A Comparative Study of the Chilopod and Diplopod
Cuticle
By GORDON BLOWER
(From the Department of Zoology, Victoria University of Manchester)
SUMMARY
I . The histology of the cuticle and epidermis of certain chilopods and diplopods
is described. Two principal layers of the cuticle are recognized, an outer homogeneous
and refractile exocuticle which is usually but not invariably pigmented and an inner
endocuticle.
2. The endocuticle and the exocuticle both contain chitin. The exocuticle is considered as a modification of the outer part of the chitinous matrix by an impregnating
substance.
3. Certain properties of the impregnating substance are described. It appears to
be a substance rich in phenolic groups, perhaps a protein, which has a stability and
resistance to acids in its own right irrespective of the presence of pigment or aromatic
cross-links. Pro-sclerotin is suggested as a name for this substance. Chemical tests
show that it is present in regions not optically definable as exocuticle.
4. The epidermis is virtually an epithelium of gland cells which appear to secrete
lipoid material. The lipoid passes on to the surface of the cuticle by means of ducts
passing through the cuticle. Here it appears to form a superficial layer and to impregnate the sclerotin and pro-sclerotin.
5. There appears to be an intimate association of lipoid with the aromatic groups
of the pro-sclerotin and sclerotin. Destruction of the aromatic groups by means of an
oxidizing agent appears to intensify the colouring of the lipoid by sudan.
6. The myriapod cuticle is shown to have many features in common with that of
other arthropods. The main difficulty in the way of extensive homology with other
arthropod types is the absence in myriapods of an outer non-chitinous and resistant
layer.
CONTENTS
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INTRODUCTION
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MATERIAL AND M E T H O D S
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T H E T W O PRINCIPAL LAYERS O F T H ECUTICLE AND THE VARIATION I N THEIR F O R M AND
EXTENT
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STAINING REACTIONS .
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T H E P H E N O L I C SUBSTANCE IMPREGNATING THE CHITINOUS M A T R I X
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ARGENTAFFIN MATERIAL I N THE CUTICLE
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T H E EPIDERMAL G L A N D CELLS AND T H EOCCURRENCE O F L I P O I D I N T H ECUTICLE
PORE CANALS
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RESUME AND DISCUSSION
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ACKNOWLEDGEMENT
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REFERENCES
[ Q u a r t e r l y J o u r n a l of M i c r o s c o p i c a l S c i e n c e , V o l . 9 2 , p a r t 2 , J u n e
161
1951.]
142
Blower—A Comparative Study of the
INTRODUCTION
D
URING recent years there has been much interest in the integument
of certain arthropods. A large part of the work in this field has been
done on the insect cuticle and the present state of our knowledge of this
subject has been reviewed in a recent article by Wigglesworth (19486).
The integument in other classes of arthropods has not been neglected,
however. Beginning with Yonge's (1932) suggestion that the two principal
layers in Homarus are common to all arthropods, there has been much discussion as to the probable homology of the described conditions in different
arthropods. Pryor (19406) pointed out the chemical similarity of the 'cuticle'
of Homarus with the epicuticle in insects. Dennell (1946, 1947a and b) made
an extensive comparison of insect and crustacean cuticles and saw a considerable correspondence in detail between the fundamental layers of the cuticle
in these two groups.
Apart from Browning's paper on the cuticle of Tegenaria (1942), a description of the epicuticle in ticks (Lees, 1947) and a paper by Langner (1937) on
the cuticle of diplopods, arthropods other than insects and Crustacea have
received little attention. It is the aim of the present paper to illustrate, by
selecting a few clear-cut histochemical reactions, the general features which
have emerged from an extensive study of the cuticle in various chilopods and
diplopods and which may contribute towards a clearer understanding of the
fundamental features of the arthropod exoskeleton.
In this paper the word myriapod is used without classificatory significance,
as a convenient term covering both chilopods and diplopods.
MATERIAL AND METHODS
The investigation has been carried out on British species of centipedes and
millipedes. Animals were kept alive in glass vessels in the laboratory. The
following of the larger species have been used for detailed examination:
Chilopoda—Lithobius forficatus (L.), Haplophilus subterraneus (Leach); Diplopoda—Schizophyllum sabulosum (L.), Tachypodaiulus niger (Leach).
Most histochemical tests, unless otherwise stated, have been performed on
frozen sections, cut at 20 ft, of material fixed in 5 per cent, formaldehyde in 0-9
per cent, saline. Fixed material was impregnated with 25 per cent, gelatine solution and the blocks hardened in 5 per cent, formaldehyde (see Carleton, 1938).
Paraffin sections have been used for a study of the histology of the epidermis
and the staining reactions of the cuticle. For chilopods, Flemming without
acetic in 0-9 per cent, saline proved the best fixative. Heidenhain's 'Susa'
was also found useful. For diplopods, fixation by neutral formaldehyde (5 per
cent.) followed by decalcification with a 30 per cent, aqueous solution of
sodium hexametaphosphate (Wilks, 1938) provided the best pictures of the
epidermis. The presence of calcium salts in the cuticle precluded the use of
an acid fixative such as Flemming, although fairly good results were obtained
with Susa which also rendered subsequent decalcification unnecessary.
Chilopod and Dipbpod Cuticle
143
Material for paraffin sections was embedded by the dioxane technique
(Carleton, 1938). Epidermal pigment in the diplopod integument was removed
by immersing sections for 24 hours in ethylene chlorhydrin (see Lea, 1945).
Mallory's triple stain and iron haematoxylin, both alone, and when combined
with ponceau acid fuchsin and light green (Masson's trichrome) have been
the most useful stains.
T H E T W O PRINCIPAL LAYERS OF THE CUTICLE AND THE VARIATION IN THEIR
FORM AND EXTENT '
In sections of the cuticle of any chilopod or diplopod two layers are
optically discernible. The inner layer is colourless and is horizontally
striated. The outer layer is homogeneous, that is to say, not obviously
horizontally striated, is highly refractile, and is usually pigmented in part
or in the whole of its thickness. In this work the terms endocuticle and
exocuticle have been applied to the optically distinguishable inner and outer
layers respectively.
