Physico-chemical studies on the epicuticle of the two-striped grasshopper, Melanoplus bivittatus Say
by William Chefurka
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Entomology at Montana State College
Montana State University
© Copyright by William Chefurka (1949)
Abstract:
The literature on the arthropod cuticle is reviewed in detail. An attempt is made at a unification and
synthesis of the findings of various investigators in this field of insect physiology.
A detailed description is given of the apparatus and methods employed in this study.
The surface area of Melanoplus bivittatus was determined and found to be expressed by S = 10.27
W72.
It was essential to the interpretation of the transpiration studies.
The epicuticle of M. bivittatus was found to consist of the cuticulin layer covered by a superficial layer
of wax. The former resists disruption by prolonged chloroform extraction, and also stains black in the
presence of osmic acid. The latter is removed by very mild Chloroform treatment.
The wax layer was found to be deposited before the old exoskeleton is shed. The insect is thus
protected against undue dessication during the moulting period.
The "critical temperature" of the wax film covering the cuticle of M. bivittatus was found to be 44°C. A
theory is proposed to account for the rapid increase in evaporation at the critical temperature. 'It
considers, the wax film as a potential barrier, the energy of which decreases at the critical temperature
as a result of an increase in the dielectric polarization of the was at this temperature. The permeability
of the cuticle of this grasshopper was found to decrease progressively with increasing time after
moulting. This is attributed to an increase in the thickness of the cuticle and tanning of the cuticular
protein.
'The variation of the dielectric polarization of beeswax was determined with increasing temperature. It
was found that the dielectric polarization of this wax increased in the temperature interval 35 - 51°C.
This was correlated with an increase in the permeability of the wax film. Paraffin films showed an
increase in permeability to water only at the melting point, i.e. change in phase. The rate of evaporation
of water through beeswax films decreased very gradually with increasing thickness beyond the
thickness of two microns.
A theory was developed which considers the wax film as a potential barrier. On this assumption, the
gradual decrease in. permeability of beeswax films to water is attributed to the non-linearity between
increasing thickness and increasing energy of the potential barrier. 
PHYSICO-CHEMICAL STUDIES ON THE EPICUTICLE OF THE TWO-STRIPED 
■GRASSHOPPER, MELANOPLUS BIVITTATUS SAY
by
WILLIAM CHEFURKA 
A THESIS
Submitted to the Graduate Committee 
in
partial fulfillment of the requirements 
for the degree of 
Master of Science in Entomology 
at
Montana State College
Approved:
J. H. Pepper_________
In Charge of Major Work
J. H. Pepper_______________
Chairman, Examining Committee
J. H» Nelson_________
Dean, Graduate Division
Bozeman, Montana 
June, 1949
2(Zo/D. 3
TABLE OF CONTENTS
Abstract 
Introduction 
Acknowledgements 
Review of Literature
Methods and Materials _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Results and Discussion
1. Determination of the Surface Area of M. bivittatus - - -
2. Determination of the presence of a Wax Layer - - - - - -
3* Time of Deposition of the Wax Layer _ _ _ _ _ _ _ _ _ _ _
4. Determination of the Absence of a Cement Layer - - - - -
5- The Effect of Temperature on Evaporation through the 
Cuticle
6. Determination of the Presence of a Cuticulin Layer
7• Changes in the Cuticle after Moulting - - --  _ _ _ _ _ _
8 . Dielectric Polarization of Beeswax - - - - - - - - - - -
9- The Effect of Temperature on Evaporation through films of 
Beeswax and Paraffin
1 0. Nature of the Transitional Point
Summary and Conclusions
Literature Cited and Consulted
- 3
- k
- 6 
- 7
53
64
64
74
83
88
94
-116
120
133
146
l6l
164
169
3
/
ABSTRACT
The literature on the arthropod cuticle is reviewed in- detail. An 
attempt is made at a unification and synthesis of the findings of various 
investigators in this field of insect physiology.
A detailed description is given of the apparatus .and methods employed 
in this study.
The surface area of Melanoplus bivittatus was determined and found 
to be expressed by
S = 10.27 W*'2 .
It was essential to the interpretation of the transpiration studies.
The epicuticle of M. bivittatus was found to consist of the cuticulin 
layer covered by a superficial layer'of wax. The former resists disruption 
by prolonged chloroform.extraction, and also stains black in the presence 
of osmic acid. The"latter is removed by very mild dhloroform treatment.
The wax layer was found to be deposited before the old exoskeleton is 
shed. The insect is thus protected against undue dessication during the 
moulting period T
The "critical temperature" of the wax film covering the cuticle of M.' 
bivittatus was found to be 440S. A theory is proposed to account for the 
rapid increase in evaporation at the critical temperature. 'Tb considers, 
the wax film as a potential barrier, the energy of which decreases at the 
critical temperature as a result of an increase in the dielectric polariz­
ation of the was at this temperature. ' ,
The permeability of the cuticle of this grasshopper was found to de­
crease progressively with increasing time after moulting. This is attri­
buted to an increase in the thicknebs of the cuticle and tanning of the 
cutlcular protein.
'The variation of the dielectric polarization of beeswax was determined 
with increasing'temperature. It was found that the dielectric polarization 
of this wax.increased in the temperature interval 35 - 51°C. This was cor­
related with an increase in the permeability of the wax film. Paraffin 
films showed an increase in permeability to water only at the melting 
point, i.e. change in phase. I
The rate of evaporation of water through beeswax films decreased very 
gradually with increasing thickness|beyond the thickness of two microns.
A theory was developed which considers the wax film as a potential barrier. 
On this assumption,.the gradual ,decrease in. permeability of beeswax films 
to water is attributed to the non-linearity between increasing thickness 
and increasing energy of the potential barrier.
4INTRODUCTION
The layer covering the external surface of the body, appendages and 
invaginated parts of insects and other arthropods is called the cuticle.
It is composed of non-cellular orgainc matter presumably secreted by the 
epidermal cells.
The cuticle acts as. an intermediary between the. insect's external 
and internal environments. It follows, therefore, that the very existence 
of the insect will be directly or indirectly dependent upon the physico­
chemical structure and properties of the cuticle. The cuticle determines 
the nature and. direction of the growth and developmental physiology of the 
insect; it.governs the insect's responses to external sensory stimuli; and 
it plays a very vital role in determining the ecological distribution of 
these animals. Consequently an elucidation and basic understanding of . 
many of the problems of insect physiology and ecology depends upon a 
thorough understanding of the nature of the cuticle of the particular 
insect.
The economic entomologist, confronted with the tremendous problem 
of controlling the ravages of insects, partly by the use of .contact in­
secticides, wants to get some understanding of the action of these toxic ■ 
sprays. Since such agents must penetrate the cuticle before they become 
effective, it is imperative that these workers know something abouttthe 
interaction between the cuticle and the penetrating material; Such in­
formation will be of great, aid to them in planning an effective method ■'
•' 5
of control with the aid of such poisons. It is generally considered 
that answers to these problems must come from the insect physiologist;
5M t ,  at the present state of his knowledge, he is unable to supply the an­
swers to such queries. His present position was well described by the 
British insect physiologist, Wigglesworth (1934), when he said "with the 
demand for increased knowledge, has come a realization of our present ig­
norance". ' . .
Realizing their predicament in this field of insect physiology, con­
siderable time and effort, has. been devoted in the past few years by some 
to the study of the cuticle. From the rapidly accumulating data resulting 
from studies of cuticles of many species of insects, certain basic prin­
ciples of cuticle structure have emerged. It is the object of this study 
to determine to what extent these principles may be applied to the cuticle 
of Melanoplus bivittatus which is one of the .more destructive grasshopper 
species in western Canada and north-western United States.
6ACKNOWLEDGMENTS
■ The author wishes to acknowledge his indebtedness to Dr. J. H.' 
Pepper, Head of the Department of Zoology and Entomology at Montana 
State College for suggesting this problem and for constant advice, en­
couragement .and criticism given during the development of it,*' to Profes­
sor E. B. Hastings for his criticisms and assistance in the work on 
"The Effect of Temperature on Evaporation through the Cuticle"; to Drs.
L. H. Johnson and C. N. Caughlan for their advice on the chemical as­
pects of the problem; to the Division of Entomology, Canadian Department 
of Agriculture for permission to use the following information fSr thesis
purposes and for-granting a transfer of work which permitted a more 
.
thorough investigation of this problem.
Sincere thanks are due t° the Bureau of Entomology, United States 
Department of Agriculture at Bozeman, Montana for the loan' Sf their rear­
ing cabinets and a grant of a.quantity of grasshoppers for this study.
The cooperation and assistance given by Mr. D. K. Scharff and Mr.
P. W. Eiegert in the translation of many of the articles is gratefully 
acknowledged.
Finally I wish to thank my wife, Patricia, for assistance in 
laboratory routine; for her constant encouragement and technical advice 
on the physical aspects of the problem. Without her help much of- the 
work here reported could'not have been accomplished.
7REVIEW OF LITERATURE
On the basis of chemical and histological studies the cuticle is 
currently divided into two layers:-
I. The outer layer is called the epicuticle. Usually, it is color­
less. It contains no chitin. Its thickness varies with the type of 
cuticle, ranging from one micron or less in the case of Rhodnius prolixus 
(Wigglesworth 1933pl9^7) to four microns in the larva of Sarcophaga 
falculata (Dennell 1946). It is resistant to cold concentrated acids and 
bases. It is also resistant to chitinases and proteolytic enzymes which 
are suspected to be present in the moulting fluid. The epicuticle is the 
first layer to be formed during ecdysis. In most insects, it is subdi­
vided into a variable number of layers. In R. prolixus and Tenebrio 
molitor four such layers have already been described (Wigglesworth 1945, 
1947, 1948) while in Periplaneta americana (Richards and Anderson 1942), 
Calliphora e'rythrocephala, Sv falculata (Dennell 1946) only two layers 
have as yet been detected.
2-. Below the epicuticle is the endocuticle. This layer is subdi­
vided into two regions. The outer region is called the "outer endocuticle". 
(Dennell 1946) or exocuticle (Campbell 1929). It is a hard, inextensible, 
amber, brittle region of variable thickness. In the case of Rhodnius 
(Wigglesworth 1 9 3 3).19^7 ) it is secreted before moulting but in the larva 
of S. falculata this region continues to be secreted even after moulting,
(Dennell 1946). The inner region is called the "inner endocuticle"
(Dennell 1946) or "endocuticle" (Campbell 1929). It is usually thick and 
elsstic': being secreted after moulting (Wigglesworth 1933, 1947; Dennell
819^ -7) • The chief chemical constituents of the endocuticle are chitin and 
protein.
The arthropod cuticle will be discussed in detail under the follow­
ing topics:-
(I) Formation of the cuticle.
(II) Structure, chemical and physical properties of the epicuticle, endo­
cuticle and pore canals.
(ill) Permeability of the arthropod cuticle to water and other materials.
Although most of the discussion which.follows will be confined to 
the insect cuticle. reference will be made in the appropriate sections to 
information available on the cuticle of other arthropods.
I. Formation of the cuticle.
In the process of moulting, the epidermis, after undergoing mitosis 
and chromatolysis, separates from the old cuticle and proceeds to secrete 
the new epicuticle. At the same time the oenocytes reach their maximum 
development and Wigglesworth (1933*"19^7) suggests that they provide the 
epidermis with the necessary substances for the secretion of the epicuticle 
Tower (1906) studied the process of moulting in Leptinotarsa 
decimlineata and observed that the cuticle was first laid down as a very 
thin structureless membrane which he called the "primary cuticula". It 
was laid down before moulting. A similar phenomenon was observed by 
Poisson "(1924) in certain aquatic Hemiptera and by Wigglesworth (1933) 
in Rhodnius. Dennell (19^6) also found that the epicuticle was the first 
layer to be secreted in Sarcophaga.
Wigglesworth (19^5, 1947, 1948) studied the deposition of the epi-
9cuticle in Rhodnius and Tenebrio and found that in the former insect, the 
cuticulin layer, polyphenol layer and wax layer of the epicuticle are de­
posited before the old cuticle is shed. In Tenebrio, only the first two 
layers are deposited before moulting; the others are secreted after moult­
ing.
After the epicuticle is formed, secretion of the endocuticle commen­
ces. Tower (1906) considered the epicuticle to increase in thickness 
reaching its maximum just before moulting. Wigglesworth (1933) showed 
that in Rhodnius, it is not the epicuticle which increased in thickness. 
Rather, a new region was secreted. This region is the "outer endocuticle". 
It reached its maximum thickness prior to the shedding of the skin. It is 
very probable that such was the case in Tower's observations. Chemical 
tests showed that this region is primarily composed of chitin and protein. 
From the very outset, this region “is striated vertically. Dennell (1946) 
recorded similar observations for the third instar larva of'Sarcophaga, 
but here the outer endocutiele contines to be secreted after moulting.
After the old skin is shed, the "inner endocutiele" commences to be 
secreted. This was observed by Tower (1906) Poisson (1924), Wigglesworth 
(1933, 1948) and Dennell (1946). Dennell (194?) reported that the inner 
endocutiele continues to be secreted not only throughout ,the third instar 
of Sarcophaga larva but even for a short time after pupation. Similar
-T
results were recorded by Kuhn and Piepho (1938)* in the case of Ephestia 
kuehniella.
* cited by Dennell (1946)
While the outer and inner ehdoeuticlesiare being-secreted, the epi- 
cuticle gradually acquires the property of impermeability to water. Lees 
(1947) found that the cuticle of the tick, Ornithodorus moubata, becomes 
progressively less permeable to water during moulting. Generally, this 
decrease of permeability is due to the impregnation of the epicuticle by 
fatty substances. This impregnation appears to be some sort of initmate 
association between the protein and fatty substances, perhaps by oxidation 
and polymerization, resulting, in the formation of a resistant lipoprotein 
called "cuticulin" (Wigglesworth 1933; 1949, 194?, 1948). Following this, 
there is an active secretion of a.wax film.
The exact mechanism by.which the cuticle is produced is not yet de­
finitely known.
Chatin (1892).considered the formation of the cuticle to be the re­
sult of a sort of peripheral differentiation of the superficial layer of 
the epidermal cells. He concluded this from his work on the larvae of 
Libellule. He observed the presence of protoplasmic filaments which 
'radiated from the nucleus. Gradually these filaments oriented themselves 
parallel to the free surface of the cell,' It was this protoplasmic por­
tion, which became differentiated into the new cuticle. This view was 
also held by such authors as Gilson (1890), Vignon (l90l), Plotnikow 
(1904)* and Eder (1940).
The school headed by Leydig (l864) and including Kofschelt (1896), 
Henneguy (1904), Tower (1906, Haas (1916)., Poisson (1924), Wigglesworth 
* cited by Schfoedef'(1928): .
10
11
(1933) and Dennell (1946) considers the cuticle to he a secretory product 
of the epidermal cells. The cuticle is secreted in liquid form around 
filiform outgrowths from the epidermal cells. These outgrowths later form 
the pore canals.
II. Structure, chemical and physical properties of the epicuticle, 
endocuticle and pore canals.
A. Epicuticle.
(i) Definition
- Since the time of Haeckel (l8 %^) much work has been done bn the epi- 
cutiele, and in the light of modern findings, the whole issue becomes 
very confusing. Most of the German authors referred to this layer as the 
ttGrenzlamellelt or the ttGrenzsaum". Plotnikow (l90k) called it the "Plas­
matic layer".*  Tower (1906) referred to it as the "primary cuticula" 
and Campbell (1929) as the "epicuticula" or the "epicuticle".
What exactly is the epicuticle? Wigglesworth (1933) gave this name 
to that layer of the Rhodnius cuticle which was resistant to solution in 
cold concentrated acids and which was not penetrated by pore canals. At 
that time it was thought, that this definition referred only to the "cuti- 
Culintt. layer which appeared to be the only consituent of the epicuticle. 
Pryor (1940b) showed that the epicuticle was not the only portion of the 
Rhodnius’cuticle that is resistant to solution in concentrated hydro­
chloric and sulfuric acids; a portion of the outer endocuticle is also 
resistant. Furthermore, Dennell (1946) found that the protein epicuticle
* cited by Schroeder (1928)
12
of Sarcophaga dissolves slowly in concentrated hydrochloric acid. In view 
of this, the question arises, is the protein epicuticle and - part of the 
exocuticle to be considered as the "epicuticle"?
Recently, Wigglesworth (19^7) has shown that in Rhodnius,the pore 
canals probably do penetrate the "cuticulin" layer of the epicuticle but 
do not penetrate the polyphenol, wax and cement layers. In view of the 
original definition, is the epicuticle to be considered as composed only 
of the latter three layers? Haas (1916) defined the epicuticle as that 
layer secreted by the dermal glands. On the. basis of Wigglesworth1s 
studies (1947) this would limit the epicuticle in Rhodnius to the cement
i '  .
layer. One cannot say that the epicuticle is the waterproofing layer, 
since this would limit it to the wax layer, although Ramsay .(l935¥i)> did 
apply this term to the oily film on the cockroach cuticle 'and Bergmann 
(1938) applied it to the wax layer on.the cast skin of the silkworm.
Many authors tried differentiating the layers of ’the cuticle on the 
basis of specific staining reactions. Dennell (l9k6) separated the 
layers in the larval cuticle of Sarcophaga on the basis of the reaction 
with the Mallory stain. The epicuticle stained red while the endocuticle 
stained blue; Recently, Wigglesworth (1948) showed that in Mallory- 
stained ventral portions of Tenebrio, the outer exocuticle took on a red 
colour while the inner exocuticle took on a blue colour.* On the basis 
of these reactions, it appears as if the outer exocuticle of Tenebrio 
should be analogous to the protein epicuticle of Sarcophaga larva, as
*- Apparently Wigglesworth divided the exocuticle of Tenebrio into two 
- regions. ,
13
described by Dennell (1946). It is ob'yious that considerable confusion 
can arise if the differences"revealed by staining techniques are not check­
ed by other methods. Furthermore, since biological material is known to 
react differently to the same stain under different conditions,, this pro­
cedure when used along, is open to question. ' ,
1
Much of the present confusion- on the limits of the epicuticle is 
■due to the facttthat -this structure was defined when very little was 
!mown about it. As more knowledge was accumulated, then, rather than 
re-orienting the definition to fit the new discoveries, it was sought 
by some to have the findings fit the old definition and by others to con­
sider only those portions of the epicuticle as the "true" epicuticle which 
had the- properties originally ascribed to it. Another important source 
of confusion is the tendency on the part of many authors to generalize.
It is possible and very probable that the properties given to the epi­
cuticle of one insect may not hold for the epicuticle of another insect. 
This should not be surprising' in view of the physiological differences 
found from species to species. Recent investigators, Wigglesworth, Hurst 
and others (1948),-showed that epicuticles do vary in. their complexity from 
species to species. Consequently before any hard.and, fast definition is 
given more work is necessary. At present, the epicuticle may best be de­
fined as that layer of the insect cuticle which is free of chitin.
■ (ii) Properties and- structure of the epicuticle
The epicuticle was observed very early in the study of the insect 
cuticle. It appears that it was first definitely recognized by Haeckel
14
In 1857.* It should he noted, however, that Biedermann (1903) cites 
Meyer (1842) as having isolated a "dark brown thin uppermost chitin 
lamella".. This lamella appeared "clearly polygonically aerolated", the 
pattern being a copy of the fiat outspread epithelial layer. On the basis 
of this description and the claim'that it contained chitin it is diffi­
cult to decide whether this was or was not the epieuticle.
Young (1932) states that Ambron (1890) and Zander (1897) identified 
an outer layer in the integuments of various insecta, Arachnida and 
Myriopoda. Later Holmgren (1902), Plotnikow (1904),'Tower (1906), 
Lecaillon (1907) and Kapzov (l91l) recognized the presence of an outer 
homogeneous layer. Since 1911 practically every investigator reported 
the presence of such a layer although Fraenkel and Rudall (1940) failed 
to observe it. This was probably due to an oversight on their part, in 
view of Dennell's findings (1946, 1947).
Aronssohn (1910) studied the composition and structure of the cast 
skins of honey bee larvae. He found them to contain 10.7$ nitrogen. This 
was probably due to the presence of some undigested endocuticle.** He 
found no sulfur. The skins did not react with, reagents in a manner similar 
to compounds which were used for comparison. It was concluded that the 
epicuticle could not be identified with such substances as albuminoids, 
collagen, keratin and fibroin.
Wigglesworth cites Haas (1916) who called the outer non-chitinous 
layer of GryIlptalpa the "Grenzsaum". Haas found that this layer is
* ■ cited by Wigglesworth (1939)
** Wigglesworth (1939) states that the cast skins of Rhodnius contain 
- approximately "14$ endocuticle. ... -
15
resistant to solution in hydrochloric acid and is a product",of the secre­
tory process of the dermal glands.
