A B
comparable to layer VI in Galleria. A cement layer
is present but the oriented lipid layer (H and III) FIGURE ~3
seen in Galleria could not be resolved. A. The arrangement exFected for helical Fore
The Sha~e of Pore Canals." In light microscope canals when cut transversely (i.e. tangential to the
studies (Richards, 18), pore canals from several surface).
B. The arrangement of pore canals found in
insects have been described as helically coiled. slightly oblique tangential sections. (cf Figs. 1.
Helical pore canals were also reported in an 10, and 14).
608 THE JOURNAL OF BIOPHYSICAl. AND BIOCHEMICAL CYTOLOGY " VOLUME 10, 1961
FmURE ~7 Diagram of the structure of the honey bee wax-secreting cuticle.
are equidistant from one another, in sections cut phase contrast, b u t after the esterase reaction they
in the set of planes parallel to the pitch of the helix. show u p clearly t h r o u g h o u t their length (Figs. 19,
M a n y sections have been cut in all planes b u t the 22). In the intersegmental m e m b r a n e s the endo-
orientation expected for helices has not been seen. cuticle is irregularly h a r d e n e d in wedges in m u c h
T h e usual orientation is as in Fig. 23B. the same way as in Galleria, a n d as in Galleria the
T h e most p r o b a b l e explanation is t h a t the pore esterase is very marked in the pore canals just
canals follow the a r r a n g e m e n t of fibres in the below the epicuticle (Fig. 21). T h e epicuticle itself
lamellae, the hole taking a curved course repeated does not color. This could be due to lack of pene-
in the same plane in each lamella. T h e shape a n d tration r a t h e r t h a n absence of esterase, as the loose
orientation of the pore canals seen in some sections textured epicuticle (VI) seen in Galleria is absent.
can only be explained in this way. I n some sec-
tions the holes are symmetrical ellipses (Fig. 35), Honey Bee Wax-Secreting Cuticle (Fig. 27)
the long axis of each hole being parallel to its
neighbours. O n the sternites of a b d o m i n a l segments 4 to 7
Helical canals, if they occur, can readily be are paired polygonal areas of thin, clear, light
explained by this observation t h a t a pore canal yellow cuticle t h r o u g h which the wax is secreted
follows the fibre p a t t e r n of the lamellae. A helical as a viscous fluid, h a r d e n i n g on the outside as a
pore canal would result if the fibre p a t t e r n were to thick scale which is later removed a n d m a n d i b u -
rotate a b o u t an axis n o r m a l to the surface, from lated to form the c o m b (Dreyling, 8).
one lamella to the next. This p a t t e r n has not been Figs. 28 and 34 show the m a i n features of this
seen in the insects studied b u t it m a y occur. cuticle including the outer p a r t of the epithelium.
The Distribution of Esterase: In unstained cuticle T h e r e are no dermal glands in adult bees (Schnelle,
the pore canals are difficult to make out even with 23) a n d certainly none in the wax-secreting
FIGURE ~9
Photomicrograph of transverse l # s c c t i o n of cuticle a n d epithelium at the height of
wax secretory activity. T h e pore canals show up as dark lines t h r o u g h the cuticle
below which there is a distinct layer before the cell m e m h r a n e . T h e bundles of wax
canal filaments extend deeply into the cell and between the cell membranes.
n, nucleus; c, cuticle; b, bundles of wax canal filaments; w, wax canal filament layer;
l, layer of wax canal filaments between cells.
( O s m i u m tetroxide and ethyl gallate).
Phase contrast. X 1,200.
FIGURE 30
Photomicrograph of a 1 /z tangential section of the cells just below the cuticle at a stage
similar to Fig. 29. T h e bundles of wax canal filaments between and projecting into the
cells show u p darkly.
( O s m i u m tetroxide and ethyl gallate).
Phase contrast. X 1,500.
FIGURE 31
Photomicrograph of a 1 # transverse section of cuticle and epithelium not at the
height of secretion incubated with 5-bromoindoxyl acetate. T h e csterase is located
t h r o u g h o u t the cell and to some extent on the inner face of the cuticle.
Phase contrast. X 1,500.