In a chilopod the exocuticle varies in its form and thickness in different
regions of the body (fig. 1, A and B). The exocuticle of the sclerites is thicker
than that of the arthrodial membranes. In the sclerites of Lithobius (fig. IA)
there is an exocuticle occupying from a quarter to a fifth of the total thickness
of the cuticle whereas the arthrodial membrane exocuticle is very thin and
lobulated. The sclerite of Haplophilus (fig. IB) is in marked contrast to that
of Lithobius; the exocuticle is here produced inwards in the form of cones.
These cones appear to occupy the field of activity of individual epidermal
cells as manifested by the hexagonal areas which are seen in surface views of
the cuticle. This prismatic arrangement is also manifest at the external face
of the cuticle by shallow convexities. Although the hexagonal areas can be
seen in surface views of the cuticle of Lithobius, no evidence of the individual
'cell-prisms' is to be seen in section. The arthrodial membranes of Haplophilus are intucked and the exocuticle from the two contiguous sclerites
gradually decreases in thickness towards the membrane, where it is not
developed at all. The region of transition from sclerite to arthrodial membrane
has a characteristic exocuticle and is here termed the 'intermediate sclerite'
condition. The inner face of the exocuticle in this region is produced inwards
as minute conical papillae (see fig. 1, A and B—'intermediate sclerite').
The pleural regions of Lithobius and Haplophilus are quite different. In
Lithobius there is very little sclerotization, the majority of the pleuron cuticle
being in the condition of arthrodial membrane whereas in Haplophilus there
is a series of pleurites developed in the pleuron, each separated from the next
by an intucked arthrodial membrane. This difference and the difference in
the form of the cuticle as a whole is characteristic of the orders to which
Lithobius and Haplophilus belong. The intersegmental membranes of both
animals are usually in the condition of 'intermediate sclerite'.
In section the outer surface of the sclerites of Lithobius and Haplophilus
144
Blower—A Comparative Study of the
appears to be bounded by a thin colourless membrane. This is in all probability a diffraction effect since a similar appearance characterizes the inner
edge of the endocuticle when this is separated from the epidermis during
tergibe
scleribe
arbhrodial
membrane
inbermediabe
scleribe'
pleuribes
stemibe
pleuro-tergal
arch of
mebazombe
arbhrodial membrane
sssssso—
scleribe k
-inbersegmental
membrane
pleuro-berqal
'arch of s
prozonite
sbernibe
FIG. I . Transverse sections of the cuticle, diagrammatized from free-hand drawings. A, Lithobius forficatus. B, Haplophilus subterraneus. c, Schizophyllum sabidosum. Exocuticle black,
endocuticle shaded.
sectioning. Verhoeff (1925) and Fuhrmann (1921) describe three cuticular
layers in a chilopod. The outermost layer in each case undoubtedly refers to
that which has just been described as a diffraction effect. The histological
validity of this outer layer will be discussed later. In the meantime a twolayered cuticle will be considered as a working hypothesis and the following
synonyms may be tentatively tabulated:
Chilopod and Diplopod Cuticle
Verhoeff
Fuhrmann
Oberflachenschicht
Grenzhautchen
Farbschicht
Lammellenschicht
Aussenlage
Innenlage
145
This work
Regarded as a diffraction effect
Exocuticle
Endocuticle
The exocuticle of the diplopods Schizophyllum and Tachypodoiulus does
not differ perceptibly in different regions of the body (fig. ic), for here it is
the presence of a calcined outer endocuticle which distinguishes a sclerite
from an arthrodial membrane, where the endocuticle is very thin or absent
altogether. The inner surface of the exocuticle is produced inwards as
numerous minute papillae as in the intermediate sclerite of a chilopod. The
sclerite endocuticle can be divided into an inner part with conspicuous horizontal striae and an outer part where the striae are not so obvious (fig. ic).
Langner (1937), besides two layers of endocuticle, distinguishes two outermost layers, an exocuticle and an overlying epicuticle in several species of
diplopod. Cloudsley-Thompson (1950) also describes an epicuticle as distinct
from an exocuticle in certain centipedes and millipedes. Even so, a twolayered cuticle will be taken as a basis for description and the presence or
absence of an epicuticle overlying the exocuticle will be discussed later.
STAINING REACTIONS
In the unhardened fore-gut cuticle of Homarus (Yonge, 1932) and that of
the larva of Sarcophaga (Dennell, 1946) there is a close correspondence of
staining reaction, and Dennell suggests, as did Yonge, that the red- and bluestaining layers differentiated by Mallory's triple stain are probably fundamental to all arthropods.
As the cuticle of the last larval instar of Sarcophaga is converted into the
puparium, the inward extension of the hardening process modifies the staining
reaction (Dennell, 1947a). The outer red-staining region loses its affinity
for the stain and develops an amber-brown colour as also does the outer part
of the endocuticle. The puparium cuticle, however, stains red at the junction of
the tanned and untanned regions. The red-staining zone is pushed inwards,
as it were, by the tanning of the outer layers.
Sections of the myriapod cuticle stained with Mallory show an outer layer
stained with acid fuchsin and an inner layer stained with aniline blue. The
red-staining layer has been 'pushed inwards', however, to various depths,
according to the extent to which the outer layers have been involved in the
hardening process. In the sclerite cuticle of Schizophyllum and Tachypodoiulus
the exocuticle stains red except for a very thin outermost layer and the whole
of the endocuticle stains blue—the inner endocuticle staining much more
deeply than the outer. In Haplophilus the exocuticle stains red except for the
outermost region of the sclerite. The cones do not stain so deeply as the
continuous part of the exocuticle. In Lithobius none of the sclerite exocuticle
stains red but practically the whole of the sclerite endocuticle takes the acid
146
Blower—A Comparative Study of the
A
exocubicle
basiphil zone
beneath exocuticle
gland ducb
endocubicle
gland cell
nucleus o f
epidermal cell
basemen b
membrane
ouber non-staining
parb of exocuticle
papillabe part
of exocuticle
gland ducb
endocubicle
gland cell
epidermal cell
exocuticle
(viewed obliquely)
gland ducb
endocuticle
gland ceil
epidermal cell
FIG. 2. Transverse sections of the cuticle of Lithobius forficatus. A, Sclerite. B, 'Intermediate
sclerite'. c, Arthrodial membrane. Camera lucida drawings. Flemming without acetic, iron
haematoxylin.
fuchsin. In the arthrodial membranes however, the exocuticle stains red and
the endocuticle blue.