The implication that the epicuticle is thin is not necessarily true. 
Poisson (1924) observed the presence of a thick "external region" in the 
cuticles of certain Wepidae. He suggested that this region was analogous 
to the outer layer observed by Lecaillon (1907). It probably represents 
a highly modified epicuticle similar to that described by Wigglesworth 
(1939, Pg- 21).
' The presence of fatty substances in the epicuticles of- grasshoppers 
and beetles was first demonstrated conclusively by Kuhnelt (1928). In 
the grasshoppers he found the quantity of lipoid material to be negligible 
but in the beetles, the cuticles showed an average loss of weight of 5$ ' 
after acetone extraction. This loss in weight was assumed to be due to 
the removal of the lipoids.
Chemical tests performed by Kuhnelt on these extracted lipoids 
showed that they were composed principally of fatty acids and cholesterol. 
Because the permeability of the cuticle was greatly increased as a result 
of the removal of this substance, he concluded that it was, to a great ex­
tent, governed by this lipoid substance.
Perhaps most of our knowledge on the nature and structure of the 
epicuticle came as a result of the brilliant investigations of Wiggles- 
worth on the bug Ehodnius. In 1933  ^ he reported that the epicuticle of 
this insect was approximately ore' micron thick, colourless and refrac-■ 
tile. It was found to be resistant to solution in cold concentrated 
hydrochloric, sulfuric, and nitric acids. He reported that it 1 '-contained
16
no chitin, protein, nor carbohydrates. Pryor (1940b) reported the pre­
sence of protein in this layer of Rhodnius and Wigglesworth (19^7)'con­
firmed this. In 1933 Wigglesworth reported negative fat staining reac­
tions indicting the absence of free lipoids, although in 19^5 he found 
that the ppicuticle of Rhodnius could be stained by Black Sudan B. When 
the cuticle was placed in hitric acid saturated with potassium cholorate 
the endocuticle dissolved first, with an evolution of gas. Further gentle 
heating caused" a disruption of the epicuticle with a liberation of drop­
lets which were shown by the Sudan' black B. stain to be oily in nature. 
This led him to the conclusion that the epicuticle is a complex fatty or
waxy substance or a mixture of these. He suggested that this fatty mater- 
' ' - ' 
ial might be closely allied to cutin and suberin found in plants, and
gave it the name "cuticulin". The presence of this layer in Rhodnius 
was also confirmed,by his later studies. It was also found by him to be 
present in Tenebrio (1948) and in a number of species of ticks. (Lees 
194-7) • Further studies showed that this fatty material impregnates the ■ 
protein in the epicuticle forming azlipo-protein (Pryor 1940b, Wiggles- 
worth 194-5, 194-7). But the lipoid is not entirely;' limited to the epi­
cuticle; there is some evidence that it extends into the outer protions 
of the endocuticle (Wigglesworth 194-2). This fatty material incorporated 
in the protein, would correspond to the bound lipoids" postulated by 
Hurst (194-3)
Ramsay (l935h) found that'the epicuticle of the cockroach, Blatta 
ofientalis, contained a mobile oily film. He. found it to be responsible 
for the impermeability of the cuticle of this insect, for if it was taken
17
up to a temperature of 30oG ., the rate of evaporation increased beyond all 
prediction, probably due to a change in phase of the fatty film. The 
average thickness of this film is approximately 0.6 jnxerons (Beament 1945).
Bergmann (1938) studying the composition^bf the silkworm cuticle 
extracts-, - found that ether or chloroform extracts consisted chiefly of 
paraffin hydrocarbons and esters. They were part of the "cuticulin"
Iyer. Efo sterols were found. He did find that the hydrocarbons had 
the formulae Cgy 5 or C2Q H^q and the ester C2Q H56 O2 . Because 
Chibnall et al (193^) state that the insect and plant waxes contain hydro­
carbons of the odd-numbered carbon series only and alcohols and fatty 
acids of the even carbon series, Bergmann concluded that his mixture of 
hydrocarbons consisted of_ 2 7 -3 1 carbon! atoms and the mixture of alcohol 
and acid esters of'26 -3 0 carbon atoms.
Wigglesworth’s results on the chemical properties of the Rhodniud 
"epicuticle were confirmed by Prypr (1940b). The latter author found that 
the epicuticles of B v prolixus, Ranatra linearis and Locusta danica did 
not stain with Sudan Black B; while those of £. eurythrocephala and 
Philsomia cynthia did staid., He therefore concluded that in the case 
where no staining reaction was obtained, the lipoids were masked in some 
way. Furthermore, he found that the lipoids were confined almost en­
tirely to the epicuticle.
On the basis of his work on the nature._of hardening of the cock­
roach ootheca, Pryor considered the epicuticle to be secreted as a pro­
tein sheet which is then hardened by quinone tanning forming a hard 
substance which he called "sclerotin". This sclerptin is then secondarily
18
impregnated by lipoids which by a probably process of oxidation and poly­
merization become insoluble in fat solvents.
Eder (19^0) also found evidence for the presence of lipoids in the 
epicuticle of several i n s e c t s A f t e r  extraction with fat solvents, the 
cuticles of Gryllotalpa and Carausius showed a loss of,weight of 4$.' 
Pepper and Hastings (1 9V7) found this same value for. the mormon cricket 
(unpublished data). In the case of some of the beetles, the loss in 
weight as a result of acetone extraction ranged from 2-10$ (Eder 1940).
No loss in weight of the grasshopper or the bug cuticles was reported, 
probably because the amount of lipoid present was very small,. As a re­
sult of this work Eder was. able to correlate cuticular transpiration with 
the presence of lipoids.
As far as could be found, the work of Richards and Anderson (1942)
was the first study of the epicuticle by means of the election microscope.
They'found the epicuticle of. P. americana to be a two-layered structure
while that o f 'CuIex pipiens only one. The outer lipoid epicuticle. of the
cockroach was 0 .0 2-0 .0 3 microns thick, while the protein epicuticle was
2 microns thick. It is doubtful whether these represent the true values
because of the possibility of volatilization of the oily film and consider
able shrinkage of the protein epicuticle in the very high vacuum required
for this type of study.- They found the thibkness of the protein epicu-
•
ti d e  of the mosquito to be about 0.03 microns after it had been dried.
It was impossible to stain the lipoid epicuticle of the cockroach 
with Black Sudan .B. or Sudan III (Richards and Anderson 1942). In later 
studies Richards -and Korda (1948) were unable to demonstrate again the
19
presence of the lipodid epicuticle in the cockroach trachaea even when the 
epicuticle was isolated. These authors speculate that, in view of Pryor's 
findings, the epicuticle in the cockroach is first secreted as a uniform 
layer of protein becoming subsequently differentiated into a thin layer 
of polymerized lipoproteins and a thick underlying layer of tanned pro­
teins without lipoids.
Although the .presence of the.epicuticle and some of its chemical 
properties were established with'a fair amount of certainty by 19V?, it 
was not until that year that the structural complexity of the Rhodnjus 
epicuticle was realized. Wigglesworth (19^5) showed that the epicuticle 
of this bug and later of Tenebrio (1948) and of certain ticks (lees 1947)_ 
is a highly complex structure consisting of the following layers from 
within outward:
I.. CDTlQj-JlW LAYER: This layer is composed of condensed and
polymerized lipoproteins. Its presence was detected by treat­
ing the epicuticle in nitric acid saturated with potassium 
chlorate. This treatment resulted in the disruption of the 
layer with a subsequent liberation of oily droplets.
2. POLYPHENOL LAYER: This layer is. composed of.phenols and was
detected by its ability to reduce silver in a solution of am-, 
moniacal silver hydroxide.
3 . WAX LAYER: This layer is probably composed of a mixture of
alcohols, acids, paraffins and esters. (Chibnall et-al 1934).
Its chief function id to protect the insect from dessication.
Its presence can be shown by fat - stains, by chloroform extrac­
20
tion or by abrasion with, a subsequent application of the 
argentaffin test.
4. CEMEEP LAYER: According to Wigglesworth (1948) this layer
is apparently composed of protein and formed in a fashion 
similar to the formation of the cockroach ootheca (Pryor 
1940a). In both Rhodnius'and Tenebrio he found it to be 
secreted by the dermal glahds. It protects the wax from 
abrasion or extraction. Often as a result of extraction 
or sectioning, this layer became detached from the one be­
neath and therefore became visible in the light microsope.
; It should be noted that the latest investigations on .Rhodnlus showed 
that the epicutidle could be stained by Sudan Black B. (Wigglesworth 1945) 
whereas the earlier investigations (Wiggleswprth 1933, Pryor 1940b) re­
ported the absence of such a staining reaction.
Wigglesworth (1945) and Lees (1946, 194%) showed that the wax layer 
is a very integral part of the epicuticle of all the insects and ticks in­
vestigated. The nature of the wax varied with the species, of the insect 
or tick and with their habitat. They found.that those living in zerophytic 
habitats have a very hard wax' which is highly oriented and closely packed 
on the cuticle surface ther^y limiting the evaporation through the cuti­
cle to a minimum. Those living in more moist environments like the grass­
hopper,. the cockroach and certain bugs ,show a moderate loss of water under 
ordinary temperatures indicating that the wax is perhaps softer, not as 
highly oriented and perhaps more -.'hydrophilic. Beament (1945) estimated 
the wax film to be about § .2 5 microns thick on all the insects studied by
him. .
citesThe preaence of a waxy film is not universal. Pryor (l$40b) 
the cuticle of Psilopia petroli as remaining hydrophilic throughout the 
life span of this insect. It cannot contain any appreciable quantities 
of wax. Richards and Anderson ( W )  state that lipoids are also absent 
from the epicuticle of the mosquito C_. pipiens.
Dennell (1946) found that the epicuticle of the Sarcophaga larvae 
stains red in the Mallory stain while the endocuticle stains blue. He 
attributed this difference in staining to differences in the isoelectric 
ppints which further indicate a differing chemical constitution of the 
two layers. After treating the cuticle in 5$ hydrochloric acid and stain 
ing with Mallory stain, the epicuticle that was left stained blue. H e - 
therefore concluded that the epicuticle in the Sarcophaga larva is a 
double-layered structure. The outer epicuticle was less than one micron ' 
thick while the inner epicuticle was about four microns thick.
By isolating the outer epicuticle and staining with Sudan Black B. 
a very positive reaction was obtained showing the presence of IipoidS in 
the outer epicuticle. If this outer epicuticle was then treated with 
chloroform it Still stained with Sudan Black B. indicating the presence 
of unremoveable lipoids. The presence of these substances was further 
confirmed by Schultz's modified Liebermann-Burchardt stain. Furthermore, 
since the outer epicuticle was not destroyed by hot potash, Dennell con­
cluded that probably contained some unsapbnifiable* sterol as well as ■ 
oxidized and polymerized lipoids which when combined with tanned proteins
21
* Pepper and Hastings found that 88$ of the wax extracted from Anabrus
simplex was unsaponif!able (oral communication).. '
22
form a very resistant complex.
He also showed, that the inner epicuticle gives a positive xantho­
proteic biuret, ninhydrin and Millon reaction, indicating the presence- 
of protein. This protein layer was soluble in hydrochloric acid hence 
was considered to be untanned. The presence of lipoids could not be de­
tected here.
From the discussion on the lipoids in the epicuticle, it is obvious 
that although their presence seems to be universal in terrestrial insects 
yet they may and do occur in various forms. Dennell (1946) points out at 
least three forms in which they may occur in the insect cuticle:-
1. The lipoids may form a relatively labile' external layer of wax 
at the surface of the cuticle.
2. They may be closely bound- to the surface of the epicuticle 
giving rise to a stable thin outer epicuticle.
3* They may impregnate the inner epicuticle"when this layer is 
tanned.
It .is suggested, and probably quite justifiably so, by Denhell that 
the thin layer previously designated as the epicuticle by many ahthors 
other than Pryor and Richards and Anderson, is acutally the outer- epicuticle.
The' cuticulin layer of Rhodnius and Tenebrio and of many ticks pro­
bably corresponds to_ the outer or lipoid epicuticle of Sarcophaga or to that 
of the American cockroach. In all these insects this layer, appears to be 
a lipoprotein. The protein epicuticle of Sarcophaga ,arid the American 
cockroach probably correspond to the■outer'exocuticle of Tenebrio. The 
basis for this suggestion is. their similarity in staining with Mallory's
23
stain. Apparently no layer has yet been found in Rhodnius.which would 
correspond to the. protein epicuticle of Sarcophaga, probably because it 
is masked when the protein is tanned.- If such a synthesis is accepted, 
then-the only difference between the epicuticles of these insects Is, 
that in some (Rhodnius, Tenebrio and some ticks) it is further modified
by the addition of a ■free.wax layer, polyphenol layer and cement layer.
- ' '
That such a plan might be basic in the structure of the epicuticle, is
'
strengthened by the findings of Dennell (1946) which indicate that the 
epicuticles of Blaps mucronata, T. molitor, Tenebrio obscums and 
Dermestes vulpinus are two layer structures.
Working on Galliphora Dennell (1946) found that it was possible 
to get a strong argentaffin reaction in the inner epicuticle whereas 
in Sarcophaga,' a strong reaction was obtained in the outer epicuticle. 
This difference might be due to the difference in the degree of quinone 
tanning of these, layers. It is important to note that the entire region 
gave the argentaffin test. Wigglesworth (1945), on the other hand, uses 
the positive argentaffin test as evidence for another layer in the epi-• 
cuticle, - the polyphenol layer. If the few stained cross-sections pre­
sented by-himiare'scrutinized, it becamezevident that the,outer stained 
region is very thick; much thicker than one would expect if the poly­
phenol layer, is to be considered as one of the four layers of the epi- 
cuticle which itself is only about one micron thick. From his photo­
graphs, it appears that the entire epicuticle took on the brown stain.
In view of this great possibility, more conclusive evidence ..is required 
before this layer can be definitely established. Furthermore'-much of
2k
his support for the existence of this, layer came from observations of sur­
face staining 'reactions. The-fact that ammoniacal silver hydroxide will
:
be reduced to a greater extent as the epicuticle is formed does not neces­
sarily mean that a layer of polyphenol is deposited. It might merely in­
dicate that polyphenols are impregnating the cuticulin layer during the 
tanning process. The polyphenol might therefore be diffused throughout 
this layer rather than forming another layer. In his latest report (1948) 
a diagrammatic sketch of the epicuticle is produced which does not include 
the polyphenols ..as a discrete layer.' In the case of Tenebrio, the entire 
outer exocuticle stained in the presence of an ammoniacal silver solution. 
This and his previous results on Khodnius merely mean, that aromatiCL. sub- 
stances are present in"these layers. Pryor (1940b) found that the exocu- 
ticles of C_. erythrocephala, E_. kuehniella, Aparia crataegi, Pieris 
brassicae and elytra of T. molitor and T. obscurus all give a positive
reaction with the argentaffin test and concludes that "there,".is no doubt
- . . i . ' ' . . .
that the results are due to the presence of phenols"..
Dennell (194-6) investigated the structure of the epicuticle in a 
softened Sarcophaga puparium. . In the early puparia he was able to detect 
a two layered epaicuticle. As the-puparia grew older, the inner epicuticle 
failed to stain red with fuschin. Sections of unsoftened puparia showed 
no signs of the two-layered structure. He assumed this to be-, due to the . 
■heavy tanning which the epicuticle underwent after pupation. Furthermore 
the' inner epicuticle could not be distinguished from' the outer endocuti'cle 
'He suggested that the resulting "exocuticle" results from a fusion of the 
inner epicuticle and outer endocuticle.during tanning of. these regions.
25
In 1948 evidence was presented in support of another concept of the 
structure of the epicuticle. Hurst, working on Calliphora, suggests that 
the epicuticle of this insect consists of a laminated lipo-protein. The ■ 
protein layers lie parallel to the cuticle surface and are separated, by 
regions of "bound" and ".labile" lipoids. These lipoids appear to be 
oriented perpendicular to the protein surface. The epicuticle is, there­
fore, a laminated multi-layered structure which is presumed to be -held 
together by a network of vertically arranged lipophylic fibrils or epi- 
cuticular rods. He states that fat solvents will remove the “labile"
- lipoids.without difficulty. In the presence of water or fat solvents 
this lamellar arrangement swells, thereby decreasing the close packing 
of the lipoids resulting in ah increase in cuticular permeability. When 
this network shrinks the lipoid packing is increased and the permeability
- decreased. The epicuticle can therefore be compared to a porous "valve" 
system;
This theory proposes a mechanism for water retention that is radi­
cally different from anything thus far advanced. In view of the physi- 
. ol'ogy and specialization of the blowfly this, should not be. surprising.
B. Endocuticle
The endocuticle may be defined as that layer of the insect cuticle 
which contains chitin. 'In no case known.to the author, has the absence 
of chitin from the endocuticle been reported, although Richards and 
Anderson (1947) reported the absence of chitin from wing scales df cer­
tain butterflies. At least in .one instance (Pepper arid Hastings 1943), 
the chitin content of the larval exoskeleton of L. sticticalis was
20
found to "be very low.
Beauregard (1885)'* was apparently the first investigator to report 
the presence of two ehitin layers, of different thickness, on the wing ' 
covers of beetles. Since then, every author has reported the same obser­
vation (see authors cited under mEpicuticle"). These two layers are now 
called the outer and inner endocuticle. In the paurometabolous insects, 
investigated, the outer endocuticle is almost invariably hard, inexten- 
sible and brittle. "When in this state it is called by"many the "exocu­
ticle". This condition of hardness appears to be the result of quinohes... 
tanning the protein (Pryor 1940b, Dennell 19^7). In the few holometabo- 
Ious larvae which have been investigated, this condition does not seem to 
exist. Only in the puparia does the hard exocuticle appear. Here it seems 
to be a fusion of the hard outer endocuticle and the inner epicuticle 
(Dennell 19^7-) •
Most authors do not subdivide■the endocutifele beyond the divisions 
already mentioned. However, at least in two instances a further subdivi­
sion has been made. Schultze (1913) divided the exocuticle in Lucanus 
into two layers as does Wigglesworth (1948),in Tenebrio. The probability 
of the "outer .exocuticle" in Tenebrio being analogous to the protein epi­
cuticle has already been considered.
(i) Laminations
Early investigations of the endocuticle revealed the. presence of
* cited by Schultze
27
laminae running parallel'to the cuticular surface. Meyer (1842)*. recorded 
the presence of lines in,the endocuticle running parallel to the surface 
in the cuticle of L. cerus. These lines were approximately 0.008 mm. 
apart. These striations and others, running at various angles to them ■ 
gave the cuticle a fibrous appearance and led to many interesting specu­
lations as to their function and probable analogy to muscle and tendon 
structure of vertebrates (Biedermann 1903).
Tower (-1906) considered these laminations, which in L. dectml ineata 
appear to be most predominant in the inner endocuticle, to be caused by 
the intermittent addition of new cuticle to the already existing one. 
Similar views were proposed by Janet (1909), and Poisson (1924). Wiggles- 
worth (1939), discussing the presence of these striationsstates that they 
are "probably the result of slight shifting of the epidermal cells as-, suc­
cessive layers of the cuticle are deposited".
Recent X-ray studies (Fraenkel and Rudall, 1940, 194?) indicate that 
the laminations in the cuticle of the larvae and pupae of Ca.11 iphora are 
due to the presence of alternate layers of chitin and protein. This sug­
gestion is based on the presence of equatorial spots of 33A° which appear 
to be due to planes lying parallel to the surface. Such-a hypothesis is 
strengthened by their discovery that the length of three amino acid resi­
dues is 10.3A°. This is also the length of one chitobiose unit. They 
therefore postulated that the cuticle is composed of alternating and 
interpenetrating lattices of chitin and protein. If the larval cuticles
* cited by Biedermann (1903) .
28
are'- "boiled, they found that the laminations disappear. The concluded • 
that the laminations, therefore, must has a real physical significance, 
and are not due merely to intermittent secretion.
Richards and Anderson (19^2) found that one. of the outstanding 
features of the outer and inner endocuticle of the.American cockroach
L *
is the presence of dense laminae. These occurred about 3-5 per micron 
and had an average thickness of 0.15 microns. After the - cuticle was 
treated with alkali the laminations were removed* but otherwise the cu­
ticle was left in a normal condition. It was therefore concluded by 
Richards and Anderson that the laminae are distinct structural components 
and can only be due-to differential molecular densities within the chitin 
framework.