610 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
M. LOCKE Pore Canals and Related Structures {ill
Electron Micrographs of Honey Bee Wax-Secreting Cuticle
FIGURE 3~
Tangential section t h r o u g h part of a cell showing bundles of wax canal filaments sur-
i o u n d e d by an envelope of plasma m e m b r a n e a n d desmosomes between two cells, b,
bundles of wax canal filaments; m, mitochondria; tr, tracheole; d, desmosomes, n,
nucleus.
( O s m i u m tetroxide, Epon). ;< 44,000.
FIGURE 33
Enlargement of three bundles of wax canal filaments from Fig. 28. The filaments a p p e a r
tubular.
(Permanganate, Araldite). X 100,000.
FIGURE 34
Transverse section t h r o u g h the outer part of the cuticle showing the outermost oriented
lipid layer, the cuticulin layer and wax canal filaments within pore canals in longi-
tudinal section.
ol, oriented lipid layer; c, cuticulin.
( O s m i u m tetroxide, Araldite). X 67,000.
FIGURE 35
Pore canals in transverse section. T h e pore canals are filled with dense contents, some
of which seem to be the cross sections of tubes with a b o u t the same dimensions as the
wax canal filaments seen in the cells.
( O s m i u m tetroxide, Epon). X 68,000.
612 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961
M. LOCKE Pore Canals and Related Structures 613
TABLE I
Approximate diameter
Insect Cement Oriented lipid Cuticulin Dense layer of wax canal filaments
616 T h E JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY " VOLUME 10, 2961
a n d alcohols (Richards, 18) which do not fix p r o t e i n / e n z y m e template shaped in the form of a
readily in osmium tetroxide. T h e layer of wax on tube which determines the shape of the wax a n d
the outside of most cuticles is relatively thick penetrates the epicuticle. T h e t r u t h m a y be
(0.1 to 1 /~, Beament, 2) b u t it does not survive in between these alternatives, the wax molecules
osmium-fixed material, n o r are the wax scales of aggregating in this m a n n e r u n d e r the particular
the honey bee m a d e insoluble by osmium te- conditions of solution of protein, enzymes, wax,
troxide like the whorls of filaments within the cells a n d its precursors. This can only be d e t e r m i n e d by
(comb wax is fixed by o s m i u m tetroxide however). future in vitro experiments a n d by a study of the
It is, therefore, by no means certain t h a t the con- genesis of the epicuticle. For example, are the
tents of the holes in the epicuticle are the same as holes t h r o u g h the epicuticle formed by depositing
the surface wax. T h e r e are three possibilities: cuticular material a r o u n d pre-existing lipid fila-
(a) T h e surface wax, even t h o u g h unfixed w h e n ments or does the hole form first a n d fill u p later
dry in bulk, m i g h t yet take u p osmium w h e n in a with wax?
particular physical state, for in potassium lino- T h e p r o b l e m of m o v e m e n t of wax across the
lenate emulsions the o s m i u m t h a t gives contrast in epicuticle remains. If the filaments are wax micelles
the microscope m a y be a deposit of reduced freely ending below the inner surface of the epi-
osmium between the hydrophilic surfaces of the cuticle, there seems no cause for the wax to flow
layers (Stoeckenius, Schulman, a n d Prince, 25). outwards.
(b) T h e contents of the holes m a y be a n unsatu-
rated precursor which undergoes a further chemi- I am very grateful to The Rockefeller Institute for
cal change at the surface. (c) T h e holes contain the the privilege of being a Guest Investigator there
surface wax mixed with a n u n s a t u r a t e d lipid which during the summer of 1960. I particularly thank
is either volatile or similar to the thin lipid layer Dr. K. R. Porter and Dr. G. E. Palade for their
(H) on the outside of the epicuticle. hospitality, and to them and other members of their
department I extend my warmest thanks for many
T h e distribution of esterase suggests t h a t at
helpful discussions. I particularly acknowledge my
least the final step in the synthesis of wax takes
debt to Dr. D, S. Smith for his technical advice and
place in or near the spaces where the filaments are Miss S. Walser for photographic assistance. I am
found. T h e crux of the p r o b l e m is w h e t h e r newly happy to acknowledge a grant from the Rockefeller
synthesized wax can form thread-like micelles Foundation which made this work possible.
spontaneously or w h e t h e r there is a pre-existing Receivedfor publication, February5, 1961.
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618 THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 10, 1961