With Masson's trichrome stain the details are similar, the acid fuchsinophil
zones of Mallory preparations taking iron haematoxylin in their outer parts
Chilopod and Diphpod Cuticle
epidermal cell
basemenb
membrane
'inbermediabe .
scleribe' exocufcicle
cone of exocubicle
gland cell
Inbermediabe „
scleribe eaocutrcfe
epidermis
endocubicle
gland cell
50]X
FIG. 3. Transverse section of an arthrodial membrane between the tergite and a pleurite of
Haplophilus subterraneus. Below are surface views of the cuticle corresponding to the three
regions indicated on the section. Camera lucida drawing. Flemming without acetic, iron
haematoxylin.
and ponceau acid fuchsin inwardly; the region staining blue with Mallory
taking the light green of the Masson combination. In the sclerite of Haplophilus, for example, the continuous part of the exocuticle stains blue-black
with the haematoxylin, save at its free edge which does not stain. The cones
stain with the ponceau and acid fuchsin, which, together with a little of the
retained haematoxylin, give a purple colour. Figs. 2, 3, 4, and 5 illustrate the
148
Blower—A Comparative Study of the
various regions of the cuticle stained with iron haematoxylin alone. It will
be noticed that the sclerite endocuticle of Lithobius stains with haematoxylin
only in its outer layers; the inner layers stain red when the haematoxylin is
outer non-sbaining
parb of eiocubide
continuous parb
of exocubicle
pnsmabic cone
of exocubicle
gland duct
endocuticle
epidermal cell
gland cell
basement
membrane
FIG. 4. Transverse section of the sclerite cuticle of Haplophilus subterraneus. Camera lucida
drawing. Flemming without acetic, iron haematoxylin.
6uber non-staining
and inner basiphif
exocubicle
swelling^ in
gland aucb
outer endocuticle
gland duct
inner endocuticle
gland cell
j^i_—epidermal cell
FIG. 5. Transverse section of the sclerite cuticle of Schizophyllum sabulosum. Camera lucida
drawing. Neutral formaldehyde fixation followed by decalcification in sodium hexametaphosphate and removal of epidermal pigment by ethylene chlorhydrin.
followed with the other stains of Masson's combination. The figures show
clearly the passage inwards of the basiphil zone (equivalent to the outer parts
of the red-staining zones with Mallory) associated with greater degrees of
tanning of the outer layers. The whole of the arthrodial membrane exocuticle
of Lithobius is basiphil. The exocuticle of Schizophyllum, which is of a more
distinct amber colour than that of the arthrodial membrane of Lithobius, fails
Chilopod and Diplopod Cuticle
149
to stain in its outermost layer. This non-staining zone is extended in the
sclerite exocuticle of Haplophilus and in the sclerite of Lithobius the entire
exocuticle fails to stain, the basiphil region occupying a large part of the
endocuticle.
In all cases, treatment with diaphanol (chlorine dioxide in glacial acetic
acid) leads to the entire thickness of the exocuticle staining with either the
acid fuchsin of Mallory or the iron haematoxylin used in Masson's combination—the endocuticle staining with aniline blue or light green respectively.
In Lithobius the intensity of blue or green staining of the endocuticle is
greater in the sclerite than in the arthrodial membrane, and similarly in
Schizophyllum the inner endocuticle still has a greater affinity for these stains
after diaphanol treatment.
Dennell (1946) has described the effect of diaphanol on the subsequent
staining reaction of the cuticle of Sarcophaga puparia. The larval condition
is restored as far as staining reaction is concerned and there appears to be
a reversal of the effects of tanning. Since diaphanol treatment in this case
restores the fundamental two-layered nature of the cuticle it might be assumed
that the endocuticle and exocuticle of the described myriapods are the homologous fundamental layers. The two layers of Sarcophaga and Homarus are,
however, further differentiated by the presence or absence of chitin, whereas
no such distinction can be drawn in the case of the myriapod cuticle. It might
be added here that the division of the thin outer layer of a diplopod into nonstaining and basiphil zones appears to be the basis which Langner (1937)
adopts in distinguishing an epicuticle and an exocuticle. Since both regions
of the outer layer contain chitin (see next section) and diaphanol treatment
removes the distinction, it is doubtful whether this particular staining reaction
is a suitable criterion to adopt.
T H E PHENOLIC SUBSTANCE IMPREGNATING THE CHITINOUS MATRIX
The optical appearance of the exocuticle and the endocuticle is, in fact, the
only criterion which constantly differentiates these two layers. Although
certain chemical properties are always associated with the exocuticle, the
same properties are sometimes shown by the endocuticle. Even such a
characteristic feature as pigmentation is not shown by all regions of the exocuticle.
A feature of both layers is the presence of chitin. This fact is made very
clear in the case of Lithobius where the exocuticle often separates from the
endocuticle during the potash treatment, but is nevertheless just as intensely
positive to the chitosan test (see Campbell, 1929) as the endocuticle. The
exocuticle is therefore to be regarded as the modification of the outer layers
of a chitinous matrix by the addition of some impregnating substance or
substances.
The exocuticle of all forms always gives intensely positive xanthoproteic and
Millon reactions, from which it can be assumed that phenolic groupings
enter largely into the composition of the impregnating substance. The
150
Blower—A Comparative Study of the
exocuticle is not, however, the only part of the cuticle positive to these two
tests. The whole of the sclerite endocuticle of Lithobius is likewise intensely
positive.
When frozen sections of the cuticle are treated with concentrated mineral
acids the exocuticle survives the treatment, as also do the ducts of the epidermal gland cells which bridge the gap vacated by the endocuticle. The
sclerite endocuticle of Lithobius dissolves much more slowly than that of
the arthrodial membranes. In the sclerite of Haplophilus the inner part of the
exocuticle, that is, the part divided into inwardly projecting cones, dissolves
in several hours at room temperature. The outer, continuous part of the
exocuticle is a typical amber qplour as is the whole exocuticle of Lithobius and,
like that of Lithobius, survives acid treatment for days. The inner discontinuous part of the exocuticle of Haplophilus is not pigmented. The foregoing observations may be summarized thus: the typical amber-coloured exocuticle is
very resistant to concentrated mineral acid. The colourless inner region of the
exocuticle in Haplophilus is not quite so resistant to acid, and finally the
sclerite endocuticle of Lithobius is even less resistant—but nevertheless shows
quite a degree of resistance when compared with the arthrodial membrane
endocuticle, which dissolves almost immediately. All regions showing any
degree of resistance are alike in giving positive xanthoproteic and Millon
reactions. It appears that the chemical difference between the conical part of
the exocuticle of Haplophilus and the sclerite endocuticle of Lithobius is one
of degree and not kind—the cones of Haplophilus being included as part of
the exocuticle by virtue of their homogeneity and refractility.