In order to take account of the presence of these laminae these 
authors offer two possibilities:-
1. The laminae represent chitin-protein polymers which are sepa- 
. rated either by pure chitin or else by chitin low in protein
content. '
2. The chitin may play a more direct role than that assigned to it
in the first postulate. It may have its micellae more tightly
packed in these dense laminae. This highly packed form is then
=
helddntact by protein but it may be destroyed by alkali treat­
ment. As a result of such treatment or drying, Richards and 
. Anderson state that these laminae "are formed probably subse-
* Dennell (1946) notes, that even after violent.treatment of the 
Sarcophaga cuticle by alkali, the laminae will not be- removed.
29
quent to secretion as a chemical phenomenon in the cuticle and. 
as such probably do not represent simply successive layers of 
secretion". A similar suggestion was recently made by Dennell 
(-194-6).
(ii) Chemical and Physical Structure.
Chemical tests performed by Wigglesworth (1933) on the endocuticle 
of Rhodnius showed that both chitin and protein were present in this 
layer. The former was shown to be present by the "chitosan" test while 
the latter by such tests as the xanthoproteic, buiret and Millon. Severe 
alkali treatment of the endocuticle removed the brown coloured portion 
leaving only chitin. Further treatment with concentrated nitric acid re­
sulted in ac.complete dissolution of the endocuticle. Similar positive 
- . ■ :
tests were reported by Dennell (1946) for the Sareophaga cuticle.
There does not appear to be any definite relationship between the 
thickness of the exocuticle and endocuticle in some of the species, in 
the orders Hemiptera, Orthoptera and Coleoptera (Eder 194©). Furthermore,
- . ' I
this author showed that the pigmentation of the exocuticle is' very slight 
in the Orthoptera, increases in amount in Hemiptera and becomes very pro­
nounced in Coleoptera. The significance of this pigmentation will be dis­
cussed elsewhe.Se. There were no discernible laminations in the exocuticle ' 
of the orders Studied. These appear to be confined to the endocuticle.
When the puparia of Ephestia and Callophora are viewed in polarized
"• i
light, the outer endocuticle is isotropic while the inner'.endocuticle 
exhibits form birefringence. The absence of birefringemce in the un-
I
treated outer endocuticle was apparently due to the presence of a
t
3©
material of index of refraction 1.54 or I . 70 in the intermicellar spaces 
of chitin [Pryor 1940b).
Hurst (1948) found that the cuticle of the blowfly, has a-' ^ ,amellar
, . " ' - ' \ 
structure. Furthermore it exhibits form birefringence. He therefore'
concluded that the lipoids are oriented in the lamellae, and that the
protein molecules lie parallel to the surface..
X-ray investigations of the larval cuticle and puparium of
/
Sarcophaga by Fraenkel and Rudall (1940  ^ 1947) showed that the state.of 
chitin organization is determined by the manner in which the cuticular 
components are laid down during development. 'When'the protein was re- 
moved, or the chitin mechanically agitated,, then a highly crystalline 
state was obtained.
Their data indicate the presence of planes which run parallel and 
perpendicular to the surface. The length of three amino-acid residues 
is 10.3A° which is also the length of one chitobiose unit. This close 
dimensional agreement between.the protein and .-.-ichitin was used as a basis 
for their postulate that the cuticle,is composed of alternating and inter­
penetrating protein and chitin layers lying parallel to the surface.
Since these planes are easily disrupted in steam or by forces of compres­
sion, they concluded that the materials which constitute the planes are 
apparently' not held firmly together in the larval cuticle. On the other 
hand, no change was seen in the protein epicuticle of cockroach trachae 
even after alkali treatment (RicharcP and Korda 1948). It appears that
the protein-protein-linkage is much stronger than the protein-chitin
I •
linkage (Richards and Korda 1948). Likewise, no change was seen in the
31
puparium when, it was subjected to steam (Fraenkel and Rudall 19^7). Here
■ /
the chitin-protein linkage appears to be strong: ;and indeed Fraenkel and 
Rudall (1947) found that the agents which tan the cuticle seem to perform 
their action by attacking the. side chains of the proteiri-chitin complex. ' 
Undoubtedly, the side chains are modified in some manner.
Powder protein extracts of the larval cuticles give diffraction■ 
patters of the fully extended polypeptide grids (Fraenkel and Rudall 
1947). The pattern was of the B-protein type irrespective of the tempera­
ture at which the water extraction was performed. Boiling the protein 
only produced more defined B-patterns and the protein remained water solu­
ble even after heat denaturation. The presence of the B-protein chain is 
significant in view of the postulate that the cuticle is composed -of"'al­
ternating and interpenetrating layers of chitin and protein (Fraenkel' 
and Rudall 1947).
■ The structure of chitin of cockroach tracheae which .were extracted 
with alkali, is a fibrous meshwork. .This was shown by Richards and Korda 
(1948). ■ The meshwork consisted of a huddle of fibres which were in turn 
composed of microfibres oriented at random. Their dimensions are'75- 
SOOA0 in diameter. Richards and Korda speculate that "perhaps these micro­
fiber diameters represent the range of micelle or crystallite dimensions". 
The above" authors believe that the chitin molecules are probably not 
normally in the arrangement as seen in the treated material. Rather they 
are oriented in some other fashion. This orientation is then stabilized 
by the protein. When the protein is removed, there is a rearrangement' of 
the chitin into the microfibres which run parallel to the surface of
32
the membrane.
As a result of the work done on the molecular structure of the cu­
ticle, Fraenkel and Rudall (19^7) postulate that the cuticle of
ophaga is composed of alternating and interpenetrating layers of 
chitin and protein. The theoretical chitin-protein weight ratio as . 
calculated by them was found to be 55:45- This ratio is considered to 
be ideal in that there would be no chitin-protein aggregation. They sug­
gested that perhaps most cuticles have this chitin-protein ratio at the 
time of their formation. Only secondarily do they acquire more protein 
or other organic material. Richards (1947) on the other hand, argues 
against such a concept. He believes that the basic component is pro­
tein in the form of a plasticized sheet. This sheet is subsequently 
modified by the addition of chitin, pigments and waxes.
(iii) Pore Canals: ,
The endocuticle contains vertical as well as horizontal stria- 
tions. The presence of these vertical striations was first discovered 
by Valentin (1831)* in the cuticle of Hydrometra paludum. It was con- ' 
eluded that these striations were canals presumably filled with air.
Poisson (1924) found that the cuticle of Hepa was very compact 
and homogeneous and showed no signs of pore canals. Their presence in 
insect cuticles is therefore not universal. Richards and Anderson (1942) 
also failed to detect the presence of pore canals in the cuticle of the 
larva of Ch pipiens. But in the cuticle of Hydrometra nagas, Poisson
* cited by Poisson (1924)
33
found numerous pore canals traversing the cuticle. This was also the 
case in many, other aquatic hemipterous studied by him. Here, they ap­
peared as little non-ramified tubes beginning in the deepest chitinous 
•layer. They were considered to be filled with air.
Wigglesworth (1933) found that the pore- canals were present from 
the outset of the formation of the Khodnius cuticle. They probably orig­
inate as filiform, outgrowths of the epidermal cells around which the new 
cuticle is-secreted. They extended from the epidermal cells, through the 
endocuticle into the epicuticle,penetrating the cuticulin layer (Wiggles- 
worth 19^7) • He also found this to be so in the cuticle of Tenebrio (19I18). 
Wigglesworth suggested that the pore canals contain cytoplasmic filaments 
or some kind of fluid for if thdchf cro'ss-sections are allowed, to dry, many 
of the canals contain air:.' due to.the drying up of the contents. A simi­
lar suggestion was made by Lees (1946, 1947).
Eder (1940) found that the pronotum, of all the grasshoppers studied, 
showed the prepence of prominent pore canals.- They appeared as winding 
striations traversing the whole inner endocuticle. In the Locustidae they 
were also seen quite clearly in the exocuticle, while in the Acrididae 
they were only seen in certain regions of the exocuticle. This is no proof 
that they are absent from this region. Eder further, found that the pore 
canals seemed to join and form bundles. This bundle arrangment appeared 
to be more clearly defined in the Acrididae than it was in the Locustidae. 
The structure of the abdominal cuticle,was the same as that of the pro­
notum, only that here the pore canals were much finer.
The cuticles of the bugs appear to be similar in structure to that
\of the orthopterons except that the pore canals are not asp prominent in 
the former (Eder 19^0). In the beetles, the pore canals were very short 
..giving the appearance of degeneracy. They never passed through the com­
plete thickness of the inner endocuticle and were very difficult to see.
The significance of this gradual decrease in size and outline will be dis­
cussed elsewhere.
Richards and Anderson (1942) found that the pore canals in the Ameri­
can cockroach follow a regular helicoidal spiral path from the underlying 
epidermis and through the endocuticle but not into the epicuticle. The 
average diameter of these canals was about 0.15 microns increasing to 
about. 0.4 microns at the basal end. This expanded portion.which is 4 to- 
5 microns-long was almost straight instead of helicoidal. The rest of the 
spiral was a fairly regular'helix with a pitChriOf about 0.25 microns. The 
length of a canal was about twice the thickness of the cuticle or 80 
microns. There were about 1.2 x IO^ pore canals:per square millimeter 
of surface area or more than 25 x IO^ for a single:adult cockroach. About 
200 pore canals were found to overly each cell. Clearly, the surface area 
of each epidermal cell is vastly increased if the canals contain cytoplasm. 
Richards and Anderson calculated that the volume of a single pore canal 
was about I.7 cubic microns. On this basis, they account for about 
5-6/0 of the total volume, of an adult cockroach. At the epicuticle-exo- 
cuticle interface the ends of the pore canals represented about 2/0 of 
the interfacial area while at the endocuticle-epithelium interface they 
represented 15$ of this interface.
Contrary to Wigglesworth, these authors present arguments to support
34
35
their contention that the pore canals of the adult cockroach do not con­
tain protoplasmic filaments. Rather, they suggest that they are filled 
with a salt solution in equilibrium with the epidermal cells. They also 
criticize Eder (1940) for her contention that the pore canals observed by 
her were chitinized. In view of_Dennell's work.(1946) such a criticism 
does not ^ seem justified.
The exact function of the pore canals has not as yet. been definitely 
established. Wiggleswdrth (1933) suggested that they may act a conduits 
for enzymes which modify the cuticle after moulting. In 19^7.and'1948 
he also suggested that polyphenols may pass along them to the cuticle sur­
face. Riahards and Anderson (194-2) suggested that the pore canals facili­
tate the penetration of water and water soluble substances through the 
cuticles. The same suggestion was made by Wigglesworth (194-2) . . Lees 
(194-6, 194-7)— found evidence, for the suggestion that the moisture.on the 
cuticle surface may : be actively taken up by the pore canals and passed 
into the-haemocoele when the ticks are in a highly humid atmosphere. Un­
doubtedly, the large surface areas exposed by the ends of the pore canals 
(Richards and Anderson 194-2) are of great significance here. Dennell 
(194-6) thinks that a very important function of the pore canals is the 
secretion of the protein epicuticle. Some support for this comes from 
the similarity in staining of the inner epicuticle and the cytoplasmic 
filimehts in the pore canals.
Perhaps no other author has done more to elucidate the structure 
and-form of the pore canals than has.Dennell (1946). Histochemical 
studies performed by him showed that in very y o u h g Sarcophaga larvae,
■fch.6 contents of the pore canals are very .fine strands of cytoplasm aris - 
ing from the epidermal cells. The strands hrea^hup into tufts of fila­
ments extending to, but not entering, the epicuticle. In older larvae, 
the coiled cytoplasmic filaments fail to reach the epicuticle. In still 
older larvae the cytoplasmic strands are very short and in larvae about 
three days old, they are no longer visible. This gradual recession ;of 
the cytoplasm in the pore canals.may be the reason for the frequent sug­
gestion by some authors that the pore canals'are filled with air.
When the pore canals were tested for the presence of chitin it was 
found that the space left as a result of the shrinking of the cytoplasm, 
is filled with chitin. In the youngest larvae no chitin was detected in 
the pore canals. In older larvae, only the distal ends were chitinized 
while in the three day old. larvae, the whole extent of the pore canals 
gave an intenstive reaction for chitin. It is not known whether the chitin 
is secreted from the tips of the receding cytoplasmic filaments or whether 
the filaments'themselves become chitinized. Whatever the process, the ’end 
result is the formation of chitinized plugs perfectly fitting the pore 
canals.
Dennell does not suggest, that the presence of chitinized canals in 
an insect cuticle, is an universal phenomenon. But it probably is true 
that in the early stages of development, the pore canals are filled with 
cytoplasm. In later stages the pore canals 'become empty or chitinized 
depending upon the direction of specilization of the cuticle. ■
The outermost third portion of the canals is broad. It ..is often 
seen split_ into many branches. The central third is very.closely coiled '
36
while the lower third is broad> uncoiled and undivided. The diameter of 
a canal is about 1 .0  -microns and the pitch of coiling is 2 -5 microns.
There are as"many as 50-JO canals per epidermal cell and at least 15,000 
pore canals per sq. mm;
As far as can be determined, Dennell was the first investigator to 
suggest a reason for the helecoidal form of the' pore canals. The particu­
lar shape of these structures is due to the mode of secretion of the cu­
ticle. In the very early stages of development, the larval cuticle not 
only increases in thickness but also in length. It was found that this 
increase in- length actually involves a mechanical, stretching of the cuticle 
by as much as 250%. Hence, as new cuticular substances are added to the 
outer endocuticle, the cuticle does not assume the maximum possible thick­
ness due to the growth in length. Consequently, although the cytoplasmic 
filaments are undergoing an extension in length due to the gradual 
thickening of the outer endocuticle, they are also involved in this over­
all compression (not a true compression) of the cuticle due to the stretch­
ing in length, and hence the pore canals assume the helicoid form. As 
the growth in length ceases after the third day, the outer endocuticle 
begins to expand due to the distribution of new cuticular components 
among those already present. The pore canals likewise increase in length 
by the uncoiling of the chitin strands present in them.
Ill Permeability of the cuticle
The cuticle maintains the distinction. betwe.en the interior of an 
insect and the external environment. ,Continued existence of the organism
37
is dependent upon the ability of the cuticle to permit passage of some sub 
stances through it and prevent that of others. Inisuch a complex animal 
as the insect, other membranes are also important in permeability. For 
example, underlying the cuticle as a layer of cells called the epidermis 
the influence of which on the permeability of the cuticle is not knownJ' 
Also there are membranes surrounding various organs.. The influence of 
these membranes on the ability of-foreign agents to pass into the organs 
after these agents' have gained entrance into the insect is not known. The 
study of these various membranes is a basic branch of physiology.
From a study of such membranes, information of two kinds is avail­
able : -
1. Knowledge of the structure of the membrane.
2. Knowledge of the mechanism which enables living bodies to 
maintain their composition and function.
Permeability measurements give information which could be used to 
predict the relative ease with which various types of chemicals could 
penetrate the cuticle, and cells under a ,certain definite set of condi­
tions. When confronted with a group of insecticides, it should be possi­
ble to predict that some will penetrate the cuticle rapidly while others 
slowly if at all. This has not been realized as yet, because too little 
is known about the structure of the membrane through which these materials 
must pass.
An agent will not necessarily kill an insect once it has gained en­
trance into the. body. The substance must act upon some vital component
■ ' ' ' ' 1 
of the cell. This immediately leads to a study of cellular activity in
38
39
relation to permeating materials and hence into cellular ^ physiology.
It is well known that certain compounds do not penetrate the cells. 
Their action must therefore be confined to the surface of the cell. Other 
compounds penetrate.the cells very rapidly: Their action may be on some
component of the cell. Furthermore some substances diffuse into the cell 
according to the laws of thermodynamics. In such instances, substances 
pass from a region of high concentration to one of low concentration, re­
sulting in an establishment of;a.dynamic equilibrium. In other cases, 
materials do not appear_tp behave according to the thermodynamic laws.
Here, there is an accumulation of molecules on one side-of the membrane 
In excess of the amount on the other side. Rather than say that the laws 
of thermodynamics are. not obeyed, it is inferred that in such cases the 
cells concerned supply energy for the transport of the molecules across 
the membrane.„ Evidence that the cell respires actively during the trans­
port of materials across the membrane: supports this contention. The be­
havior of the cell membrane to the penetrating substances may therefore 
be either active or passive and. probably is intimately connected faith its 
structure.
Undoubtedly such phenomena occur in the insect, but insect physi­
ologists have not as yet been entirely successful in their efforts to ex­
plain many of these phenomena associated with permeability to insecti­
cides . . .
The following is a review of some of the factors studied to date 
which have an important bearing on the permeability:of the•cuticle.
A. Chemical Factors
(i) Wax. Layer
Ero'bably the greatest chemical factor governing the permeability 
of the insect cuticle is the presence of lipoidal material in the epi- 
cuticIe in the majority of terrestrial insects. Kuhnelt (1928) was prob­
ably the first to realize the great importance of this material, He 
showed that in mapyinsects it was composed of fatty acids and choles­
terol. When it was.removed'from 'the cuticle by acetone extraction, ■ the 
cuticle of some beetles lost about jjo of its weight as well as its im­
permeability. Kuhnelt's results were confirmed by Ramsay (1935b), Klinger 
(1936), Bergmann (1938), Eder (1940), Richards and Anderson (194-2), 
Alexander, Kitchener and Briscoe (1944b) and more recently by Wigglesworth 
(194-5), Beament (194-5),.Lees (194-7) and Pepper and Hastings (unpublished 
data). These authors indicate thatvthe. cuticle' studied by them owes its 
impermeability to a layer of wax. When this layer is disrupted then the 
permeability of the cuticle is increased.
It has been demonstrated that the wax layer can be'disrupted by ab­
rasion or by extraction in' a fat solvent. Zacher and Kunicke (1931) found 
that.when a cuticle was dusted, its permeability to water was greatly in­
creased. They postulated that the dusts promote an increase .in the water, 
loss by capillary action similar to the actiorrof blotting paper. Germar 
(1936) attributed the increase in water loss through the cut idee as a re­
sult of similar treatment, to the penetration of the particles into the 
intersegmental membranes. This-,".allowed for an. increase in the rate of 
diffusion into the air. Chiu (1939) stated that the action of such dusts
kl
lies in their power to cause lessication by' conducting water away from 
the body surface and by mechanical irritation. Alexander et al (1944b) 
criticize the work of Zacher and Ktinicke by calling attention to the fact 
that dessication is brought about by both Vhydrophylic and hydrophobic dusts
'■j ■
even though water will not rise by capillary action through hydrophobic 
powders. Alexander et al suggested that the increase in permeability is ■ 
due to a breakdown, of the lipoid layer over submicroscopic areas. The' 
breakdown is presumably brought about by the preferential attraction of the 
fatty film by the crystalline forces at the surface of the solid particle.
The simplest and most probable explanation is that offered by Wiggles- 
worth (1945). He showed that the permeability of dusted cuticles increases 
only when the insect is in motion. Insects which have been dusted but 
remained motionless did not lose any more moisture than'did undusted ones. 
Hence the probable explanation for the increase in evaporation through 
dusted cuticles is mechanical abrasion of the wax. Since physicists 
probably cannot supply an explanation for "abrasion", then it is possible 
that the mechanism proposed by Alexander et al (1944b) might also be 
operating here.
The cuticles of different insects vary in their permeability to water, 
even under ordinary conditions. Wigglesworth (1945) and Lees (1947) 
showed that the rate of transpiration through the insect and tick cuticle 
varies with the species. This variation is evident both below and above 
the critical temperature of the wax, and is probably due to differences 
in the type of wax covering the cuticle surface and to morphological 
differences differences of the cuticles. That the-waxes are different is
42
seen in the variation of their "critical- temperature". The differences
may be in their orientation on the cuticle surface, in the close-packing
of the wax molecules and in their hydrophilic properties (Hurst 194l).
Hurst (1948) showed that the lipoids are oriented at tight angles to the
J
cuticle surface, the lipoids are crystalline in form probably associated 
with the protein surface although the nature of the linkage is.not as yet 
established. It is speculated by him that the union resulting in a lipo­
protein '.complex occurs, through the polar groups of the side chains.
The variation in the permeability of the cuticle of various insects 
above the "critical temperature" is probably due to morphological varia- . 
tions because the orientation of the wax above this temperature is prob­
ably destroyed.
In at least some insects, the effect of the wax film on cuticular 
permeability is very subtle. Hurst (l94l) and Beament (1945) observed 
that the" cutidle of Calllphora and exuvia of Rhodnius respectively are 
asymmetrical with respect to"the permeation of water through them. ■ Such 
an asymmetry is apparently al&o present in the skins of frogs (Kfiger 
1942). In insects there is a one hundred fold increase in the permeability 
of the membrane-' in the direction of lipoid layer to chitin layer (Hurst 
194l). He suggested that this asymmetry is partly regulated by the lipoid 
layer and further states that it has the special function of maintaining 
a physiological balance in each particular cuticle. This•balance may be 
destroyed when the wax is disrupted or when the wax of one cuticle comes 
into contact with that of another. Yonge (1936) observed a similar pheno­
menon. He showed that the passage of O.IZ Ba (OH)g through untreated
k3
cuticles of the foregut'of Homarus is inhibited'in comparision with that 
of KOH, by almost twice as much in the direction epicuticle to chitin 
layer - than in the reverse direction. The passage of chloride ions was 
also more inhibited than that of divalent cations in the direction epi­
cuticle to endocuticle. When such cuticles were suspended in air, he 
found that fat solvents penetrated them faster in the epicuticle to endo­
cuticle direction.
(ii) Aromatic substances
Although the wax layer appears to be one of the more important '• 
chemical factors governing the permeability of the cuticle, it is by no 
means the only factor. Undoubtedly, factors not as yet too well known 
are also important. Pryor (1940) first suggested the mechanism by which 
the insect cuticle hardens. This appears to be brought about by the in­
corporation of phenolic substances into the cuticle. He also suggested 
that "a reduction in the extent to which the epicuticular proteins are 
'tanned' is probably one of the factors responsible for the hydrophilic 
nature of these epicuticles". Since part of the endocuticle is also 
tanned, this suggestion would also apply to the endocuticle. Eder (1940) 
has found considerable variation in the amount of sclerotization with 
the species of insect. To what extent this factor influences the per­
meability of the cuticle*when all other morphological factors are held 
constant is not known. Wigglesworth (1948) believes that water is pre­
sent throughout the endocuticle and in many insects, this water occurs 
even in the epicuticle up totthe wax layer.
B. Morphological Factors
(i) Pore canals
1 , ■ .
It has long been suspected that the pore canals might be impor­
tant factors, in cuticular permeability. Since it is known that some cu­
ticles have chitinized pore canals (Eder 1940, Dennell 194-6) then ob­
viously only those which are unchitinized are of interest here.
Kuhnelt (1928) and later Eder (1940) found that orthopteron and 
hemipteron cuticles show a high degree of permeability under natural 
field conditions. These cuticles were further characterized by the pre­
sence of well-developed pore canals. Eder (1940) found that when the 
pore canals were poorly developed as in the beetles, then the cuticular 
permeability was likewise' decreased. The beetles were also more heavily 
pigmented. One is therefore at a loss as to how much of this decrease 
in permeability should be attributed to the poorly developed pore canals.
Wigglesworth (194-2) found that when the cuticle of Rhodnina is 
stretched, the penetration of pyrethium.is increased. During'the process 
of stretching the diameter of the pore canals is increased. This layer 
diameter presumably accounts for the increased permeability. Accompany­
ing the increase in pore diameter is a decrease in cuticle thickness. 
Consequently, the increased permeability may not be due entirely to. the 
former factor.
(ii) Cuticular thickness
Several authors have suggested that the thickness of the cuticle 
influences its permeability although Kuhnelt (1928) and recently Bozkurt ■ 
(1948) found no such correlation.
45
Sodium fluoride particles can penetrate different parts of the 
cuticle of the cockroach at different rates (Hockenyos 1933). The 
rate of. penetration was greatest where the body wall was thinnest. 
Klinger (1936) found that the' resistance of the last iristar of 
Dendrolinus pirii to toxic agents is increased by three fold when the 
thickness of the cuticle is increased. O'Kane et al (1933) found that 
the sclerotized regions of- the thorax and abdomen of Tenebrio were less 
penetrated by pyrethrum extracts and nicotine than were theVLnterseg- 
mental regions.- Usually these regions were much thinner than the 
sclerotized ones. In come insects it is possible to stretch the cuticle 
by givingLbhem a blood meal (Wiggleseorth 1942). In such cases the cu­
ticle become more permeable to toxic agents. Robinson (1942a) found 
that larvae of ticks respond more slowly to toxic agents as they 
grow older, probably due to the increased thickness of the cuticle.
(iii) Pigmentation of the'endocuticle 
Pigmentation of the outer endocuticle is another factor which has 
been thought to influence cuticular permeability. It has been shown in 
the last section that Hockenyos (1933) and O 'Kane et al (1933) found that 
sclerotized regions' of the cuticle offer more resistance to. the penetra­
tion of toxic agents than do intersegmental membranes. Although these 
observations can be attributed partly to■differences in thickness they 
might also be due to differences in amount and extent of pigmentation. 
Eder (19^0) found that the orthopteron and hemipteron cuticles show very 
little pigmentation of the outer endocuticle. They also have a highly
46
permeable cuticle under normal temperatures and pressures. This author 
also'found that cuticles of beetles are only slightly permeable and have 
a highly pigmented outer endocuticle.
C. Effect of the epidermis on cuticular permeability
As has alre'ady been pointed out, very little attention has been 
paid to the role of the epidermis in cuticular permeability. Some 
authors have found that the epidermis is important in this respect and 
a review of their findings is given below.
Kuhnelt (1928).found that injured insects had a reduced cuticular 
transpiration. This was attributed to the displacement of the lipoids 
in the cells. Similar results were obtained by Eder (1940). Ivanova 
(1936) working with the larvae of P. brassicae and sawfly Ptemnus rabesii 
found that the epidermis was penetrated very slowly by liquids when in 
its normal condition. If a 0,5$ solution of anabasine is first applied 
to the cells, then their selective action is greatly diminished and various 
substances penetrate readily. No reason was given for the loss of this 
selective permeability.•
Danielli (19-35) found that the rate.of passage of water and water 
soluble substances is determined by the proportion of monovalent ions to 
polyvalent ions in the' membrane•• If the concentration of Caf ^  increases 
to a critical point, the lipoid1film breaks down and there is a sudden 
irreversible increase in the permeability. Davson (1941) found that if 
erythrocytes are exposed to glycolytic poisons,their permeability is 
apparently due to an accumulation of an intermediary metabolite which 
appears to modify - the ,membrane structure sufficiently to increase the
47 , .
permeability of the cell. Danielli (l94l) found that if a nerve cell is 
stimulated, its permeability is increased. This increase was attributed 
to a revision of the close-packed lipoid film resulting in a production 
of gaps between the micelles of the close-packed molecules. These gaps 
became large enough to permit the passage of ions. It is well known that 
substances which depress the metabolic activity of the cells will in cer­
tain instances increase the cell permeability but no direct relationship 
has yet been established.
D. Characteristics of the penetrating materials 
It is doubtful that the penetration of materials through the cu­
ticle will be wholly determined by its physicochemical and morphological 
structure. It has" been found that the physical and chemical properties 
of the penetrating materials and the conditions under which penetration 
occurs is also very important.
(i) Variation'in the rate of solution of the lipoid 
materials
If a flea is immersed in refined■petroleum oil, there is a' libera­
tion of minute droplets of water from the surface of the cuticle (Wigglesr - 
worth- 1942), If lighter oils are used, the appearance,of water droplets 
occurs much faster. This difference in the ease with which water is 
'drawn out of the cuticle was partly accounted for on the basis of dif­
ferences in the rate of solution of the lipoid material. He also found 
that the entry of dissolved pyrethrum can be increased by using oleic or 
other fatty acids. The.mechanism by which oleic acid acts upon the wax 
is unknown. Wigglesworth suggests thatthe partition coefficient of the
substance between oil and water is very important in determining the rate 
at which it will penetrate the insect tissues. Gregarine protozoa in the 
gut of mealworms are apparently more permeable to substances of high 
lipoid solubility (Adcock 1940, Hutzel 1942) even when these substances 
have large molecules.
If the silkworm is first exposed to oleic acid the rate at iwMch 
alkalis penetrate the integument is increased (Klinger 1936)'. O 1Kane et 
al (1940). found that various liquids penetrate the cockroach integument 
at different rates. There was considerable speculation on the part of 
these investigators as to the effect of these penetrating substances on 
the fatty covering and also as to molecular size upon the rate of pene­
tration. Different types of oils vary in their ability to disperse the 
wax layer. Sulfonated oils were found by Fulton and Neale (1942) to be 
very effective in increasing the penetration of derris into the, cuticle 
of'the harlequin bug. This effect was accelerated if acetone was added 
to the oil. Robinson (1942a) found mineral oils to be more efficient in 
this respect than vegetable oils.
(ii) Dissociation of the penetrating materials 
Most authors report that undissociated materials penetrate the cu­
ticle much more readily than do dissociated substanc.es. Hoskins (1932) 
placed mosquito pupae in a solution of sodium arsenite which was adjusted 
to acidic and basic conditions. This was equivalent to ..'Comparing equal 
concentrations of solutions of arsenious oxide and' sodium arsenite. The 
arsehious oxide was found to be four and one half times more toxic thah. 
sodium arsenite'. This was true over a range of 0.01 M. - 0.03.M. ’ This
48
phenomenon was explained by Hoskins by assuming that undissociated mole­
cules penetrate the cuticle faster than ions of strong electrolytes. 
Alexandrov (1934, 1935) also found that undissociated molecules such as 
acetic acid etc. penetrated the cuticle of certain aquatic dipterous larvae ■ 
more readily than hydrochloric acid and sodium hydroxide. Richardson and 
Shepard (1930) found that the toxic action of pyridine, methyl pyrrolidine 
in aqueous solutions to the larvae of C. pipiens is directly related to 
the concentration of. undissociated molecules. In 1945 Richardson showed 
that the. rate of penetration of nicotine through the cuticle of_the Ameri­
can cockroach is greater from molecular than, from ionic solutions;
Hurst (1940) found that slightly dissociating compounds with a high 
dielectric constant penetrate the lipoid layer of Calliphora much more 
readily in the presence of apolar substances of low dielectric constant.
This increase in the permeability of the cuticle to these substances occurs 
in both directions, but more in the lipoid to endocuticle direction than 
in the reverse.
The only deviation from this general trend is that reported by Yonge 
(1936) who found that the rate of penetration of fatty acids and hydro- 
choloric acid through untreated cuticles of'the foregut of Homarus was in 
the order of their degree of dissociation. Yonge also followed the pene­
tration of glucose and found it to depend upon the isoelectric point of 
the epicuticle and chitin. In fresh untreated cuticles, the penetration 
was notable lower at the isolectric point of the epicuticle Alexander and 
Trim (1946) found the cuticle of Ascaris to be impermeable to colloidal 
particles and soap micelles of 5OA0 in diameter.
50-
In determining;-.; the effect of the variation of pH of a solution on 
the ability of this solution to penetrate the cuticle, most of the above
I '
authors immersed their insects into the solution. Since it is not known 
to what extent the variation in pH of a solution disrupts' the cuticle, 
the above result must be taken with some reservation.
(iii) Detergents
The wax layer "of most insects can be disrupted by specific deter-, 
gents and emulsifiers. Wigglesworth (1945) and Beament (1945) found this 
to be true. These authors point out that the efficiency of a detergent 
in this respect depends upon a proper balance between its hydrophobic and 
lipophilic characteristics. If the detergent is too hydrophobic 
it might supress the rate of permeation of water through the membrane.
On the other hand, it might be too hydrophilic but’may not have suffi­
cient affinity for the lipoid layer.
Their work suggests that in general, it is the oilrsoluble substan­
ces with certain hydrophilic properties that are most effective. This was 
found to be true in the case of a series of polyglycerol esters of the 
coconut fatty acids. This does not always hold true, however. Other cases 
indicate that detergents of particular chain, length may associate with the 
long chain of the wax thus bringing about its dispersion. Beament (194-5) 
suggests that it is the highly lipophilic detergents' that are most ef­
fective' since they can accommodate a greater concentration.of was. All 
of these suggestions are merit'ous and the effectiveness of detergents 
can probably'be attributed to all of them rather than to any one in.par­
ticular.
51
(iv) Association between toxic agent and wetting agent 
The investigation of Alexander and Trim (1946) has thrown consider­
able light on the mode of penetration of toxic agents in the presence of 
surface active agents. They found that the penetration of hexyl resor­
cinol in the presence of a soap solution through the cuticle of Ascaris 
depended upon the concentration of the soap. The penetration of the 
toxic substance was inhibited above a certain concentration of soap.
Measurements of interfacial tension by,these investigators at a 
nujol oil-soap solution interface showed that maximum penetration occur­
red at the minimum interfacial tension. At-this, value of .interfacial ten­
sion, there is a micellar aggregation of soap molecules which combine with 
the active agent to form a complex. The drug at this soap concentration 
distributes itself between the micelles and any other interface (here 
Ascaris/water). The maximum biological activity occurred at the soap . 
concentration.which is critical for micellar formation. They suggested 
that this simple picture of competition between micelles and a biological 
interface will probably be applicable to all biologically important com­
pounds when present in aquedus soap solution. Furthermore, in' making a 
general application it is important to remember that modifications will 
occur due to variations in interaction between chemical groupings on the 
biological interface and the soap solution or drug molecules.
(v) Temperature -•
Temperature at which the orgainism is kept appears to be very im­
portant in determining the rate of penetration of the material into the 
orgainsm. Hastings and- Pepper (1939) found that the toxicity of meta
7
52
sodium arsenl-te to Mormon crickets increases with increasing temperature. 
They suggest that this is due to a change in the permeability of the 
cuticle. Furthermore, this effect was more pronounced in the case of 
adults than in nymphs. Fan, Cheng, and Richards <1948) found that the 
low concentrations of D.D.T. penetrated the arthropod cuticle more effect 
ively at low temperatures. The penetration of the cuticle is apparently 
due to adsorption of the D. D. T. out of solution by the body wall.
53
METHODS M D  MATERIALS
All of the experiments 'described herein, were performed on grass­
hoppers , M. hiYittatus, in the- various stages of development or on por­
tions of the cuticle of this grasshopper.
• • • The eggs were collected in the vicinity of Billings, Montana.
These were brought into the laboratory and, after being in contact 
with moisture for 2k hours at room temperature, they were placed in a 
constant temperature cabinet at approximately 100% R. H. at 2 J . O ^ . ^ C . 
The rearing chamber, 12" x 6" x 4", was made of Plaster of Paris. The 
open top of this chamber was covered by,a black plate of glass. Two. 
glass tubes, approximately 6" long and 2" in diameter and closed at'one 
end, were fitted into two holes in one side of the chamber. As the . 
grasshopper's hatched, they congregated in these glass tubes.
When the.grasshoppers hatched, they were transferred from the 
above small tubes into glass tubes approximately 18" long and V  in 
diameter. Both ends were closed with a screen cap. These tubes were 
kept at a temperature of 85.0^.5°F.. and approximately 35% R. H. This 
temperature proved to be quite satisfactory because a low mortality and 
a rapid development was obtained. Under these conditions, adults were 
obtained in about 30 days. After the grasshoppers reached the fourth 
instar, they were transferred to screen cages, the dimensions of which 
were 12" x 6" x 6". The" animals were fed once a day on a mixed diet of 
dandelion and grass when available and lettuce when the former Were un­
available . r
When the supply of grasshoppers was depleted, additional quantities
were obtained- from the Grasshopper Research Centre, U.S.D.A., at Bozeman,
-Montana. These grasshoppers were reared in a manner similar to the one
already discussed. In no case, was it possible to detect a physiological
!
difference which could be attributed to differences in the source of 
grasshoppers.
The rate of evaporation of moisture through the„ intact cuticle and 
of distilled water through portions of a cuticle, used as a membrane, wa,s 
measured in dry air in an apparatus similar to the one described by 
Wiggleswof fch (19*1-5). Only those grasshoppers which had passed their 
third day after moulting> were used. This was found to be necessary due 
to the continued secretion of the endocuticle and hardening of the cuticle 
during the first few days after ecdysis. ' -
The grasshoppers were killed in hydrogen cyanide to which was added 
a few drops of ammonium hydroxide. This insured rapid death and prevented 
the loss of body fluids by regurgitation. The'grasshoppers were then 
placed into a small basket made of metal gauze. The basket and dead 
grasshoppers were weighted on a calibrated analytical balance and then 
suspended in a conical flask, the bottom of which was covered by approxi­
mately one-half inch of phosphorus pentoxide. This large dessicating 
surface kept the atmosphere quite dry around the insects ^ The flask was '■ 
immersed into a constant temperature bath. The temperature within the 
flask was measured by a thermometer the bulb of which was near the basket 
containing-the grasshoppers. To minimize the effects of a humidity grad­
ient in the immediate vicinity of the insects, the air in the chamber was 
constantly kept in motion by a mechanical glass stirrer propelled'by
figure I. Photograph of apparatus used in measuring water evaporation.
. ..,v T? '.'AVri-"."'.'" “
compressed air. Although the speed of this stirrer was not controlled 
precisely, it was possible, after some experience, to maintain its motion 
fairly constant during the course of the experiment. A photograph of this 
apparatus- is seen in Figure I.
In the transpiration study to. determine the "critical temperature", 
i.e. the temperature at which the rate of increase of rate of evaporation 
was a maximum, a quantity of dead grasshoppers equivalent to 0 .8  - I gram 
was used. This meant that less grasshoppers were used as they grew older. 
For example, about 50 first instar grasshoppers were used, and only 2 - 3 
adults. The difference in the weight of the grasshoppers before and after 
exposure to the dessicating atmosphere for ajoeriod of time was assumed to 
be due to the loss of , moisture by evaporation, predominantly through the 
cuticle. Such an assumption! is justified in the, light of the work done by 
Mellanby (1932) and Gunn (1933)• The rate of loss of body, moisture was 
expressed in mg/sq. cm/hr. .
The method used to determine the surface area was similar- to that 
described by Simanton (1933)• The insects were weighed in a weighing 
bottle to the nearest tenth of a milligram. After they were killed, the 
head, legs, and antennae were removed. The abdomen and thorax were cut 
open and the viscera removed. The fat body was cleaned away by swabbing 
with cotton. The cuticle was then but up into various shapes and placed 
between two glass slides. The slides were inserted into a microprojector 
and the image, which was reflected on a sheet of. paper, was traced out. 
Since only one side of the legs, antennae, and wing pads was measured, the 
area of these parts was doubled. The apparatus, is seen in Figure 2.
56
57
I
Figure 2. Photograph of microprojector as used for measuring surface
areas.
WrrTBMsVW^' -
58
The area of all the images, traced out on paper, was determined by- 
means of a planimeter. The planimeter reading was reduced by a magnific­
ation value thus giving the actual area. The sum of all the areas of the 
parts gave the total area of the grasshopper.’ In this manner, a total of 
sixty-six grasshoppers was measured.
In order to avoid this laborious procedure in all subsequent■work, the
data already obtained were fitted into Meeh's formula which relates the
surface area to the weight, i.e. S = k Wn where
S = surface area in sq..cms. ¥ = weight in grams
k = constant for the species ‘ ir= power of the weight 1
Evaporation from a free water surface was measured by noting the am­
ount of water lost from a small cell when the latter was exposed for a per­
iod ,of time in dry air. The cell was made from aluminum, so only a small 
quantity of. heat, was required to heat up the ceirjjn comparison to that 
used to heat- up the water in the cell. The cell was fitted with a screw 
■ cap, the centre of which had a hole of the same diameter as the inside of 
the cell. This cap with its narrow flange fitted tightly against the brim 
of the cell. Thus it was possible to place a membrane over the open end of 
the cell and have it held securely in position by the screw cap. The area 
of the evaporating surface was 0 .$l6 cm^.
The electrical circuit used for the measurement of the dielectric con­
stants of beeswax consisted of a capacitance bridge, type Tl6-C, to which 
was connected an audio-oscillator, Hewlett and Packard model. The ampli­
fier and null detector, type 1231-A, used to detect the balance point, was 
connected to the audio-oscillator. The balancing capacitor was a, Precision
Condenser of type 722-D.
59
The 716-capacitance bridge is essentially a Schering bridge which 
over a frequency range from 30 cycles to 300 kilocyles is a direct read- 
ing type. Here, it was used for substitution measurements by the addi­
tion of a variable balancing condenser. All the measurements were ob­
tained by the substitution method. The error in the capacitance was the 
larger of ^0.2% or ^2u.u.f. These accuracies held for the frequency range 
30 cycles to 300 kc. provided that the difference between the operating 
frequency and the range selecting frequency were not greater than a fac­
tor of three. The circuit wiring is presented in Fig. 3 .
O  GEN O
Schematic diagram of circuit used in dielectric 
measurements.
Figure 3.
6o
The generator was connected to the posts marked generator detector. The 
voltage was applied to the generator terminals from a power line of ap-. 
proximateIy H O  Volts. At I' kc., a maximum of one watt could be applied 
This allowed for a maximum of 300 volts. The amplifier .and .null detect, 
tor was connected to the posts marked detector. Both were connected by 
means of the Type 274-KE Shielded Conductors. The shielded side of the 
conductors was connected to the terminals marked G. The Precision Con­
denser used as the variable balancing, capaciter was connected across the 
terminals marked,Unknom Direct. Its ground side was connected to the 
terminal marked G.