It appears from these facts that the process of sclerotization consists in the
impregnation of a chitinous matrix by a substance rich in phenolic groups
and a subsequent.process which renders this material resistant to acids. The
development of refractility and, later, of an amber colour seems to be correlated with the development of this resistance, although resistance is developed by the phenolic substance present in the inner, discontinuous, part
of the exocuticle of Haplophilus without the assumption of an amber colour
and in the sclerite endocuticle of Lithobius without even the assumption of
refractility.
As suggested in a preliminary communication (Blower, 1950) it seems
possible that this phenolic impregnating substance is a protein in contrast to
the alcohol-soluble phenol which is one of the precursors of sclerotin in the
cockroach ootheca (Pryor, 1940a). Evidence in favour of this view arises from
a comparison of the arthrodial membrane endocuticle of Lithobius, on the
one hand, with the sclerite endocuticle of this same animal where the phenolic
substance appears to be present in a simple form, since here the substance
has conferred a degree of resistance on the endocuticle but has not rendered
it homogeneous or refractile. In the first place it will be remembered that
the sclerite endocuticle gives positive Millon and xanthoproteic reactions
whereas the arthrodial membrane endocuticle does not. This, and the following histochemical details, even if their absolute meaning is not clear, give a
Chilopod and Diphpod Cuticle
151
comparison under closely controlled conditions since each test has been performed on a transverse section where both regions under consideration are
present together.
The iso-electric points of the two regions have been determined by the
method employed by Yonge (1932). The value for the sclerite endocuticle is
between pH 5-4 and pH 5-6 whereas that for the arthrodial membrane endocuticle was between pH 2-8 and pH 3-0. It follows from this that the substance responsible for the difference between the two regions is probably
amphoteric such as an amino-acid or protein. This difference in iso-electric
points is reflected in the staining reactions of the two regions. With Mallory
the sclerite endocuticle stains red whereas the arthrodial membrane endocuticle stains a light blue. The substance differentiating the sclerite endocuticle
from the arthrodial membrane endocuticle is still present after treatment
with water, alcohol, xylene, &c, and remains demonstrable by its staining
reaction. It seems on this account that a simple amino-acid is not responsible
and that a protein is probably involved. The fact that the additional substance
in the sclerite endocuticle of Lithobius confers a degree of resistance to acids
would also suggest that the substance concerned is something other than a
free phenol or amino-acid.
The resistant nature of the substance impregnating the sclerite endocuticle
is manifest during the application of the chitosan test. In sections of
potash-treated material the sclerites are still noticeably different from the
arthrodial membranes. The two layers of the sclerite are quite obvious and
the exocuticle is still homogeneous. The arthrodial membranes appear to
have separated into individual laminae. At the first application of the iodine
and sulphuric acid only the arthrodial membranes assume the violet colour.
The sclerite endocuticle assumes a much lighter and more delicate tinge of
violet, the exocuticle remains colourless. A few moments after flooding the
slide the sclerite endocuticle and exocuticle split up into individual laminae,
expand to the extent of the arthrodial membranes and assume the deep-violet
colour first shown by these membranes.
If the temperature of the potash during treatment has fallen below that
specified by Campbell (1929), i.e. 1600 C , or if the solution has not been
fully saturated, the initial reluctance of the layers of the sclerite to assume a
deep-violet colour is prolonged. Pieces of cuticle in this condition immersed
in 3 per cent, acetic acid did not completely dissolve. The residue still gives a
strongly positive Millon test.
Lafon (1943), on treating the cuticle of a scorpion with 10 per cent, potash
at ioo° C , found that two layers survived the treatment—a colourless layer
composed of chitin and an outer very thin amber-coloured layer. Lafon
suggests that this resistant outer layer is comparable with the cuticulin of
insects. This layer bears similarity to the exocuticle of Lithobius, which
remains as a discrete layer after potash treatment. This layer in Lithobius
survives even more brutal treatment than that which Lafon used, still remaining homogeneous after 2 hours at 1550 C. in concentrated potash. It may be
152
Blower—A Comparative Study of the
that in the scorpion this outer layer contains chitin but shows a reluctance to
display itself similar to that of the exocuticle of Lithobius, and the unsuspected
presence of chitin would account for the anomalous figures obtained from
analysis of this layer. Lafon subjected Lithobius to the same treatment, but
in view of the fact that he was dealing with whole pieces of cuticle, the small
amount obtainable from Lithobius may have led to his overlooking a surviving
outer layer in this case.
As has been suggested (Blower, 1950) the substance impregnating the
sclerite endocuticle of Lithobius and the exocuticle of all forms may be a
protein with a high tyrosine content, since it is very rich in phenolic groups
and has an iso-electric point similar to that of tyrosine. If this were so it seems
possible that there is a development of cross-links by the oxidation of the
side chains of this tyrosine-rich protein. Brown (1950) has suggested that
this method of tanning may take place in certain regions of Mytilus and in the
egg-capsule of Fasciola. Whatever the mechanism of tanning, it seems possible from the foregoing facts that the substance differentiating the sclerite
endocuticle of Lithobius from the arthrodial membrane is a protein rich in
phenolic groups and probably represents the precursor of typical sclerotin.
This substance will accordingly be termed pro-sclerotin. The prismatic cones
of the exocuticle of Haplophilus may then be regarded also as consisting of
pro-sclerotin.
In the diplopod Schizophyllwm there is a thin exocuticle, only the outer
part of which is amber coloured. The endocuticle is optically divisible into
two distinct layers. The outer layer is impregnated with calcium salts—a fact
ascertained by the application of the alkaline pyrogallol test (Lison, 1936).
The inner layer is much more conspicuously laminated and appears to be
impregnated with pro-sclerotin. It gives positive xanthoproteic and Millon
tests and survives acid treatment for much longer than the outer endocuticle,
but is not.so resistant as the exocuticle. As would be expected, the inner
endocuticle has a higher iso-electric point than the outer (pH 3 6 compared
with pH 2-8).