Since there are considerable disadvantages in making measurements 
by the Direct Method Reading, the Substitution Method was used. This 
meant that the variable balancing condenser was alwa^p in the circuit 
and the unknown capacitor was substituted for part of it. The Method 
switch was set attsubstitution and the Range Selector either at the fre­
quency of the audio oscillator or at one near it. In this case, the 
frequencies used for M = I  was such that the,Range Selector frequency 
corresponded tti the one of the audio oscillator. When.M = I the capaci­
tance was read directly. The Capacitance and Dissipation factor dials
were then alternately adjusted until a sharp balance was obtained. Then 
’ '
the unknown capacitor was connected into the terminals marked Unknom 
Substitution. The bridge was re-balanced and the capacitance was read 
directly. Other data on .this bridge can be obtained by consulting the 
"Operating Instructions for Type rJlG-O Capacitance Bridge General Radio 
Company".
6l
The cell used for the measurement of the dielectric constants of 
beeswax consisted of three concentric brass cylinders held rigidly in 
position by small ceramic spacers. The cylinders were gold coated and , 
the inner and outer cylinders were connected together and grounded, 
while the middle cylinder remained at a high potential. The outer 
cylinder was the containing vessel. Through it water was circulated,
.at a constant temperature, from a thermostat.. The volume of this cell 
was 100' ml. All measurements were made at a frequency of I kc.
The apparatus used for the determination of the surface areas of • 
grasshopper cuticles was also used, with slight modification, for mea­
suring the contact angles of droplets of distilled water placed directly 
on the cuticle surface. It is similar to that used by Ebling (1939).
■ The apparatus consisted of a lamphouse, attached to an optical 
bench which contains a groove permitting tie lamphouse to be adjusted 
in any desired fashion.. The system which condensed the light rays, 
emanating from a six volt bulb in the lamphouse, was attached to the 
optical bench in front of the lamphouse. For coarse focussing, the whole 
condensing system was moved, but finer focussing was achieved by opera­
ting a small handle in a helical slot. A glass slide was glued to a 
cork which was held in position by a clamp connected to the verticle 
rod of a ring stand. This was placed in front of the condensing system. 
Onto this glass .slide was placed a cork board platform with two vertical 
sides, each having a groove. The grasshopper was placed into this groove 
and, because of the angualarity of the groove, the surface of the grass­
hopper upon which the droplet of water was placed, was level. The
v
62
Figure 4. Photograph of microprojector as used for measuring contact
angles.
. ........ . ... ..- .............
63
droplet of water was placed upon the cuticle by means of a micro-pippette 
The outline of the droplet was traced out on a sheet of paper and 
measured. Careful observation of the drop image showed that there was 
no detectable.evaporation during the time necessary to trace the image. 
The temperature of the air.in the immediate vicinity of the drop, as 
measured by a thermometer was .750F • at all times. There was negligible . 
heating of the drop by the light source because the latter was adjusted 
to be about 12" from the object, and because the light source was fund-.' 
tioning only during the very short time when the drop image was traced. ■ 
A photograph of the apparatus is seen in Figure 4.
6k
r e s u l t s mu Discussion
I ’ Determination of the Surface Area of M-, Mvittatus.
Much of the recent insect physiological work dealing with metabolic 
rates and transpiration has been expressed in terms of the body surface 
of the insect. Such studies necessitate the calculation of the insect's 
surface area. This is usually carried out by direct measurement with a ■ 
subsequent application of Meeh1s formula or some statistical treatment.
The determination of the surface area of M. bivittatus was'-under- 
taken because it was very essential to the interpretation of the trans­
piration studies. The general procedure has already been outlined. Once 
the surface areas were calculated, they were fitted into Meeh's formula 
,S-= kWn .
. The use of the formula depends upon acknowledge of the values of 
the constants k-and n. Consequently, for a particular value of "n", the 
average of "k" was calculated by using the previously determined surface 
areas and weights. This was done for "n" from 0.4 to 0 .9 . The standard 
deviation of the ave. value of "k" was then obtained for each value of
"n" Dy using the formula (T" =T/ £  (d2) , where d = deviation of a single
• V a(n-l)
observation from the mean and n - number of observations. This deviation
was then expressed as a percent of the average value of "k", i.e-.
(T x 100 where ■ a-standard deviation for each value of "n". This 
k
gave a value which determined the degree of variation of each individual 
value of "k" from the average. The percent standard deviation was then 
plotted against the exponent value^"n". The value'of "n" for which the 
standard deviation, expressed as a percent of the average "k", was a
'65
minimum^ was "bhe n which, best fitted the data; The coarresponding value 
of "k" was likewise the most appropriate- value.
Once these constants were determined and the formula established, 
it was only necessary to weigh the insects and then calculate the sur­
face areas by the established formula. The determinations reported here 
were carried out on grasshoppers in the various stages of development.
The results are presented tabularly in Tables I, II, and III, and graphi­
cally in Figures 5, 6, and rJ.
It is clear from Table II and Figure % that the smallest percent 
standard deviation of the average value "k" occurred for the exponent 
value O'.71 and 0.72: Probably the best value would be O .715 but it is
felt that such accuracy is unwarranted. Consequently, the value which 
was chosen was O .7 2 with the corresponding average' value for "k". This 
was done merely for convenience. All subsequent surface areas were cal­
culated by S = 10.27 W'72.
Using this formula and the grasshopper weights already available, 
the surface qreas were recalculated and compared with those obtained by 
dissection. The data are presented tabularly in Table I and graphically 
in Figures 5 and 6 .
It is obvious that considerable discrepancy exists, in. some in­
stances, between the observed and calculated surface areas. This is 
attributed to the technique by which the surface areas were originally 
obtained. Although the variation is large in some instances, it is 
felt that the formula S - 10.27 . is a suitable expression of the data
in view of the large sample used.
66
; ' .Table I
Comparison of Calculated and Observed Surface Areas 
of the 'Two-Striped Grasshopper.
Hopper Weight of Grass- Observed Surface I Calculated Sur- I
No. hopper in mgms-„ Area in sq. mm. • S face area 70 - 
I S= 10.27 W <2 I
i . . 82.1
2 27.2
3 : . 2 6 .3
I h . - 28.U
I ' 3 1U .9
6 UU.6
7 ' ' 3 3 .9
8 _ .6 .U
9 . UU.3
\ io 10.0I 11 .10.2
j ■ - 12 .73,U
I . '13 . .3 2 .8
lit ' 3 6 .3
I ' 13 " 3 9 .8# 16 8 2 .3
j ' '17. 2 7 .8
I 18. 137.1
I
19 3 3 .3
20 7U.3I ■ 21 ' 7 9 .9
| ,22 ; 7 9 .8
I  ■
.23 ,113.7
2k . 119.7
I 23 ...?U.8
I 1 26 101.8
I 27 7 3 .9
I
28 ' 10.0
29 10.0
30 7 3 .U
31 3 2 .8
32 3 6 .3
; 33 . 3 9 .8
\  3b 8 2 .3
\ 33 2 7 .8
36 . 137.1
' 37 3 3 .3
38- 7U.2I7: '39 ■ 80.1
•1 ■ 'bo . .7 9 .8
. Ul 113.7
2 1 2 .6 2U3.3
9U ,37 110 .8
71.86 108.1
90".28 11U.2
.7 2 .UO 8 i'.9 3
/ 1UU.3 1 3 8 .3
/  1 3 8 .6 1 81 .3
, U2.2? 3 9 .0 8
' 1 3 8 .9 1 3 7 .9
33.U7 3 3 .9 0
33<38 3U .63
191. U 2 2 ^ .2
1 7 8 .8 178.6
1 3 9 .7  ' 1 3 6 .9
'1 9 3 .3  ' - 1 9 3 .3
2 0 3 .2 . . 2U6.3
'113 . '3 ' 112.3 ■
3 9 8 .9 3 3 3 .0
' 129.7 • - 1 3 3 .6
2UU.3 - - 2 2 8 . 3
210...9. 2U0.7  '
2 6 3 .3  . . 2U0.U
.3 2 3 .3  - 3 1 0 .2
2 9 3 ,0 3 2 1 .9
' .283 ,1 2 7 2 .1
2 9 0 .6  . 286.U
1 8 9 .7 2 2 7 .3
3 3 , U6 # . 8 9
3 3 .3 8  ' . 3 3 .7 9
. 191.U 226 .3
.1 7 8 .8 1 7 8 .6
- 1 3 9 .7 1 3 6 .9
19U.3 ■ •. 1 9 3 .3
\ 2 0 3 .3 ' 2U6.3
- .H 3 .3 .  . . 1 1 2 .6
3 9 3 .3 " 3 3 U .9
1 2 9 .7  . 1 3 3 .6  .
. 2j;U.3 2 2 8 .2  .
2 1 0 .9 2U1.1
2 6 3 .3 2U0.U
3 2 3 .3 310.1.
. 61
Comparison of Calculated and Observed Surface Areas 
of the Two-Striped Grasshopper.
Hopper 
Ho. ■
Weight of Grass­
hopper in zngms.
Observed Surface 
Area in sq. mm.
Calculated Sur­
face .area 
S = 10.27 W ° u
ii2 119.7 295.0. 321.9
W 94.8 283.1 272.1
M 101.8 290.6 286.4
h$ 73.9 189.7 227.4
k 6 82.3 254,5 245.8
hi 70.5 168.9 219.9U8 65.2' - 273.4 .- 207.8 «
U9 lU l.6 .334.2 363.3
£o 183.8 '438.8 438.4
hi 274.9 ■ 643.1 585.7
hz 119.5 . 385.6 321.5
.S3 ' 111.9 ' 299.8 306.6
Sh 229.0 533.1 513.6
SS 227.2. 576.7 510.6
214.9 584.0 490.6
Si 157.3 355.9 391.8
58 186.7 473.9 443.359 87.8 383.0 . 257.5
60 100.0 350.5 282.861 130.3 358.6 342.1
62 134.0 373.9' 349.1
63 633.7 . 922.2 1066.0
6h 212.1 417.1 486.0
65 ' ' 378.6 739.6 737.565 226.8 550.3 510.0
I
I ■
\
\
S
u
r
f
a
c
e
 
a
r
e
a
 
i
n
 
sq
.
700 - -
5oo --
0 IiO 80 120 160 200 2li0 280 320 360 h00 IiIiO IiSO $20 560 600 6Ii0
Weight in mgms.
Figure 5» Plot of observed surface area vs, weight
of grasshoppers.
S
u
r
f
a
c
e
 
a
r
e
a
 
in
 
sq
. 
m
m
s
1000 --
900 --
800 - -
700 - -
600 --
Soo =•-
I4OO -■
300 - •
0 I4O 80 120 160 00 2h0 280 320 360 I4CO I4I4O I4BO $20 560 600 SI4O
Wei hi in mgras.
Figure 6 . Plot of calculated surface area vs. weight
of grasshoppers.
s
10
0
X
a *
Figure
13 -- 
12 - •
11 -- 
10 ■ *
9 -- 
8 - -  
7 ■■
6
5 - 
Ii ■■
3 "
2
I ••
0 —
0 .1  .2 .3 oh .5 .6 .7 .8 .9
Exponent value
7. -Iethod of determining the best value of the 
exponent.
71
Table II
Determination of the' Best Value of the’Exponent
Exponent 
Value - n
Average
k
I  Standard Deviation!
I ( Cf) of k I x ioo
O.lt :k5.82 . 5.^7 '■ 12.16
0.5 26.89 2.62 9.7k0 . 6 17.3k 1.19 6 .8 6
0.69 - 11.70 . 6k 7 ■ .5.53
0.70 ■ 11 .2 2 .595 5.30
0.71 10.71 .565 5.27
0.72 ■ 10.27 .5k2 5.27
0.7k 9.k2 .507 . 5.38
0.8 - 7.30 / «kl6. ; 5.65
0.9 k.76 .k07 . 8.55
I
72
Although only two adults were originally used in the calculations 
because of the lack of insects in this stage of development, the estab­
lished formula is applicable to the adult stage also. This can be seen 
by taking the surface areas tabulated in Table III and locating.them on 
the graph in Figure 5•
If these results are now located on the original curve, (Figure 5),
/
the points will fall about the line already drawn. Thus, the curve shown 
in Figure 5 and represented by S = 10.27 W *?2 best represents the varia­
tion of surface area of M. bivittatus with weight throughout the various 
stages of development.
The method used here for the- determination of surface area is prob­
ably applicable to surface area calculations of most insects. If the in­
sect can be cut up into pieces, then its surface area can be obtained, 
and if sufficient numbers are used, the data can be expressed mathemati­
cally with a fair degree of reliability. The difficulty of measuring the
1
surface areas will vary from species to species. Those with a smooth 
exoskeleton would lend themselves readily to such treatment. On the other ■ 
hand, those with a convoluted exoskeleton present considerable difficul­
ties for it is an extremely difficult undertaking to account for all the 
convolutions of the exoskeleton.
For most species of insects, the value of "n" probably lies in the 
neighborhood of .6 6. Simanton (1933) found that for the German cockroach 
n = .63 and for the bean aphid n = .6 0. The value of k, on the other 
hand, will vary with the species, depending upon its shape, size and other 
pecularities of body structure. For example, Simanton (1933) found that
. : '-r U1 ■■
. ' 73
• Table III
;
Some weights of adult grasshoppers with the corresponding surface area
Surface area from
Weight in mgms. S = 10.2? W-72 in
sq.- mm.
L86.2 883.0
487.3 . 884.3
347.8 . 693.8
434 <; 4 ' 813.0
336.2 644.4
217.0 923.0
723.7 1271.0
663.3' 1104.0
603.6 - / 1032.0
262.2 / - 984.4
717.7 . ; 1168.0
I
I
7k
for--the German cockroach k = 12.17 while for the bean aphid it- is 3 .2 8. 
Furthermore, some authors, especially those calculating the surface area 
of mammals, have introduced another factor by which the surface area is 
multiplied. This factor deals with the degree of fattiness and state- of 
nutrition of the animal and is usually denoted by the symbol "f".- Un­
doubtedly, this factor 'is important but no attempt was made here to use 
it, mainly because no method was available by which the state of nutri­
tion of the insects could be assessed. Furthermore, such an' undertaking 
is beyond the scope of this study and would probably constitute a sepa-' 
rate problem.
II. Determination of the presence of a-Wax Layer.
This experiment was undertaken to determine whether the cuticle of 
M. bivittatus is covered by a su;p;e--rficial lipoid layer as it is in so 
many terrestrial insects and other arthropods (Kuhhelt 1 9 2 8, Eder 1940, 
Wigglesworth 1949, 194?, 19-48 & Lees 1946, 1947). The grasshoppers used 
in this Study were in the adult stage and were reared in the laboratory,
As has been shown on numerous occasions, the-waterproofing capacity 
of the insect cuticle resides mainly in the superficial lipoid layer cover 
ing the cuticle. It was reasoned, therefore, that the destruction of this 
lipoid layer, if present in this insect, should result in an increased 
permeability of the cuticle, to body moisture. To check this, the loss 
of body moisture through the cuticle.of dead insects, in a completely 
dry atmosphere was determined after various treatments.
The insects were killed as previously described. Their ...mouthparts 
and anus were occluded with cellulose paint, and after being-weighed,
75
they were exposed to dry air for fifteen minutes at a constant temperature. 
This particular period of exposure was'-chosen for convenience, since it 
was found that, within limits, the rate- of evaporation through the cu­
ticle was approximately the same, regardless' of the length of the period 
of exposure. This is seen in Table IV. These data indicate that the 
evaporation per minute through the cuticle is approximately constant re­
gardless of the length of exposure to the dry air. Apparently, equilib­
rium is achieved very rapidly. The procedure of converting all evapora­
tion data to an hourly basis is therefore justified. This linear re­
lationship between evaporation and time.becomes even more striking in 
Table V.
In the experiment, the results of whidh are given in Table V, a 
new lot of adult grasshoppers was used for each trial. This prevented 
any undue depletion of body moisture as the period of exposure was in­
creased. When the data are converted to an hourly basis, it is seen 
that the percent loss of moisture per hour is constant.
A limited area of several cuticles was abraded very.gently with a 
sharp razor blade and the■cuticles then exposed to a dry atmosphere. The 
loss of body weight increased greatly over that of the untreated cuticles. 
When several cuticles were treated with cold chloroform for ten minutes 
and then exposed to the.dry air, the permeability of the cuticle was 
likewise greatly increased. In view of the■electron microscope studies 
by Richards and Korda (1948), it is highly improbable that the trans­
piration^ as a result of the latter treatment, could be attributed to 
the general disruption of the matrix' of the. .cuticle.
>
II
76
Table IV-
Loss of weight of < untreated adult grasshoppers at i;0oC exposed. to dry- 
atmosphere for varying periods of time
Length of Expo­
sure period in 
minutes
Initial Wgt. of 
2 grasshoppers 
in gms„
Surface area 
of grasshoppers 
in sq. cms.'
Decrease in 
Wgt.
1.5075 1 9 .9 5 .do5o
10 1.5995 ' 1 9 .8 7 .0075
15 1.5920 1 9 .8 0 <0103
30 1.5817 19.70 .0222
Rate of Eva­
poration i 
mg/cm2/min.
Decrease in 
Wgt./hr. in 
gms. ■
Percent loss 
of moisture 
/hr.
..5o .o58o 3.2
.37 .0555 3 .0"  -
.35 .0512 2 .8
.3 8 .o555 3 .0
I, 77
'
■ ■ Table. V '
• Loss of weight of untreated adult grasshoppers at 30°C exposed
to dry air for varying periods of time'
' Length of ex- I Initial wgtJ Decrease in'Percent loss ’Percent loss '
; posure period I in gms. Wgt. in gms, in wgt. in weight
< in hours
I  ' per hour
I 1.2073 .OlliO 1.13 ) .1 .0 3 1 0 .0110 1.03 ) 2.13'1 .1 7 6 8 ' .0122 i;o3 , )
I ■ 1.3267 .0272 2.03 2.03
k 2.8696 .2316 8.07 ')
h 3.06^3 .2931 9.37 ) 2.20
S
I78
Table VT
I
I
Percent loss of weight of dead adult grasshoppers due to exposure to
■ ' ■ ■ '
dry air at ItO0C for 15 minutes following various treatments
I
I
I
I
Treatment
i i I
Initial Weight! Decrease . in Wgtj Percent loss,1. 
in gins,________; in gms» • ■______of body wgt.
Untreated •
Abraded l„li920
Cold Chloroform ■ I „5812
„0121 0,76
.0250 1.7
. 09^3 - 6 .0
Ji
i
I
\
.79
The data in Table VI show that as a result of abrasion there oc- 
cured a two-fold decrease in body weight of the abraded over the unab­
raded grasshoppers. Treatment of the cuticles with cold chloroform gave 
a percent loss of weight of 6.0, which is approximately an eight-fold 
increase in evaporation over that of the untreated, cuticles. It is as­
sumed that this decrease in weight is due to an increase of evaporation 
through the cuticle-.as. a result of the removal, of some protective layer.
It has been established that the insect cuticle contains polyphenols, 
and quinones, (Pryor 1940, Hackman et al, 1948, Dennell 1946, 1947). 
Furthermore, these substances reduce ammoniacal silver hydroxide to a 
deep brown stain (Lison 1946). Following a method proposed by Pryor 
(1940) it was found that the cuticle of M. bivitt.atus contains pyfocate- 
chol of 1:2 dehydroxybenzene (Huntress and Mulliken).. This compound re- 
duces ammoniacal silver hydroxide. Advantage was ..taken of this property 
in subsequent experiments.
Untteated grasshoppers were immersed in a 5$ solution of ammoniacal 
silver hydrokide. No staining was detected over the body surface. ' Only 
the tibial spurs stained a very deep brown. When the cuticle was abraded 
or treated with chloroform for a short period of time, or treated with a 
suitable detergent and then washed in distilled water and immersed in the 
solution, of ammoniacal silver hydroxide, deep brown stains appeared over 
the treated areas within ten minutes after immersion. This is seen in 
Figures 8 and 9*
80
Figure 8
A. Untreated abdominal cuticle
B„ Abraded with razor blade
C. Extracted with chloroform at 2$°C
Surface view of abdominal cuticle of adult M. 
bivittatus stained with ammoniacal silver hydroxide.
81
A» Untreated abdominal cuticle
mm
B. Treated with detergent sodium flLorol" 
sulfate
C. Treated with .detergent. Amine-0
Figure 9» Surface view of abdominal cuticle of adult M„ blvittatus 
stained with ammoniacal silver hydroxide.
82
It is interesting to note that in the case of certain detergent 
treatment, the wax was not removed completely. Rather, it was removed 
in patches, so that upon staining with ammoniacal silver hydroxide, a 
mottled cuticle was obtained. Wigglesworth (I9h5) notes the same thing 
for Rhodnius. Apparently not only do species vary in the ease with which 
the wax could be extracted but also certain portions of the cuticle of 
the same species, will resist extraction with detergents.
Often grasshoppers were kept for long periods of time in screen 
cages. When these grasshoppers were killed and immersed in a solution 
of ammoniacal silver hydroxide, a brown streak appeared very quickly 
along the ventral portion of the thorax and abdomen. This was anparently 
caused by the removal of the protective layer by abrasion by the screen 
surface. .
Abraded and chloroformed cuticles were immersed in concentrated 
nitric acid for 2 - 3  seconds and then in concentrated ammonium hydrox­
ide. Brownish stains appeared over the treated regions. Untreated cuti­
cles did not stain. The extent of staining in a treated cuticle is seen 
in Figure 10.
-3TrE=* '-E!
A. Abraded with razor blade. B. Treated with chloroform at 25°C.
Figure 10. Ventral view of abdominal cuticle of adult M. bivittatus
stained with concentrated nitric acid.
83
It is evident that the euticular materials which will reduce ther 
silver or which will be nitrated by nitric acid, are separated from these 
agents by an intervening protective layer in the untreated cuticle. When 
the cuticle is treated in a manner which will result in a removal of 
parts or all of this layer., the aromatic substances below are exposed. 
They, then, reduce the silver or become nitrated.
The protective layer is waxy in nature. This was shown by immer­
sing a cuticle in a one-half percent aqueous solution of osmic acid, by 
exposing the cuticle to fumes of osmic acid or by immersing it in an 
aqueous solution of Sudan III, which was dissolved in methylal (Dufrenoy 
and Reed 1937). Treatment with osmic acid resulted in the cuticle taking 
on a black Stain, while in the presence of Sudan'III, it absorbed the red 
dye. Either.reaction, is characteristic for fats. The presence of wax 
was also extablished by extraction in chloroform.
It is therefore concluded that the cuticle of M. bivittatus is 
covered by a protective layer of wax, which, when disrupted by abrasion, 
solvation in chloroform or detergent, results in an increased permea­
bility of the cuticle to body moisture.• The.evidence here' accrued sug­
gests that the wax layer is the seat of the property of euticular imper­
meability.
III. Time of deposition of the Wax Layer.
It has been’found in Rhodnius and several species of ticks, that 
the impermeability of the cuticle is well established before the old 
exoskeleto.n is shed (Wigglesworth 19^7, Lees 19^7) • In the case of 
Tenebrio the wax is not secreted until after the insect mounts:
(Wigglesworth 1948). Evidence was presented in the last section that a 
wax layer is also responsible for the impermeability of the cuticle of 
M. bivittatus. It would be interesting to know, therefore, when this 
layer is secreted.
Wigglesworth (1947) and Lees (1947) investigated this problem Tgy 
periodic staining of the new cuticle with ammoniacal silver solution. 
This enabled them to work out the details of wax secretion with consider­
able exactness. Since the immediate interest here was not concerned with 
such details, it was decided to gather information on this subject by 
determining the variation in the permeability of the cuticle during 
moulting and by measuring the contact angle of droplets of distilled 
water on the new cuticle.
Fourth instar grasshoppers which were moulting were used in the 
following experiments. They were killed as previously described. Since - 
the skin had not been completely shed when the insects were killed, it 
was necessary to peel off the old exoskeleton. The new cuticle was then 
blotted to remove any traces of .^ he mounting fluid. The insects were 
weighed and exposed to dry air, for twenty minutes at 25°C. and re­
weighed. They were then treated.with cold chloroform for a few minutes 
dried, weighed.and again exposed to dry air for twenty minutes. These 
results were compared with grasshoppers twenty-four hours, after moulting. 
The latter were treated in a manner similar to the former.
The data in Table VII show that transpiration through the new cu­
ticle is of the same order of magnitude as that through a cuticle of a 
grasshopper 24 hours after moulting. When the cuticles were treated in
84
Table VII
Percent loss of body weight during 20 minutes in dry air at 2£°C 
of the fourth instar nymphs of M„ bivittatus 
at different developmental stages.
Treatment Age
Initial Wgt, 
in gms.
Decrease 
in wgt. '
Percent 
loss of 
wgt.
Control moulting' .1295 o 0006 0.1i6
Cold Chloroform moulting .13U2 .0390 -33.3
Control 21; hours .1085 o OOOli 0.37
Cold Chloroform 2Li hours .1128 . .0378 33.5
86
cold chloroform for a short time, their permeability increased approxi- ■ 
mately one hundred fold. This phenomenon is attributed to the removal of 
the wax layer as a result of the chloroform treatment. Consequently, the 
wax appears to be deposited on the new cuticle before the old cuticle is 
shed. Thus, the impermeability of the new cuticle is well established, 
probably many hours before moulting, giving the insect adequate protection 
to undue dessication during the moulting process.
Further evidence for this conclusion was obtained by staining the 
intact cuticles in ammoniacal silver hydroxide solution and concentrated 
nitric acid during the moulting process and at various periods after 
moulting. In no case did obtaining of the new cuticle occur. This indi­
cates that by the time the old exoskeleton is shed, the aromatic sub­
stances, if present, are covered by a layer of wax. Consequently, the 
time of wax secretion in this insect is quite different from that in the 
bug Khodnius. In Khodnius, the secretion of the wax layer is not comple­
ted until several hours after moulting (Wigglesworth 19^7).
Some moulting fourth instar grasshoppers were killed at the time 
when the old skin had just split on the dorsal portion of the pronotum.
The old skin was peeled off, the new cuticle dried and the grasshoppers 
placed in the microprojecter. A droplet of distilled water was placed 
on various portions of the new cuticle with a micropipette. After the 
droplet achieved equilibrium, the contact angle was measured. The aver­
age contact angle was found to be 105.9°. This compared favorably with 
an angle of contact of 105.0° on cuticles which were several days old. 
Since it would probably require the.grasshopper about one-half hour to
8?
moult from this particular stage, it is assumed that the wax is laid 
down at' least one-half hour before moulting.
Contact angle measurements were also made on a cuticle treated in 
chloroform. The data showed that the cuticle, in the absence of wax, 
possesses considerable hydrophilic properties. As a result of twenty- 
five measurements, the average contact angle was 78°. It is therefore 
assumed, that the newly secreted cuticle has similar hydrophilic proper­
ties. The low contact angle indicates that the surface tension of the 
chloroformed surface is greater than that of the sum of the tension of 
the liquid and interfacial tension of the surface and liquid.. This large 
surface energy of the protein epicuticle perhaps resides in the polar 
groups of the side linkages of the protein chains which are exposed at 
the interface at this stage of cuticle development. As the, wax is se-' 
creted, the cuticle assumes a hydrophobic character and the contact angle 
rises to 105.9°• At this value, the wax presumably covers the entire 
surface.
The data presented on the similarity of contact apgles of new and ■ 
old cuticles is significant from another point of view. It is reason­
able to assume that the secretion of a waxy film upon the- protein surface 
initiates profound surface reactions.. The reactions probably occur be- . 
tween the side linkages of the protein chains and the few monolayers of 
wax at the protein surface..-, (Pryor 1940 and Hurst 1948).. However, it has 
not been definitely established as yet, whether such reactions affect the 
orientation of only the wax monolayers nearest the protein epicuticle, 
or whether all the wax molecules are affected by these surface inter­
88
actions. Wigglesworth (19-45) found that the contact angles between 
water and the cuticle of Rhodnius increased from 102° to 120° during 
the forty-eight hours after the wax was secreted. Furthermore, the con­
tact angle, increased from 102° to 117° twenty-one days after moulting.
He attributed the latter phenomenon to secretion of extra quantities of 
wax. It is difficult to see, however, how the contact angle could be 
changed by a further addition of supposedly the same kind of wax. This 
could happen if the additional secretions were of different composi­
tion, or, 1-f they covered up' submicroscopic polar region's. The factt ■ 
that the contact angle changed during the forty-eight hour period fol­
lowing moulting'might be the result of a gradual reorientation of the. 
wax molecules on the cuticle surface, as a result of the interactions at 
..the wax-protein'interface. If such reactions do occur in M. bivittatus 
then the contact angle should change. The fact that the contact angle 
-did not change indicates that there is no further reorientation of the 
upper layers of the wax film.-. Consequently, the interactions - at the pro 
tein-wax interface of this cuticle/ if such occur, do not seem to infIu-■ 
ence the state of orientation of•the wax molecules at. the wax-air inter­
face. Furthermore the high contact, angle of distilled water on the wax 
layer indicates the absence of polar groupings at this interface. Since 
it ia !mown that polar groupings are present in insect.wax, then they 
must be oriented away from the - air interface .- 
IV. Determination of the Absence--of the Cement Layer.
It has been shown by several Investigators that the,wax layer 
covering the cuticle of some insects and ticks is protected by a cemmrfc
layer (Wigglesvorth 1945, 194?, 1948, Lees 1947). This cement, layer is' 
apparently a tanned, protein secreted over the vax layer by the dermal 
glands (Wigglesworth 1948). When insects having a.cement layer were 
treated with cold chloroform, the permeability of the cuticle was not 
increased to any marked degree. But when' it was treated in hot chloro­
form at a temperature below the critical temperature of the wax, then 
the cement layer was disrupted thereby permitting extraction of the wax 
(Wigglesworth 1945, 1947, 1948).
The above criterion for the presence of the cement layer was used 
in the following experiments. Twenty dead adult grasshoppers were used. 
Their mouthparts and anus were sealed with cellulose paint. The grass- 
hoppers were divided into lots of four. ' One lot was used as a control. 
The rest of the lots were treated by exposure to vapours of cold chloro­
form at 25°C., by immersion in chloroform at 2'5°C. and immersion in- 
chloroform at 40°C. All treatments were- carried out for thirty ,minutes. 
After the treatment was completed, the chloroform was allowed to evapo­
rate and the grasshoppers, after being weighed, were exposed to a dry 
atmosphere for'one hour at 30°C.
These results, show that the average loss of moisture through the 
cuticles treated with chloroform at 25°C was 11.03%. Furthermore, when 
the cuticles- were treated with chloroform at 40°C, which is a few degrees 
below, the cutical temperature, the percent loss of moisture was 1 1.8l. 
Thus, the permeability of the cuticle was not increased as a result of 
the latter treatment, indicating that the wax in M.- bivittatus is not 
protected by any other layer. If it were, then the permeability of the
89
90
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I
Table VIII -
Loss of moisture from adult grasshoppers which, have been treated, in'
chloroform for 30 minutes and subsequently exposed
to a dry atmosphere for one hour at 30°C
I . ■ . . .
I
I
Treatment
Temp„ of 
Jgeatment ^
Initial Wgt. 
in gms.
Decrease 
in wgt./ 
hr.
% loss of ■ 
Wgt./hr
j Dead - Controls 22.6 3.2308 .0703 1.99
I Chloroform vapors 22.0 2.2137 .1228 - 2.76
i Chloroform 22.0 2.7997 .6212 11.12
i
Chloroform 22.0 .97k3 „106U 10.92
\ Chloroform W .o U.W27 .2298 ' 11.81
i
'
3
!
I
■
*
I
j
cuticle ought to have been increased by the treatment at the higher tem­
perature. It is therefore concluded that the wax is not protected by a ■ 
cement layer.
It is also obvious from the data that if the wax is to be removed 
then it must be in intimate contact with the chloroform, because the per-. 
meability of the cuticle was much less when it was exposed to chloroform 
vapors than when it was immersed in chloroform. In fact, the percent 
loss of water through' cuticles exposed to chloroform vapors is approxi­
mately equal to that of controls. Chloroform vapors do not seem to dis­
turb the orientation of the wax molecules to any extent. ■
In order to determine whether prolonged treatment of the' cuticle by 
chloroform would remove more wazc, a series of experiments were set up to 
test this point. Two freshly moulted adults were used "in each trial.
These grasshoppers were taken from a different culture than were those 
used in the previous experiment. The grasshoppers were killed and then 
immersed in chloroform at 25°C for varying times. They were then dried, 
weighed and exposed to dry air for twenty minutes, at 30°C.
The constancy of the percent loss of body weight indicates that
treatment of the cuticles in chloroform at 25°C for two hours did not re-
_  -
move any more wax than did the treatment for 15 minutes. If the cement 
layer were present, then the long exposure to chloroform ought to have 
caused it disruption to a greater extent than the 15 minute treatment.
This would have-been reflected in an increase of cuticular permeability 
at the longer period of exposure.' . Since this did not happen," these
91
F?i
J,V^-=LZA;-. ;.■ '.r.:..;.;;:7„.'
■■■■92 ;.; .■
' Table IX
Loss of body weight of freshly-moulted adult grasshoppers treated in 
chloroform at 2^0G for varying periods of time and then 
exposed to dry air at 30°C for 20 minutes
Length of 
Treatment
Initial Wgt. 
in gmsO - " ,
Decrease in 
Wgto per hr.
Percent loss.of 
body weight
Gohtrols 1.2073 - .0280 2.32
V~> minutes .9820 .3072 31.3
I hour' .95:98 .2812 29.3
2 hours .9119 . .0901 29.6
■ — .
\
\
\
t\
93
results substantiate the conclusion already made, namely, that in M. 
Mvittatus the wax is not protected by a cement layer.
Thus the absence of the cement layer in this grasshopper falls in 
line with the finding that insects and ticks have a wax of low critical 
temperature are not covered by a cement layer (Wiggleswoith 1945, Lees 
1947).. The data of Wigglesworth and Lees indicate that the cement layer 
is present in those insects which have a wax of high critical temperature; 
i.e. a wax that is hard and highly oriented. Thus, those insects.which 
are already protected from undue dessication by v i r t u e o f  the nature of 
their wax are still :further protected by the cement layer. Meanwhile, 
those insects, having a wax of low critical temperature, that is, a soft 
and less highly oriented wax are deprived of any additional protection. '
It seems to the - author.;; that the evidence presented by Wigglesworth 
(1945) and Lees (1947). for the presence of a cement layer can be given an­
other interpretation. If is highly probable that at the time of the for­
mation of the new cuticle, there are many exposed free side linkages' of 
the protein chains which must be satisfied in some way or another. Since 
the type and strength of these linkages will vary with the species, .then 
it follows that in some species these interfacial reactions, between the 
exposed polar groupings and the wax would be stronger than in others.
The stronger such a linkage, the'more difficult it will be to break it. 
Consequently, drastic treatment such as hot chloroform would be neces­
sary before any appreciable quantity of wax would be removed. Wiggles- 
worth (19^5) and Lees (1947) have further, found that once this wax was 
removed, it passed very readily into solution in- cold chloroform. This
supports the above theory for in the absence of any strong binding sur­
face forces, the wax would dissolve very.readily. Furthermore, Wiggles- 
worth and Lees found that Rhodnius and some ticks are capable of regen­
erating the wax after it had been removed. This newly secreted wax is
I
removeable by cold chloroform. This would be expected in view of the 
above discussion. A removal of any surface wax;undoubtedly results in a 
disruption, and reorientation of the side linkages of the protein chains.
I
It is conceivable that, once the wax ,is removed, these side linkages 
would react with each other in an effort ,to reduce the, free, surface energy 
to a minimum'. This means that the number of free linkages which'would be 
available for interaction with the wax polar groupings would be very small. 
This, would result in a very loose binding between them and the wax. Con­
sequently, less drastic treatment would be required to remove this re- . 
generated wax.
V. The EiEfect of Temperature on Evaporation through the Cuticle.
Ramsay (-1935) > Wigglesworth (1945), and Lees. (1947) have shown that • 
when dead insects or ticks were exposed to dry air at different tempera­
tures for short periods of time, the rate of evaporation, through the cu­
ticle increased slowly up to a certain temperature above which the rate' ' 
of loss increased beyond-all prediction. This temperature was called the 
“critical temperature". Beament (1945) observed.a similar^phenomenon when 
insect waxes were deposited, on artificial membranes. Furthermore, he 
found that the critical" temperature of the wax lies a few degrees below 
the -true melting point of the wax. Beament -^1945) was also able to cor­
relate Coptical and various other physical changes in the wax with the '
94
95
critical temperature. All these investigators agree that this, sudden in­
crease in the permeability of the cuticle at the critical temperature is 
due to some physical change in the wax. ■'
Similar experiments were carried out on M. bivittatus in the vari­
ous stages of development. The apparatus used here was similar to that 
used by Wigglesworth (19^5) and has already been described. The quan­
tity of grasshoppers used was equivalent to 0.8 - I gms. This means, 
that less grasshoppers were used as they grew older, but it gave approxi­
mately the same surface area throughout the course of the experiment. The 
same lot of grasshoppers was used for one complete run. However, each 
curve presented herein, was the result of two separate trials. No attempt 
was made to seal <the spiracles because it was found that the difference 
in the rate of evaporation through cuticles of grasshoppers whose spir- . / 
acles were sealed and those whose spiracles were not sealed, was very' 
small. It was felt that such a small difference would not affect the 
results seriously. However, the mouth parts and anus of the third, fourth 
fifth and adult stages were sealed. This was not done- in the case of the 
.first and second instars because of the size of the insects.
The temperature interval used was usually five degrees. In the 
region of the critical temperature, the. interval was usually 2j=r degrees. 
All the. trials.were performed on dead insects.
The grasshoppers were killed as previously described. They were 
weighed on a calilirated analytical balance and then exposed to dry air 
for fifteen minutes. After this exposure the insects were reweighed, 
the difference in weight being attributed to transpiration through the
cuticle. The original weights of the grasshoppers permitted a calcula­
tion of the surface area by using the formula S = 10.27 W*?2 , Since the 
curve relating the surface area,to weight is not linear, then the sur­
face area for one insect was calculated from the weight of a single in-, 
sect-. This surface area was then multiplied by the number of grass­
hoppers used. Once the total surface area was obtained, it was possible 
to express the rate of evaporation in mg. per sq. cm. per hour.
The evaporation from a free..water surface was determined by-expos­
ing the previously described aluminum cell, which Was three-fourths 
filled with distilled water, to dry air at various temperatures for twenty 
minutes. The rate of evaporation was calculated by dividing the loss of 
weight per hour by the area of evaporation which was .916 sq. cm.
It was stated b y -Wigglesworth (19^5) that "provided the spiracles 
are sealed, there is no difference in the rate of evaporation from dead 
and live insects.".. It would be interesting to know what difference ex­
ists in the rate of evaporation from dead grasshoppers whose spiracles 
were closed and those whose spiracles were open. The results .of efforts 
to gain information on this point are presented in Table X,- and Bigure
11. ,
~ \  • _
It is seen that the rate of transpiration in the case of the grass­
hoppers with i.spiracles open is slightly higher than with spiracles closed. 
This indicates that some of the body moisture is also escaping via the 
tracheae, besides the cuticle sufface. Since the difference is small, 
and because it was difficult to seal the spiracles in the grasshoppers 
in the earlier stages of development, it was decided to leave the
96
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4
Table ' X
Hate of Evaporation from two adult grasshoppers 96 - 120 hours 
old with spiracles open and closed 
________________________ Spiracles Closed___________ .
Wgt. of a grasshopper: .^2^6 gms,0 
..Surface area of a grasshopper-- 9.3 cm^ 
_____Total Surface Area = 18*6 so. cm. .
Temperature
0C
Initial Wgt„ 
in gms„
Decrease in . 
Yfgt./hr.
Rate
mg/cra2/hr;
23-0 • 1.6310 .0080 .13
30.0 l„0li90 .0161 .8 8
33.0 • .1.0119 .0161 .8 8 -
ItOoO ■ 1 .0 1 0 8 .0 2 2 1 1.36
U2.3 — — -O *■—
U3e0 '1.0332 „0318 • l-„ 8 7
■ 3o.o 1.0263 .0 6 3 6 3.12
33.o ■ 1in 1
6o.o .9916 .1 6 7 6 . 9.01
63.o .9327 .2600 1 1 .0
Spiracles Opened
Wgt0 of one grasshopper „6131 .gms. ' - 
Surface Area of one grasshopper - IOolilcm^ 
_____ Total Surface Area ~ 20*9 sq„ cih.,
Initial Wgt. in 
Gms „
Decrease in 
Wgt./hr.