The behaviour of the cuticle towards diaphanol casts a little more light on
the nature of sclerotin and pro-sclerotin. The effect of this reagent on a tanned
cuticle as described by Dennell (1946) has been mentioned. The fully tanned
puparial case of Sarcophaga is bleached by diaphanol and the condition of
the cuticle in the last larval instar appears to be restored as is evidenced by
the staining reaction of the diaphanol-treated cuticle. Its effect on the chilopod and diplopod cuticle is to remove all traces of the amber colour resident
in the exocuticle, but it has little effect on the resistance of this layer to acids.
Theoretically it would be expected to remove the cross-links of sclerotin by
destroying the aromatic nuclei by oxidation. That it does in fact destroy the
rings is evidenced by the fact that a diaphanol-treated cuticle gives no trace
of a positive xanthoproteic or Millon reaction in any region. The fact that it
does not, however, destroy the resistance of the exocuticle to acids, points
again to the fact that pro-sclerotin itself is a substance with a degree of
Chilopod and Diplopod Cuticle
153
resistance and stability which does not depend on the presence of aromatic
cross-links. It will be recalled that the sclerite and arthrodial membrane endocuticles of Lithobius still stain differently after diaphanol treatment—with
Mallory the sclerite endocuticle stains a deep purplish-blue in contrast to the
arthrodial membrane which stains a very pale blue.
ARGENTAFFIN MATERIAL IN THE CUTICLE
It is stated (Lison, 1936) that a positive argentaffin reaction (reduction of
an ammoniacal silver nitrate solution) indicates the presence of polyphenols,
amino-phenols, or polyamines. This reaction has often been employed to
demonstrate the distribution of phenolic substances in the cuticle. Hackman,
Pryor, and Todd (1948) describe phenolic substances in the epicuticle (here
referred to as 'exocuticle') of Tachypodoiulus on the evidence furnished by
this test.
When sections of a chilopod or a diplopod are treated with a 5 per cent,
solution of ammoniacal silver nitrate there is a browning of the exocuticle.
The sclerite endocuticle of Lithobius eventually reduces the silver solution,
but only after a much longer period in the reagent. The arthrodial membrane
never reduces the silver. Ammoniacal silver has also been used in order to
determine whether a polyphenol layer covered by a wax layer lies external to
the exocuticle as described in Rhodnius and Tenebrio (Wigglesworth, 1947,
1948a). A specimen of Lithobius was allowed to crawl in carborundum powder
for several hours and then plunged into silver solution for 24 hours. The
cuticle was then cleaned and washed well, and a portion embedded in wax and
sectioned. Another portion of the cuticle was mounted whole. The whole
mount revealed numerous criss-crossing brown lines on the surface of the
sclerite representing the scratching of its surface by the carborundum particles. In sections of the sclerite these brown scratch lines were represented
by lens-shaped areas at the surface of the cuticle which had reduced the
silver more strongly than elsewhere. Beneath the exocuticle in the outer
third of the endocuticle numerous vertically disposed filaments were stained
—much more intensely than the exocuticle itself. Furthermore, the contents of
the epidermal glands and their ducts had reduced the silver nitrate solution
and appeared almost black.
It will be remembered that the whole of the sclerite endocuticle of Lithobius
is intensely positive to Millon's reagent, and the whole has a higher isoelectric point than the arthrodial membrane endocuticle. If the argentaffin
reaction indicates the presence of phenolic substances one would expect the
whole of the sclerite endocuticle of Lithobius to darken evenly. This it does
not do. As will be seen later the epidermal glands appear to secrete a lipoid
material, particularly evident in Haplophilus; and since these and the contents
of their ducts reduce the silver solution in Lithobius and in Haplophilus, it
seems possible that the lipoid itself may be argentaffin.
Although Lison (1936) does not include lip'oids amongst argentaffin substances, it seems theoretically possible that unsaturated fats at least would be
154
Blower—A Comparative Study of the
capable of reducing ammoniacal silver nitrate. To ascertain whether there is
a possibility that cuticular lipoid is argentaffin, animals were cut into two
pieces, the cuticle freed from underlying tissue and one piece boiled for
2 hours in chloroform. The untreated piece of cuticle was retained as a
control. Both pieces were embedded in paraffin wax and sectioned. Two
ribbons, one from each piece, were mounted on a slide, freed from wax and
the slide immersed in 5 per cent, ammoniacal silver nitrate for 24 hours. The
sections were then fixed in a 1 per cent, solution of sodium thiosulphate (Lee,
1937). The intensity of the silver staining of the exocuticle was definitely less
in the chloroform-treated cuticle. Furthermore, the extent of the argentaffin
zone beneath the sclerite exocuticle was considerably reduced. The chloroform used for the extraction was allowed to evaporate in a watch-glass. An
orange-coloured material remained which stained readily with sudan black.
As will be made clear in the next section the distribution of lipoid in the
cuticle appears to follow the distribution of sclerotin and pro-sclerotin. It is
possible that the results of an argentaffin test may indicate the presence of
other material besides phenolic substances.
T H E EPIDERMAL GLAND CELLS AND THE OCCURRENCE OF LIPOID IN THE
CUTICLE
The cuticle of all the myriapods examined is pierced by numerous ducts
arising from glandular cells in the epidermis. The various types of gland and
duct are shown in figs. 2, 3, 4, and 5. In general there are more glands
beneath the sclerites than elsewhere, or, in other words, those regions of the
animal most likely to come into contact with its solid environment are well
supplied with glands. The sclerite epidermis is in fact an epithelium of these
gland cells. Their ducts pass through the cuticle and open to the exterior
between the cuticular prisms. The staining reaction of the glands in the
epidermis leads to a supposition that they show an asynchronous activity—
some glands having dense basiphil inclusions whilst others have not.
In frozen sections of Haplophilns stained with sudan black the contents of
the glands and their ducts are coloured blue. In Lithobius the whole of the
epidermis is weakly sudanophil, but sometimes inclusions of a strongly positive material are to be found within the gland ducts. In Schizophyllum and
Tachypodoiulus one can only say that the epidermis generally is strongly
sudanophil—the epidermal pigment granules making the precise location of
the lipoid material difficult to see.
The location of the lipoid material in the cuticle is not made very clear by
sudan staining. In Lithobius only the arthrodial membrane and the intermediate sclerite exocuticle shows signs of the blue colour. There is, however,
a very thin sudanophil layer external to the exocuticle. In Haplophilus the
cones of the exocuticle are slightly sudanophil and again there is a very thin
sudanophil layer external to the exocuticle. In the intermediate sclerites of
this animal the exocuticle is strongly positive, and over the intucked arthrodial
Chibpod and Diplopod Cuticle
155
membranes there is always quite an accumulation of sudanophil material.
In Schizophyllum the outer part of the exocuticle is sudan positive over the
entire animal.
Treatment with diaphanol has an interesting effect on the subsequent
staining with sudan black, in so far as it definitely intensifies it. In a frozen
section of diaphanol-bleached material of Schizophyllum or Tachypodoiulus
the exocuticle is coloured almost black with sudan black and this colouring
extends inwards for about a quarter the thickness of the outer endocuticle
as numerous very fine filamentous processes. There appears to be a penetration of lipoid material down the pore canals (see next section).
The mechanism by which treatment with diaphanol leads to the lipoid
material becoming sudanophil is perhaps connected with the fact that it
destroys the aromatic links. Possibly the lipoid is in some way intimately
attached to these groups and on their destruction is more readily available
to the sudan stain. This intensification of sudan staining is also evident in
Haplophilus. The effect of diaphanol on the subsequent staining with haematoxylin may possibly be explained on similar lines. It will be remembered
that in the sclerite of Lithobius after diaphanol treatment the exocuticle stains
with haematoxylin, whereas in the untreated cuticle the exocuticle does not
stain at all but the outer third of the endocuticle is stained with haematoxylin
(see fig. 2A). Here it appears that the diaphanol, on destroying the aromatic
groups in the exocuticle, has made available some basiphil substance originally firmly attached to these groups. By analogy with the above facts concerning the intensification of sudan staining by diaphanol and the fact that
the contents of the gland ducts and glands are basiphil, it might be suggested
that the substance made available to the haematoxylin is in fact a lipoid. The
haematoxylin staining substance present beneath the exocuticle of Lithobius,
however, does not remain after diaphanol treatment.
Pieces of cuticle warmed gently in a saturated solution of potassium chlorate in concentrated nitric acid show first a dissolution of the endocuticle
and then a breaking down of the exocuticle into an oily material which stains
readily with sudan black. This appears to be an oxidative process, similar to
that produced by diaphanol, but more vigorous, and presumably it leads in
the same way to a liberation of the lipoid by a destruction of the aromatic
groups with which it seems to be associated. Nitric acid alone can effect this
process. If whole pieces of animals are immersed in cold concentrated nitric
acid the process can be studied more critically since the oxidation proceeds
much more slowly. Pieces of Haplophilus thus treated show first a dissolution
of the endocuticle. After an hour nothing remains but the exocuticle. Before
the amber colour has disappeared oily droplets float to the surface. These
droplets, smeared on to a slide, colour with sudan black. The outer ambercoloured part of the exocuticle is still intact at this stage and therefore the
droplets have probably come from the dissolution of the cones of the exocuticle. If the amber-coloured portion is left overnight it is bleached and on
washing and immersing in sudan black it takes up the colour rather unevenly.
156
Blower—A Comparative Study of the
This latter observation applies also to Lithobius, although no oily droplets
free themselves from the cuticle as in Haplophilus. In Lithobius, during treatment with cold nitric acid, the intermediate sclerites and the arthrodial
membranes dissolve completely before the sclerites have lost their amber
colour. In both cases several days' treatment with acid results in the complete
solution of all but the exocuticle of the sclerites.
As was pointed out in the preceding section it seems possible that the
cuticular lipoid is capable of reducing silver nitrate. The vertically-disposed
argentafEn filaments beneath the exocuticle of Lithobius are thus possibly
lipoid in nature. Here it seems possible that the lipoid material penetrates
down the pore canals, as is indicated by sudan colouring in Schizophyllum
and Tachypodoiulus. It is not clear, however, if this be the case, why sudan
colouring in Lithobius does not give the same picture.
PORE CANALS
When a section of Tachypodoiulus or Schizophyllum is immersed in concentrated mineral acid the outer endocuticle dissolves rapidly. Between the inner
endocuticle and the exocuticle, besides the persistent ducts of the epidermal
gland cells, there are to be seen numerous fine filamentous processes emerging
from the inner endocuticle and continuous distally with the inwardly projecting papillae from the underside of the exocuticle. By analogy with other
arthropods these structures appear to represent the solid contents of the pore
canals. No such clear demonstration of pore canal filaments has been seen in
a chilopod. It will be remembered, however, that the under surface of the
intermediate sclerite exocuticle in Lithobius and Haplophilus is produced
inwards as minute papillae, and these may represent the distal ends of original
pore canals into which exocuticular material has penetrated. Furthermore,
treatment with silver has revealed filaments beneath the sclerite exocuticle of
Lithobius. Unfortunately no developmental history of what appear to be the
pore canals is available. Plotnikow (1904) figures pore canals in the form of
conical tufts with their apices arising from the epidermis in the developing
larval cuticle of Tenebrio. The* fully formed cuticle of this larva has the inner
part of its exocuticle in the form of cones as has Haplophilus. If the surface
of the cuticle of Haplophilus is examined over the region of transition from
intermediate sclerite to arthrodial membrane (fig. 3, A, B, c), the impression is
obtained that each cone of the exocuticle is formed by the merging together
of exocuticular material in the pore canals. Possibly the original pore canals
of Haplophilus are tufted and give rise to the conical arrangement of the
inner region of the exocuticle.
RisuMi AND DISCUSSION
A two-layered cuticle has been taken throughout as a working hypothesis.
It has been seen, however, that the chemical reactions of the two layers do
not neatly arrange themselves on each side of the optical dividing line between
Chilopod and Dipbpod Cuticle
157
endocuticle and exocuticle. This division is one of convenience only. By
considering different regions of the same animal, and different animals, the
possible changes attendant to the formation of a typical amber-coloured and
highly refractile exocuticle may be inferred. In the animals studied each
region of the cuticle can be placed in an ascending scale of conditions approaching a typical exocuticle (see Table I).
TABLE I
1. Arthrodial membrane endocuticle of Haplophilus and Lithobius and outer endocuticle of
Schizophyllum.