Hate
mg/cm2/hr„
1 .2 1 8 6 .0 236  ' 1.13'
1 .2 1 2 7 .0268 1 .2 8
I .2 0 6 0 .0292 I. hO
1 .1 987 .0361  - 1.74 ■
1 .1 8 9 6  ' .0 136 2 .0 9
1.1777 .0822 3 .9 3
1 .1 6 1 0 ■ .1 296 6 .2 0
1 .1 1 2 1 .2 106 ■ 1 0 .1
R
a
t
e
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s/
s
q.
 
cm
/h
r.
spiracles open
spiracles closed
11 --
I -  .
10 15 20 2.5 30 35 ho i;5 50 55 6o 65
Temperature 0C
Figure 1 1. Comparison of rates of evaporation from dead grasshoppers 
with spiracles open and sealed at different temperatures.
99
spiracles opened in "the rest of the experiment. The error caused hy the 
slight increase in evaporation due to the opened spiracles would there­
fore he a constant error throughout the course of the experiment.
Table XI shows the results obtained when M. bivittatus in the various 
instars was exposed to dry air for fifteen minutes at various temperatures. 
Each reading represents a mean value from a quantity of grasshoppers equi­
valent to 0 .8  - 1 .0 grams.
The results presented in Table XI are plotted in Figures 12 - 18. 
Figure 19 shows that there is no significant difference in the rate of 
evaporation from dead grasshoppers in.the various stages of development. 
Wigglesworth (1I^j) reported similar find jpgs for the nymphs of Ehednius.' 
However, he reported that the rate of evaporation was dependent upon the 
stage of development in the case of holometabdbous insects. The slight 
variations, in the. rate of evaporation, from instar to instar, seen in 
Figure 1 9, might be due to variations in the thickness of the cuticles 
because of the varying ages of the insects. Furthermore, as has.been 
pointed out before, the surface area used in the calculations might be 
quite different from the actual surface area of the insects in the vari-
I
ous instars. This difference might partly account for the variations.
The data show that the rise in the rate of evaporation is linear
with temperature up to 40°C. In the region 40 - 420C the rate began to
increase.• This was true in all the instars. This is attributed to a 
breakdown in .the structure of the w a x l a y e r . More conclusive evidence' 
for this postulate will be presented later. The temperature at which
the rate of increase of rate of evaporation is a maximum will be called
100
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>
k
v
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f
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Table ZI '
Rate of evaporation into dry air from grasshoppers in various stages
of developmento The period of exposure was 1$ minute's ■
'____________________ First Instar ■ ■ ■ ■ ~_____________  ■
Trial I
Wgt. of one grasshopper = .0065) gms. 
Surface Area of I grasshopper = .1+0 sq. cms. 
' Total Surface Area - .1+0 x ^O ^ 20.0 sq. cm.
Temperature
0C
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
Eate
mg/cm2/hr„
15.0 .3261+ .0061+ ' .32
2 5 .0 .321+8 ■ „0122 „60.
3 8 .0 .3218 .021+8 1.21
1+2.0 .3 156 ' .0 276 1 .3 8
.1+6.0- .3087 „01+00 2 .0 0
5 o .o .2 987  ' .0612 3 .0 6
Trial II
Wgt. of one grasshopper = .0073 gms., 
Surface Area of I grasshopper = .Ii3 sq. cms. 
Total Surface Area = .1+3 x 1+8 .= 20.6 sq. cm.
Temperature
OC
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
j Rate
j mg/cm2/hr„
3 0 .0 .3519 „0181+ - .8 9
3 5 .0 .31+73 .0188 ' .9 1
1+1+.0 .3 126 .032% 1 .5 7
5 3 .0 ..331+5 .0772  - 3 .7 5
5 8 .0 .3152 .1320 6.1+1
6 3 .0 .2819 .2072 1 0 .0 6
101
■Second Instar 
Trial I
Wgt. of one grasshopper - .0200 gmse 
Surface Area of 'I grasshopper ™ .80? sq, cm. 
Total Surface Area. = .,8'87 x 2It = 21.3 sq. -ctns.
Temperature
0C
Initial Wgt. 
in gms.
Decrease in 
Wgt./ hr.
Rate
mg/ cm2/hi
17.0 .4800 .0 0 & .30
2 5 .0 .4784 .0160 ,75
3 5 .0 .4744 .0200 .9 4
1|.0.0 .4694 .0248 1.16
U3.0 .4632 .0340 1.60
4 6 .0 .4547 .0408 1.91
5 o .o .4445 . .0708 3 .3 2
5 5 .0 .4268 .1400 . 6 .5 7
6 o . o . .3918 .2276 1 0 .8
- 6 5 .o .3349 .2 844 1 3 .3
Trial II
Wgt. of One grasshopper - .0265 gms. 
Surface Area of I grasshopper = 1.01 sq. cm. 
Total Surface Area. == 1.01 y,20 ° 20.2 sq. cm.
Temperature
°C:
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
Rate
mg/cm2/hr.
3 0 .0 .5304 .0232 1.15
3 7 .5 .5246 .0236 1.17
4 5 .0 .5187 .0404 2 .0 0
5 2 .0 .5 086 . .0988 4 .8 9
5 8 .0 . .4839 .1524 ■7.54
6 5 .0 .4458 .2528 1 2 .5  "
102
Third. Instar 
Trial I
Wgt. of one grasshopper = .OliPS gms. 
Surface area 6f I grasshopper = 1.70 .sq. cm. 
Total Surface Area 1.70 x l S  ~ 2^.? sq. cms.
Temperature 
, oG
Initial Wgt. 
in gpis.
Decrease in 
Wgt./hr.
Rate '
mg/cm^/hr. J
. 12.0 . .7390 .0084 .33
22.0 .7369 .0164 .64
32.0 .7328 .0264 1.03
ko.o .7262 .0344 1.32
42 .o .7176 .0428 1.68
20 . o ,7069 .0732 2.87
22 .o .6886 .1426' 2.71
6o .o .6222 ' . .2264 ' 8.88 '
______________________________ \
Trial II '
Wgt. of one grasshopper= .0^07 gms. 
Surface area of, I grasshopper - 1.73 sq. cm. 
Total Surface Area = 1.73. x 15 = 26.0 sq. 'cms.
Temperature
■ OQ
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr. •
Rate
mg/cm^/hr.
30.0 .7609 ' .0196 ’ .72
42.2 .7260 .0260 1.00
47.2 .7492 ' .0596 2.29
22.2 - .7346 .1072 4.12 '
. 62.0 .7078 .3498 13.4
103
■ Fourth Instar
: Trial I
- . ■ Wgt of one grasshopper ” .1135 gms.
Surface Area of I grasshopper;= 3.09 3q. cm.
Total Surface Area - 3.09 x 7 = 21.6 sc cms.
Temperature Initial Wgt. Decrease in Rate
0C in gms o Wgto/hr„ mg/cm^/hr.
20.0 .7941 '.0112 .52
30.0 ' .7916 .0216 1.00
IiOoO .7862- .0296 1.37
U5.o - .7788- .Oil 72 2.18
5o .o .7670 .0832 3.85
55.o .7^62 .1632 . 7.55
60.0 .7054 .2544 11.8
70.0 .6418 .4764 22.1
:Trial II
• Wgt, of one grasshopper = .IOli6 gms.
Surface Area of I grasshopper =2.92 sq. cm.
Total Surface Area = 2.92 x 7 = 20.Ii sq. cms.
Temperature Initial Wgt. Decrease in I Rate
0C in gms. Vfgt ./hr. m g/cm.2/hr,
25.0 .7322 .0160 .78
35.0 ' .7282 .0216 I .0 6
42.5 .7228 .0264 1.29
47.5 .7162 .0544 2.66
6Ii„0 .7026 .370k 15.1
73.0 .6100 .5616 27.5
I
i
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I
I
I
I
IOk
Fifth Instar •
________________________ ' Trial I
Wgt. of one grasshopper.= o3!?9h gms„
■ Surface Area of I grasshopper = 7.10 sq. cm. 
Total Surface Area.= 7.16 x h = 2 8 . sq. cm.
Temperature
0C
Initial Wgt. 
in gms.
Decrease- in 
Wgt./hr.
Hate
mg/cm2/hr.
20.0 I . 1373 .0112 .39
j o . d I.ii3li6 .0164 .38 .
U2.3 1.L303 .0272 .96
k 2 . o - , 1,4237- .0320 1.13
2 o .o 1.4137 , .0664 2.34
. 3U..0 1.3991 .1236 . 4.33 -
62,0 ' 1.3682 „2340 8.94
I
__________________ Trial II _________ ■ ■
Wgt. of one grasshopper = .382!? gms. 
Surface Area of I grasshopper = 7.U3 sq. cm.
Total Surface Area = 7.43 % 3 = 22.3 5q. cm.
Temperature Initial Vfgt. Decrease in Rate
°C in gms. I Wgt./hr. mg/cnr/hr. x
23.0' 1.1473 .0144 .64
33.0 1.1439 .0192 - .86
4o.o 1.1391 .0208 .93
. • 47.3 . 1.1339 .0472 2.12
' 38.3' ' 1.1221 .1416 6.3
64.0 1.0867 .2324 11.3
I
I
^ y , . . ■ >'
I
I
I
Adult 
Trial I
'105
Wgt. of one grasshopper = .6131 gms. 
Surface Area of I: grasshopper = ioJ4.l1. sq. cm, 
: Total Surface Area = IQ0It-lX '2 - 20;9 sq, cms»
Temperature 
I' 0C
Initial Wgt. 
in gms.
Decrease in
I Yfgt./hr.
I  Eate
I mg/cm^/hr.
3 0 .0 1 .2 1 8 6 .0236 1.13
3 5 .0 1 .2 1 2 7 .0268 1 .2 8
liO.O .1 .2060 . . .0292  . ■ I.ItO
- I i2 .5  . 1 .1 987 ' .0361: - 1.714
.1 5 .0 1 .1 8 9 6 .0L36 2 .0 9
' 5o ,o ,1 .1 7 77 .0822 .3 .9 3
55.o . I.I6I4O / . .1 296 6 .2 0
6 0 .0 • I.1I4.2I4 .2 1 06 1 0 .1
Free Water Surface
Surface Area of CeILI ™ 0916 sq„, ditts.
.Temperature Initial Wgt. Decrease in Rate
0C in gms 0' Wgt./hr. mg./cm2/hr0
10.0 .5 1 3 6 .0198 2 1 .6
1 5 .0 .5020 0 0210 2 2 .9
2 0 .0 - .5000 o02lt6 2 6 .8
2 6 .0 olt9i8 .0369 ItOo 2
. 3 0 .0 .It 795 .0 li26 lt6. It
3 5 .0 .lt653 .0537 5 8 .6
ItOoO .ItltTlt .0621 6 7 .7
it5Lo .1:267 .0822 8 9 .6
5 o .o .3993 - .0987 107.6
R
a
t
e
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
mg
ms
/
s
q
.
 
cm
/h
r.
110 -
100 -
20 - -
10 -L
20 30 ItO 50 60
Temperature 0C
Figure 12. Rate of evaporation from a free
water surface at different temperatures.
R
a
t
e
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s
/
s
q
.
 
cm
/h
r.
107
12 • -
11 r
10 • -
5 io  15 20 25 30 35 IiO i;5 5o 55 6o 65
Temperature °C
Figure 13. Rate of evaporation of water from dead grasshoppers in the
first instar at different temperatures.
12 - -
1 1  - -
5 io 15 20 25 30 35 bo U5 5o 55 60
Temperature uC
Figure lb. Rate of evaporation of water from dead grasshoppers in the
second instar at different temperatures.
109
Temperature 0C
Figure 1%. Rate of evaporation of water from dead grasshoppers in the
third instar at different temperatures.
H O
11  ■ -
. IO -
O 5 10 1$ 20 25 30 35 Uo 50 55 6o 65
Temperature 0C
Figure 16. Rate of evaporation of water from dead grasshoppers in the
fourth instar at different temperatures.
Ra
te
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s
/
s
q
.
 
cr
a/
hr
111
10 i s  20 2S 30 35 ho li5 so SS 6o 65
Temperature 0C
Figure 1,7 Rate of evaporation of water from dead grasshoppers in the
fifth instar at different temperatures.
13 J-
12 - -  
11 •-
10 -h
i :
O 4
T
6 A —
g
cd
i 1*
0)
O 
Qj
^  2
5 --
3 jT
cd
Cti
I -
Figure 18.
H---- 1---- 1---- 1---- 1---- I---- t---- I---- '---- 1---- I---- 1----
5 10 15 20 25 30 35 ho 15 5o 55 60 65
Temperature 0C
Rate of evaporation of Trater from dead adult grasshoppers
at different temperatures.
— «.— first instar 
second instar
----- third instar
—  fourth instar
---- fifth instar
---- adult
131 --
.10 - -
S8 -I
10 15 20 25 30 35 IiO HS 5o 55 6o
Figure 19. Comnarison of rates of evaporation of water from dead 
grasshoppers in various instars at different temperatures.
114
the critical temperature . This temperature is not obvious from the 
graphs. It was found by reading the values for the rate of evaporation 
off the graphs for each degree between W  and 49°. Then.the difference 
between the successive rates was taken.. The difference between the lat­
ter values was again taken. This corresponded to a "rate of increase of 
rate of evaporation . It was found that the "rate of increase of rate 
of evaporation" increased to a maximum and then decreased again. The tem­
perature corresponding to the maximum "rate of increase of rate of evapora 
tion" was taken to be the critical temperature. This process is equiva­
lent to finding the second derivative of the function representing the 
curve, evaluated at each temperature. In Table XII is presented the pro­
cedure used for the second instar. ■ The data for the other instars was 
treated similarly and similar results were obtained.i
From the Table XII, it is seen that the maximum occurs at 44r'46°C 
.or approximately 45°C. Accordingly, the critical temperature is taken to 
be approximately 45°C. This was true for all the'instars.
Wigglesworth (1945) and Lees (194?) used as a further method of 
comparison among species, the temperature at which evaporation into dry 
air equals 5 ™S•/crn^/hr. Lees states that.this is-"a.more accurate stan­
dard of comparison"-. This will be true only if the work was done under 
rigorously controlled conditions.. If this is hot done, then the uncon­
trolled variables such as the age of the insects and hence thickness of
’ ‘
the cuticle, mightmaterially affect the value of this temperature. This
^WL
ii£
.' ' Table XII
Calculation of the Critical Temperature
Temperature
0 C
Hate of 
Evaporation. Differences'
16.0 1.30
.08'
Ul.O : " i .3 .8 .02
'.10
h2.0 i.i*8 : . b H
.11 ■
1*3.0 1 .2 9 .0 1
'.12
1*1*.o H .03
.13
. ; 1 .8 6  - x . .ol*
.1 9  '
1*6.0 2 .0 2
-a*  '• • *
1*7.0 • 2 .2 9 .00
'.21*
1*8.0
4 9 .0  -
2 .2 3
2 .7 8
.0 1
.2 2
method of comparison will also be influenced by the accuracy with which 
' the surface area was determined. Since the rate of transpiration depends 
upon the surface area, then, depending upon the accuracy of the surface 
area, it is possible to get an infinite number of transpiration curves,• 
all of which will be displaced vertically. Consequently, it is possible 
to get any number of temperatures for a particular species at which the 
rate of transpiration will be 5 mg/sq.■cm/hr.
116
For example, such variations as read off Figures 13 - 18, are presented 
below in Table XIII.'
The above Table shows that the temperature at which the rate of 
evaporation into dry air is 5 mg./cm2/hr. varies with the instar. There 
is an overall 4.5 degree r;ange and yet all the instars showed approxi­
mately the same critical temperature. These variations might be attri­
buted to a number of causes, the most obvious one being the variation 
in cuticle thickness as a result of the variation of the age of the grass 
hoppers both within the same instar and between different instars. In ' 
view of these possibilities, this method of comparison has "very little 
meaning unless the proper precautions are taken to control all the vari­
ables .
A comparison of the rates of evaporation from the grasshopper and , 
from a free water surface (Fig. 12) shows that even at the critical tem­
perature when the wax is presumably totally disruped, the rate of evapora 
tion from adult grasshoppers is still approximately l/40 of that from a 
free water surface at the same temperature.
VI. Determination of the Presence of a dutlculin Layer.
Wigglesworth (1933) observed the presence of a brownish or amber 
layer'in the Bhodnius epicuticle.• He concluded at that time that it was 
a "complex fatty or wax substance or mixture". In 19^7 he showed that 
this fatty substance was combined with a protein, forming, as a result 
of oxidation and polymerization, a resistant lipoprotein complex. This 
layer was called by him the'"cuticulin layer".
If the cuticulin layer forms a resistant lipoprotein, it should
117
-e
Temperature at "which the rate of evaporation into 
dry air equals 5> mg./cm^/hr.
Table XIII
Instar Temperature
I ■ 33 o 3
2 . 33.3
3 32.3
k 31.3
3 ■ 36.0
Adult 32.0
3
118
not be broken down, by prolonged chloroform treatment. To test this hypo­
thesis, ten cast skins from the fifth instar of M. bivittatus were taken 
and divided into lots of two. The first lot was used as a control. It
was exposed to fumes of an aqueous solution of osmic acid. After a per-
"
iod of exposure the skins assumed a black color, indicating the presence 
of fatty material. The other lots were extracted with chloroform at 
53°C in a Soxhlet extraction for two hours, seven hours, three days and 
eight days. After each period of extraction, the chloroform was allowed^ 
to evaporate and the skins were then exposed to fumes of osmic acid solu­
tion. In every instance, the skins stained black,. indicating the pre­
sence of unremovable lipoids. '
McClung (1937) states that the osmic acid in the presence of cer­
tain fats is reduced to black metallic osmium. These fats are then 
blackened by the precipitated osmium  ^ He also states that saturated fats 
do not reduce osmic acid. Nath- Vishwa (1933) states that the time re­
quired by osmic acid to blacken fats or lipoids depends upon the amount 
of oxidation the fat has undergone and its degree of unsaturation. Satur­
ated fats will not blacken in an osmic acid solution. According to this, 
the fats present in-the cuticle of M. bivittatus are™perhaps partly un- 
sat'urated. It is interesting:; to noteothat Wigglesworth (19A7) suggests 
the presence of unsaturated- fats in.the newly secfeted epicuticle; of 
Khodnius.
When cuticles were treated for varying periods of time in chloro- . 
form at 25°C and then exposed to dry air for twenty minutes at 30°C, the 
results obtained are presented in Table XIV.
119
Table XIV '
Loss of body-weight of freshly moulted adult grasshoppers treated in 
Chloroform at 2Z0C for varying periods of time 
and then exposed to dry air at 30°C for 20 minutes
Length of 
Treatment
Initial Wgt. 
in gms„'
I Decrease, in 
Yfgt. per hr.
Percent loss j  
of body Wgt. j
Controls 1.2073 .0280 2.32
13 minutes .9820 .3075 31.3
I' hour .9598 .2812. 29.3
2 hours .9119 .0901 29.6
J
120
It Is seen from the Table that the chloroform treatment removed 
only the superficial layer of wax. Prolonged chloroform treatment does 
not increase the permeability of the cuticle any more than does the 
fifteen minute treatment. The constant permeability of the cuticle in- 
dicates that prolonged treatment did not cause any further breakdown 
of the lipoprotein layer. In fact, as was already pointed out, this 
layer is apparently not disrupted even after-eight days of extraction.
It is concluded that the epicuticle of M. biyittatus contains a 
lipoprotein layer which is resistent to prolonged periods of chloro­
form extraction. This layer will be called, according to - the termin­
ology of Wigglesworth (19^7) the "cuticulin" layer.
VII. Changes in the Cuticle after moulting.
It has been shown by Wigglesworth (1933) that the endocuticle of
' ,  ' v -  . . .
Rhodnius continues to be secreted after moulting. Furthermore, he showed 
that there is a continuation of the secretion of wax after moulting. 
Dennell (1946). found that the endocuticle of the larva of Sarcophaga is 
partly secreted after moulting,. In an attempt to ascertain the changes 
in the grasshopper cuticle after moulting, the permeability of the cu­
ticle was’ measured at various periods after moulting by exposing the 
'adult grasshopper to dry air at various temperature for 15 minutes.
The data in Table XV and Figures 20 - 24 show that the rate of 
transpiration tom grasshoppers‘depends, within limits, upon the time
. ' 1 ” Vj
after moulting. Figure 20 indicates that there is very" little differ­
ence between the evaporation from Grasshopper 48 - 54 hours and 96 W .
120 hours after moulting. .
•121
• Table XV-
Rate .of transpiration through the cuticles of dead adult grasshoppers, 
at various periods after moulting. The adults 
were exposed to dry ,air for 1$ minutes at various temperatures
Immediately after moulting
Trial I
Weight of Grasshopper = «U873 gms,
Surface Area = 8,8# sq, cms,
Temperature I Initial Wgt, Decrease in Rate
OC I in gms, \ Wgt„/hr. mg/cm2/hr.