Sclerite endocuticle of Lithobius and the inner
endocuticle of Schizophyllum.
3. Prismatic cones of Haplophilus. Inner layers of
exocuticle of Schizophyllum.
< co
I"
4. Stainable portion of the continuous exocuticle
of Haplophilus. Arthrodial membrane exocuticle of Lithobius. Inner portion of the exocuticle
of intermediate sclerite in Lithobius and Haplophilus.
T
*4
P
§
^
O
W
o
§
OS
o
•<
-52
5. Whole of sclerite exocuticle of Lithobius and
the non-staining outermost regions of the exocuticle of Haplophilus and Schizophyllum.
-
«
s
The process of exocuticle formation seems to be heralded by the appearance
of a substance, possibly a protein, rich in phenolic groups, in the chitinous
matrix of the cuticle. This substance has been given the name 'pro-sclerotin'.
It is resistant to acids even before the more characteristic features of an
exocuticle are manifested. The substance eventually renders the impregnated
region homogeneous and refractile, by which time the region may be called
exocuticle on the definition here adopted. Later there is a development of
even greater resistance, the development of an amber colour and finally the
loss of staining reaction.
158
Blower—A Comparative Study of the
The process of sclerotization as described in detail by Pryor (1940 a and b)
in the cockroach ootheca and in insects, and by Dennell (1947a) in the puparial case of Sarcophaga, is regarded as a tanning of cuticular protein attended
by the development of an amber-brown colour. Pryor, however, states that
a water-soluble protein is passed into the cuticle to form the basis of the
sclerotin whereas Dennell considers that the pre-existent cuticular protein
may be involved in the tanning process.
It does not seem that the whole of the process of exocuticle formation as
suggested in this work is comparable with the tanning process as described
by Pryor and Dennell. Neither in Sarcophaga nor in the cockroach ootheca is
there a substance described,similar to pro-sclerotin. In the cockroach ootheca
neither of the precursors of sclerotin appears to have any degree of resistance
before they are linked together as sclerotin.
Browning (1942) describes a colourless exocuticle in certain regions of the
spider Tegenaria, and Brown (1950) records the fact that the precursor of
sclerotin in certain structures of Mytilus and in the egg-capsule of Fasciola
is not water- or alcohol-soluble but is probably a protein. Brown also suggests
that the deamination of the phenol destined to tan the protein as described by
Pryor (1940 a and b) is possibly a derived condition. Both the cockroach
ootheca and the puparium of Sarcophaga appear to be special cases. In Sarcophaga the sclerotin of the puparium is formed at the end of the instar, and it
may be that there has been a modification of the precursors of sclerotin to
diffusible substances since they have to pass to a region of the cuticle distal
to the epidermis.
A feature invariably associated with the exocuticle of myriapods is the
presence of lipoid material. This lipoid appears to be secreted by the gland
cells of the epidermis through ducts which open on to the surface of the
cuticle. At the surface it appears to form a thin film. It appears also that the
exocuticle is impregnated with lipoid where there appears to be an intimate
association of the lipoid and sclerotin or pro-sclerotin. It may be that the
lipoid in the exocuticle is responsible for this layer (and adjacent regions of
pro-sclerotin) assuming an avidity for iron haematoxylin.
Pryor (19406) suggested that the sclerotin in insects is impregnated with
lipoid. He pointed out that sclerotin and any material rich in aromatic groups
is very lipophil. In myriapods this observation is consistent with the fact that
both sclerotin and pro-sclerotin appear to be impregnated with lipoid. In
Haphphilus, it will be recalled, there is a dense accumulation of sudanophil
material over the arthrodial membranes. This region of the cuticle is unmodified and no demonstrable exocuticle is formed at all. This may account for
the whole of the lipoid being stainable at the surface, since in this region
there is no lipophil layer to absorb it.
It is interesting to consider the possible explanation of the scratch-pattern
effect obtainable by treating the cuticle with an abrasive dust followed by
immersion in ammoniacal silver nitrate. It may be that the outer surface of
the lipoid film covering the exoeuticle undergoes a process similar to the
Chihpod and Diplopod Cuticle
159
drying of a coat of oil-bound paint. Perhaps the abrasive agent removes this
inert layer and reveals the reactive lipoid beneath which may reduce the
silver solution. Wigglesworth (1947, 1948, &c.) explains scratch patterning in
insects on the basis of removal of a wax layer and exposure of a polyphenol
layer which lies beneath the wax layer. In view of the fact that there appears
to be evidence, in myriapods, that the lipoid itself will reduce the silver
solution and that no developmental details are available for myriapods, it is
unwise to speculate further on this point.
In the myriapods studied there appears to be no cuticular layer corresponding to the cuticulin layer described by Wigglesworth in Rhodnius and Tenebrio
(1947, 1948a). Cloudsley-Thompson (1950) on the basis of treatment with
chlorated nitric acid suggests that a similar layer does exist in certain species
of myriapods which he has examined. From this evidence,. however, there
seems nothing to differentiate between a layer of sclerotin impregnated with
lipoid and a cuticulin layer. Just as Langner (1937) speaks of an epicuticle
and an exocuticle in the outer part of the cuticle of a diplopod on the basis
of an outer non-staining and an inner basiphil zone, the differential solubility
of the outer and innermost parts of the sclerotin of the cuticle is apparently
taken by Cloudsley-Thompson as evidence for the presence of an epicuticle.
Wigglesworth (1947) has been careful to distinguish between cuticulin as
a lipo-protein subsequently tanned with quinones, and Pryor's sclerotin
secondarily impregnated with lipoid. Only developmental evidence can settle
this point, but in view of the chemical similarity between sclerotized cuticulin
and lipoid-impregnated sclerotin the difference in time relation may not be
of fundamental importance, for pro-sclerotin may be impregnated with
lipoid before tanning as generally understood takes place. In this case there
would seem to be nothing to distinguish between lipoid-impregnated prosclerotin (e.g. the exocuticular cones in Haplophilus) and cuticulin.
If the sequence of events during deposition of the cuticle is the same in
myriapods as in Rhodnius and there is no essential difference between sclerotized cuticulin and lipoid impregnated sclerotin or pro-sclerotin, then the
whole of the myriapod exocuticle and parts of the endocuticle (sclerite endocuticle of Lithobius) can be considered as being impregnated with cuticulin.
Even so, these regions still cannot be homologized with a cuticulin layer,
since they contain chitin.
The only layer of the myriapod cuticle which appears to be in any way
homologous with the insect epicuticle as described by Wigglesworth is the
thin layer of lipoid at the surface. This layer may be responsible for the
diffraction effect at the surface of the cuticle which lead Verhoeff and Fuhrmann (see page 144) to speak of an outermost colourless layer (Oberflachenschicht, Grenzhautchen). As regards the possible homology of the layers of
the myriapod cuticle with those of Homarus (Yonge, 1932) and Sarcophaga
(Dennell, 1946, 1947a), there appears to be close correspondence of the
myriapod exocuticle with the 'cuticle' of Homarus and the epicuticle of
Sarcophaga larva. Here again the main difficulty of comparison arises from
160
Blower—A Comparative Study of the
the fact that the exocuticle of myriapods contains chitin whereas the similar
layers in Homarus and Sarcophaga are described as being free from chitin.
As has been seen, extensive sclerotization of the sclerite of Lithobius has led
to the whole of the exocuticle being unstainable. The epicuticle of Sarcophaga
larvae stains red with Mallory but loses its affinity for the stain on being
converted into the sclerotin of the puparium. Diaphanol treatment restores
the larval staining reaction. Similarly in Lithobius diaphanol treatment leads
to the exocuticle staining red with Mallory—the two cases seem quite comparable. Then again, where sclerotization does not seem to be so advanced,
as in the exocuticle of the arthrodial membrane of Lithobius, this layer stains
red without previous diaphanol treatment.
The outer layer of the epicuticle of Sarcophaga larvae (the outer epicuticle
—see Dennell, 1946) is much more resistant than the bulk of the epicuticle
and is lipoid in nature. Were both layers of the epicuticle not described as
being free from chitin it would be natural to compare, the inner and outer
epicuticle with a region of pro-sclerotin underlying a region of completed
sclerotin such as obtains in the sclerite exocuticle of Haplophilus or the whole
of the sclerite cuticle of Lithobius.
The suggested condition in myriapods where the fat is believed to impregnate the whole of the lipophil outer layers and to originate from epidermal
gland cells recalls the condition in Homarus in which the 'cuticle' contains
lipoid material. Thomas (1944) records that the 'cuticle' of Lepas contains
lipoid. This latter author also records a fat reaction in the tegumental glands
of Lepas. If the glands of Homarus secrete the lipoid of the 'cuticle' then it is
not surprising that the glands show a periodicity in relation to the laying
down of the new integument and that Yonge believed that these glands were
responsible for the secretion of the whole of the 'cuticle'. Pryor (1940&)
suggested that the lipoid which he believed to impregnate the sclerotin of
insects might be secreted from epidermal gland cells. Lastly, on this same
point, it may be mentioned that Langner (1937) records a fat reaction in the
epidermal glands of the diplopods she studied.
It has been suggested (Blower, 1950) that the myriapod cuticle might be
considered as a chitinous matrix impregnated to varying extents by prosclerotin which may or may not be tanned, that is to say, which may or may
not have the amber colour and inertness usually associated with complete
sclerotin. To this generalization may be added the fact that lipoid material
appears to be secreted on to the surface of the cuticle from glands in the
epidermis and also to impregnate the regions of sclerotin and pro-sclerotin.
Here, there appears to be an intimate association of the lipoid with the aromatic groups of the sclerotin (evidenced by the fact that destruction of the
aromatic groups by diaphanol or nitric acid renders the lipoid available to
sudan colouring agents).
The myriapod cuticle, then, is characterized by the absence of an outer
layer which is both resistant and non-chititious, by the presence of a material
allied to sclerotin but peculiar in being stable and resistant independent of
Chilopod and Diphpod Cuticle
161
tanning, as usually understood, and by the apparent presence of lipoid in all
the modified regions of the cuticle. These characteristics, in the absence of
detailed information as to the mode of development of the cuticle, make
extensive comparison with other arthropod cuticles a matter of some difficulty.
ACKNOWLEDGEMENT
My sincere thanks are due to Professor R. Dennell for the original suggestion of this problem and for the help and encouragement that he has freely
given to me at all times.
REFERENCES
BLOWER, J. G., 1950. Nature, 165, 569.
BROWN, C. H., 1950. Ibid., 275.
BROWNING, H. C , 1942. Proc. Roy. Soc. B, 131, 65.
CAMPBELL, F. L., 1929. Ann. Ent. Soc. Amer., 22, 401.
CARLETON, H. M., 1938. Histological technique. Oxford.
CLOUDSLEY-THOMPSON, J. L., 1950. Nature, 165, 692.
DENNELL, R., 1946. Proc. Roy. Soc. B, 133, 348.
1947a. Ibid., 134, 79.
19476- Ibid., 134, 485.
FUHRMANN, H., 1921. Z. W1SS. Zool., 119, I.
HACKMAN, R. H., PRYOR, M. G. M., and TODD, A. R., 1948. Biochem. J., 43, 474.
LAFON, M., 1943. Ann. Sci. nat. zool., 5, 113.
LANGNER, E., 1937. Zool. Jb. (Abt. Anat.), 63, 483.
LEA, A. J., 1945. Nature, 156, 478.
LEE, A. B., 1937. The microtomist's vade-mecum. London (Churchill).
LEES, A. D., 1947. J. exp. Biol., 23, 379.
LISON, L., 1936. Histochimie animate. Paris (Gauthier-Villars).
PLOTNIKOW, W., 1904. Z. wiss. Zool., 76, 333.
PRYOR, M. G. M., 1940a. Proc. Roy. Soc. B, 128, 378.
19406. Ibid., 393.
THOMAS, H. J., 1944. Quart. J. micr. Sci., 84, 257.
VERHOEFF, K. W., 1925. 'Chilopoda' in Bronn's Klass. u. Ordn. Tierreichs, 5, Abt. 11.
WIGGLESWORTH, V. B., 1947. Proc. Roy. Soc. B, 134, 163.
1948a. Quart. J. micr. Sci., 89, 197.
19486. Biol. Rev., 23, 408.
WILKS, R. A. C , 1938. Nature, 142, 958.
YONGE, C. M., 1932. Proc. Roy. Soc. B, 111, 298.