2^ ,0 .4873- .0100 - ' 1.14
3 5 .0 . W ' .0148 1 .6 8
ItO.O .4 811 .020Q 2.27
1 5 .0 .,4761 .0388 '4.41 ,
5 o .o .4664 .0800 9 .0 9
5U .o »4464 ~ .1 053 1 1 .9
Trial TI'___________ ___________ __  |
Weight of grasshopper = .3478 gms, -
Surface Area » 6.93 sq. cms.. ,
■Temperature Initial Wgt. Decrease in I Rate
0C in gms. Wgt./hr. I mg/cm^/hr.
. 2 0 .0 .3478 .oo44 .64
3 0 .0 .3467 '.0084 TL .22
4 7 .5  ■ =3446 .0 468  - 6 .7
5 2 .5 .3329 .0892' 1 2 .9
122
Six hours after moulting
Weight of grasshopper = .3170 gms. 
Surface Area = 9.23 . sq., cms.
Temperature Initial Wgt. Decrease in Hate
0C in gms. Wgt./hr. mg/cm^/hr.
2 3 .0 .3170 .0068 • o7h-
3 3 .0 .3133 . .0108 1.17
IlO.O .3 126 .0112 1.23
it3 .o .3098 .0198 . 2.13
3 o .o .3 063 .0372 It.Oit
3 3 .0 .3003  . .0 9 06 9 .8 3
6 0 .0 .1832 .1338 l i t .  3
AS - 31t hours after moulting
Trial I
Weight of grasshopper = ,,ItS62 gms,
Surface Area of I grasshopper = 8 ,8 3 sq, cms„ 
Total Area = 8 ,8 3 x 2 = 17°7 sq» cmso
Temperature 
• 0C
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
Hate
mg/cnr/hr.
13.0 .9723 .0092 ‘ .32
2 3 .0 .9692 ■ .OlltO .79
3 3 .0 .9637 . .020lt 1.13
itO.O .9606 .0236 1 .3 3
1 3 .0 .9317 .03lt8 1 .Y 7
3 o ,o .9139 .0616 3.3
3 3 .0 - .9 303 ' „ll6lt 6.6
1|8 - hours after moulting 
'Trial II
123
‘Weight of Grasshopper = J4.3I1.Ii gms, 
Surface Area of I grasshopper =8.13 sq.' cms. 
Total Area r- 8.13 x 2 ~ l602 sq. cms.
Temperature
I 0C
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
i Bate - .
I mg/cm2/hr.
20.0 ■ .8688 .OlJid .86
30.0 .86$3 .0192 1 .1 8 .
12.$ .8602 .0320 1.96
U7.3 .8222 .Oii22 ■ 2.8
60.0 .8I412 .2126 13.2
96 - 120 hours after moulting
Average Weight of grasshopper = .6131 gms. 
Area of grasshopper = lO.J4.li sq. cms. 
Total Area - 10 JiL' x 2 - 20.9 sq. cm..
I
Temperature
I 0C
Initial Wgt. 
in gms.
Decrease in 
Wgt./hr.
Rate-
mg/cm2/hr„
3 0 .0 1 .2 1 86 .0236 1.13
3 2 .0 1 .2 1 27 .0268 1 .2 8
JiO.O 1 .2 0 6 0 • .0292 1.40
l i2 .2 1 .1 9 87 .0364' 1 .7 4
b 2 .o 1 .1 8 9 6 .0 4 3 6 ' 2 .0 9
2 o .o 1.1777 .0822  . 3 .9 3
2 2 .o 1.1640 .1 296 6 .2 0
6 0 .0 1.1424 .2 1 0 6 1 0 .1
— *•— immediately after moulting
— ----6 hrs. after moulting
---- hrs. after moulting
-----96-120 hrs. after moulting
0 5 10 15 20 25 30 33 ho US 30 33 60
Temperature 0C
Figure 20. Comparison of rates of evaporation of water at different
temperatures from dead grasshoppers at various stages
after moulting.
11
10 J-
15 20 25 30 35 Uo 15 5o 55 6o5 io
Temperature 0C
Figure 21 Rate of evaporation of water from dead grasshoppers immed­
iately after moulting at different temperatures.
126
12 -L
11 - r
1 0  - -
o S 10 IS 20 2S 30 32 Lo LS So S2 6o
Temperature °C
Figure 22. Rate of evaporation of water from dead grasshoppers 6 hrs.
after moulting at different temperatures.
Ra
t
e
 
o
f
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s/
p
q,
 
cm
/h
r
.
127
12 - -
11  - -
' ' * 1 • » - -----r~~" ■ —  K— —  (—
o 5 10 15 20 25 '30 35 Uo U5 5o 55 6o
Temperature 0C
Figure 23- Rate of evaporation of water from dead grasshoppers 
U8-5U hrs. after moulting at different temperatures.
R
a
t
e
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s
/
s
q
.
 
c
m
/
h
r
128
0 $ io  15 20 25 30 35 Lo L5 #  55 6o
Temperature 0C
Figure 2L. Rate of evaporation of water from dead grasshoppers 
96-120 hrs. after moulting at different temperatures.
129
The rate of transpiration from grasshoppers taken immediately 
after1 moulting rises rapidly with increasing temperature and in"the 
vicinity of 40°C the rate changes very rapidly becoming approximately 
linear again.above k^°C. It is important to note that the very rapid 
change in the rate of transpiration occurred within an interval of five 
degrees or less. This gives a good indication of the sensitivity of . 
the wax to temperature changes. Apparently the wax becomes completely 
disoriented within five degrees. In view of this, a grasshopper in 
this stage of development would be very susceptible to high tempera-
I . ■
tures or any type of abrasion. It would be interesting to correlate 
this with bahavior reactions of grasshoppers in this stage of develop­
ment to such environmental stimuli as temperature in the. field.
It has been shown by Wigglesworth (1933) that the cuticle of 
Rhodnius, at the time of moulting, consists of the thin epicuticle and 
and. a very limited amount of untanned endocuticle. If it can be as­
sumed that the same is true in the grasshopper, then the only effective 
barrier to evaporation at this stage of its development is'the wax 
layer. Consequently, the most reliable value for the critical tempera­
ture is obtained from the data taken on grasshoppers immediately after 
moulting. The procedure for obtaining the value of the maximum'"rate 
of increase, of rate of transpiration" was. the same as already outlined. 
Table XVI. indicates that the maximum lies at a temperature of 44°C.
I
This value is taken to be the true "critical temperature".■
As the grasshoppers grow older, the rate of transpiration be-
s - .  ,
comes recuded, although the transition region -is not shifted; it is
130
Table XVI
Calculation of the critical temperature from data obtained 
from grasshoppers immediately after moulting
j Temperature °C | Rate Differences
ho . 2.28
hi 2.U3
h2 ' 2.65
h3  ^ 3.00
hh 3.5o
U5 k.25
h6 ' 5.20
hr 6.15
.15
.22
.35
.5o
.75
.95
.95
.07
.13
.15
.25
.20
.00
)
V''
131
flattened out considerably. The graphs indicate; that the region of 
rapid change of rate of evaporation now extends over a range of ten 
degrees. Obviously some other factors are beginning to affect the 
permeability of the cuticle. The rate, of transpiration above the -cri­
tical temperature is unaffected by the wax layer for it has been rendered 
ineffective in this respect by the high temperature. Yet it is clear 
that at-i- 52.5°C the rate of transpiration from grasshoppers taken six
■ i  '  ■ ■
hours after moulting was 6 .6  mg/cmp/hr., while from, grasshoppers taken 
immediately after moulting the rate was 11.6 mg/cm.2/hr. Thus/ the 
rate of transpiration has been reduced by approximately bjfo, within 
six hours-:after moulting at this temperature. In. the case of grass­
hoppers taken H Q - ^ k  hours or 96 - 120 hours after moulting, the 
rate of transpiration was b.9 mg/cm2/hr. or a decrease of 58/0, at - 
92.g°C._ The graphs and these calculations indicate that a very,pro-, 
nounced decrease in the perms ability of the grasshopper cuticle oc­
curs, within six hours after moulting.
Since there is no positive evidence as to the nature of the pror 
cessess which are taking place in the grasshopper cuticle at these 
various times after moulting, it is necessary to form a few postulates 
which would account for the above phenomena and yet be in line with 
what is already known about cuticlular permeability. Numerous authors. 
have shown that the thickness of the cuticle does not affect its per­
meability. Wigglesworth (1933) and Dennell (1946) have shown that the 
greatest portion of the cuticle is laid down after moulting. This 
process would increase the thickness and hence■contribute to the
132
resulting decrease in transpiration through the ctiticle.. Simultaneous- - 
Ij, the outer endocuticle undergoes a process of tanning and hardening. 
Pryor (1940) Hackman et al (1948) and Dennell (1947) have shown this to 
he accomplished by phenolic substances. These agents attack the side 
linkages of the protein and chitin, which then form a very resistant 
complex (Fraenkel and Rudall .1947). Pyyor (1940) suggested that the 
action of these agents is on the imino or amino side linkages of the 
proteins. This process of tanning begins shortly after moulting. Eder 
(1940) found Eu definite correlation between the amount of sclerotiza- 
tion (result of tanning) and permeability of the cuticle. Evidence for 
the presence of at least one of the tanning agents has been found in 
this grasshopper.
In view of the findings of the above authors, it is here postulated 
that the profound changes in cuticular peremability as seen in Figures 
20.-24 are due to the following two factors:
1. Gradual increase in the thickness of the grasshopper cuticle.
2. Gradual tanning and hardening of this cuticle.
Both of these factors seem to exert their greatest influence within six 
hours after moulting.
Sone interesting speculations, can be made if the portion of the- 
curves in Figure 20 below the critical temperature is examined. It is 
obvious that there is a gradual decrease in the' rate of transpiration. 
This decrease is assumed, to be due partly to the factors already dis­
cussed. But below the critical temperature the wax is still present 
on the cuticle. It is possible, therefore, that some physical changes
133
in the structure and orientation of the wax might also he partly respon­
sible for the decrease in the permeability of the cutucle. However, the 
measurements of contact angles indicate that the .contact angle does not 
change with time after moulting. Consequently, reorientation of the wax 
molecules at the wax-air interface is unlikely. If any change in orien­
tation does occur, it must-, therefore, be confined to the protein-wax 
interface. It is. also ,doubtful if any change in molecular close-packing 
occurs after moulting, for if such were the case, then it ought to have 
been detected in a change of contact angle.
It is important to note that there is very little difference be­
tween the 4 8 - 5 4  and $6 - 120 hour; curves. This indicates that all the
'i' "
processes which would tend to-.chahge the morphological aspects of the 
cuticle, affecting its permeability have been completed. -The majority of 
the cuticle is probably tanned by six hours after moulting and definitely 
.'by fifty hours. Probably very little new cuticle is secreted after fifty 
hours after moulting.
VIII. Dielectric Polarization of Beeswax.
In an effort to explain the phenomenon responsible for the critical 
temperature, the variation of the dielectric polarization of beeswax was 
determined with increasing temperature. Muller. (1932) found that the 
lattice structure of.certain paraffins increased, with increasing tem­
perature, a few degrees below the true melting point. In 1937 he was 
able to establish at this temperature a structural change of the crystal 
lattice in the form of rotation of dipoles around the axis of the chain.
He found that the variations in polarization of compound is a good
134
manifestation of this structural- change.
The cell used in this experiment was filled with beeswax and al­
lowed to. settle and harden. Air bubbles were eliminated by stirring 
during, hardening and also by reheating when the wax .had hardened. Dvhen 
the wax hardened and reached the temperature .of the room as'recorded 
by a thermometer fitted into the cell, the temperature of the cell was 
raised periodically and the capacitance of the filled cell, when in the 
circuit, was determined. To take account of any variations in.the cell 
which would be .caused by the increasing temperature, the capacitance o f ■ 
the empty cell at various temperatures was determined first. ..Then the 
cell was filled with benzene, freshly distilled over sodium, and its 
variation in terms of capacitance was determined'with increasing tem­
perature. The data presented are those accrued during heating of the 
wax.
The cell constant was calculated at various temperatures by the 
formula: C
<pA • where
C = cell constant
V  ^ B = Capacitance of cell out of the .circuit - capacitances' • of cell 
filled with benzene. i.e. Cp - Cp
V Q e = Capacitance of cell put of circuit - capacitance of empty cell 
in the circuit, i.e. Cp - Cg 
£  B =' Dielectric constant of benzene 
g A = Dielectric constant of air
The values used in this equation were read off the graphs at the re­
quired temperatures.
Once the cell constant, was calculated, the dielectric constant of
135
beeswax was obtained by the formula
fB = ^ cBW - ^ gE Z z- , .  where 
C
v Cb w = Capacitance of cell out of the circuit - capacitance of cell 
filled with beeswax.
' X a  = I..
The data and graph in Figure 29 indicate that the capacitance of 
the empty cell decreases with increasing temperature. On the other 
hand, the capacitance of the cell filled with benzene behaves in an er­
ratic fashion with increasing temperature, as is shown in Figure 28.
This erratic behavior is probably due to some unknown interaction be­
tween the cell and the benzene. The same is true for the cell constant 
(Figure 2j). .
The graph in Figure 25 shows that the dielectric polarization of 
beeswax decreases with increasing temperature up to '350C. At this tem^ ■ 
perature it began to increase and continued to do so till 5 1-5°!• after 
which the polarization decreased again. The melting point of this bees­
wax was found to lie between 63 - 650C-. Hence the increase in dielec­
tric polarization began at about 28° below the true melting point.
The curve for the polarization ".of beeswax with increasing tempera-, 
ture'bears some resemblance to the ones given by Muller (1937, 1938) for 
some ketones. He found that certain ketones showed an increase in polar! 
zation about 20° below their true melting .points. ..Furthermore, he showed 
that such an increase in polarization could be correlated with an in­
crease in lattice volume. The curves presented here show considerable 
variation from his curves, perhaps because beeswax is composed, of many.' 
components, some of which have': not yet been isolated. This chemical
. .y, CJ~. ~  .^ T .> f.-  r v , r f ^ ;y fr > *<< '< -% , L^vl'^.,
1]6
Table XVII
Variation of the capacitance of the empty cell -with'temperature
Temperature Capacitance of
Q b - Q _ _ _ _ _  I  emntv Cell
25.0 1:36.1
'31.2- 1:35.6
1:2.9 1:31:. 7
#.5 1:33.6
61.5 1:32.3
Table XVIII ' . '
Variation of Capacitance of cell filled with benzene with temperature
Temperature
°C
Capacitance of Cell 
filled with benzene
21:.2 370.9
31:.3 36)4.08
hhoO 365.7
52.9 367.):
62.5 363.7
;S
I
J!
-
■137
] Table XIX
'Variation of cell constant "with temperature „
I
I
Capacitance of cell out of.the circuit = 2 0 3 .7  u.u.f
' ■ I
• C1
I
I '
.
Tempera­
ture . °C 0B V 0B ■ .cE ' VCE 6 -benzene
! Cell Con-I 
stant I! 2^ .2 3 7 0 .2 '133 .2  ' .k 3 6 .1 6 7 .6 2 .2 7 2 2 1 .8 1
3 0 .1 3 6 7 .3 136 .k k 32 .8 6 7 .9 2 .2 6 2 2k .28
j 3 k .2 3 6k .8 1 3 8 .9 k 3 2 .2 6 8 .2 2 .2 2 3 2 6 .k 2  .
3 9 .3 3 6 2 .2 1 3 8 .2 k 3 2 . i 6 8 .6 2 .2kk 26 .1 9
I k l .2  ‘ .362 .k 1 3 8 .3 k3k .9 6 8 .8 2 .2k 0 2 2 .6 9
I A 2 .8 3 6 2 .6 .1 3 8 .1 k 3k .7 6 9 .0 2 .2 3 7 2 2 .8 6$
j U 3.1 3 6 2 .6 1 3 8 .1  ' k3k .7 6 9 .0 2 .2 3 6 2 2 .9 1
I U2.1i 3 6 6 .0 1 3 7 .7 k 3k .2 69 .2 2 .2 3 2 2 2 .6 oI k 8 .7 3 6 6 .6 1 3 7 .1 k3k . I 69.-6 .2 .2 2 2 5 2 .1 0  -
I 2 1 .6  . 3 6 7 .0 1 3 6 .7 .k33 .7 70 .0 2 .2 1 9 2 k .722 k .8 3 6 6 .6 1 3 7 .1 k33 .2 7 0 .2 2 .2 1 3 2 k .9 l
\ 6 0 .0 3 6 k .6 1 3 9 .1 _k32.2 70 .8 2 .2 0 3 ' 2 6 .7 7
Ij 6k . 2 3 6 3 .3 iko.k k 3 l .8 7 1 .9 2 .19k 2 7 .3 7  -
Table XZ
Variation of dielectric constant of beeswax and its
dielectric polarization -with temperature
CapacitancexOf Cell out of the circuit =  203 .8  u.u.f =C1
J Tempera- Cell Con-
arizationII ture 0C 0BW V 0BW V=E stant
2 2 .2 2kk .9  ' 1 2 8 .9 67.6 2l.8l 2 .7 62 ,370
I 3 0 .1 3 k 3 .9 1 2 9 .9 6 7 .9 2k .28 2 .6 9 2 .361
3 k .2 3 k 2 .9 1 6 1 .1  • .6 8 .2 26 .k2 2 .6k 7 .32k
I 3 9 .3 3 k 0 .7 1 6 3 .1 68.6 56 .19 2 .6 82 .329k l .2 3 3 9 .7 16)4.1 6 8 .8 2 2 .6 9 2 .7 1 1 .363
I . .k 2 .8 3 3 9 .7 16k. I 6 9 .0 2 2 .8 6 2 .7 0 k .362
• _k3.1 3 3 8 .8 162.0 6 9 .0 2 2 .9 1 2 .7 1 7 .36k
k 2 .k 3 3 7 .1 1 6 6 .7 69 .2 2 2 .6 0 2 .7 2k .369
I k 8 .7 3 3 2 .3 1 8 8 .7 6 9 .6 2 2 .1 0 2 .7 9 2 .37k
i 2 1 .6 3 3 3 .k 170. k 7 0 .0 2 k .72 2 .8 3 2 .380
t \  2 k .8  . 3 3 2 .8 1 7 1 .0 7 0 .2 2 k .91 2 .8 2 0 .379
i \ 6 0 . 0 3 3 3 .k 1 7 0 .k 7 0 .8 2 2 .7 7 2 .7 $k .369
i
IJ
6k .^ 3 3 2 .8 1 6 8 .0 7 1 .9  ' 2 7 .3 7 2 .6 7 2 .358
V
138
.380-
.370"
O
•360-
I
rH
• 3Uo
Figure 2$.
--1----1----1----1----t----!----!----1----1----1----1----1-
10 15 20 25 30 35 ho 1*5 5o 55 60 65
Temperature 0C
The dielectric polarization of beeswax at different 
temperatures.
139
Temperature 0C
Figure 26. The dielectric constant of beeswax at different temperatures^
15 20 25 30 35 ho h$  50 55 60 65
Figure 27* Cell constant at different temperatures.
59 09 $5 05 5% 0% 5C 52 OS 51 OI 5
4 OZC
Ib2
h 3>&-
k 3$-
h 3 k -
133-
Q)
H03-P
•H
C d
&
O
k 3 2 -
131
k30 -4--- 1-----1----- 1--- 1-----1----- 1---- :--- 1------I---- 1---f-
10 i s  20 2S 30 3S ko a s SO SS 60 6S
Temperature 0C
Figure 29. The capacitance of the empty cell at different temperatures.
Ilt3
£ 2.21-
5 10 15 20 25 35 ko 45 5o 55 6o 65
Temperature 0G
Figure 30. The variation of dielectric constant of benzene with temperature.
144
complexity probably provides for considerable dipole interaction. Such 
interactions might explain the decrease in polarization with increasing 
temperature up to 35°C and also the decrease in polarization after 
51.5°C. The picture might be further complicated by such a phenomenon 
as torsional flexibility. That such phenomena figure prominently in 
both long and short chain compounds was well shown by Muller (1-937).
In fact, FrBhlich (1946) states that for molecules with more than one 
dipole, “effect of twisting should be of major importance because it 
may affect the value of the total dipolar moment of such a molecule".
It is generally agreed by workers in this field that under ordinary 
conditions of temperature and pressure, the molecules in a solid are . 
held, in a definite position by Van der Waals forces and dipolar forces. 
However, these molicules do-perform small oscillations due to the elas­
tic restoring forces. Muller (1932) divides the forces.holding the 
molecules in the crystals^ into two groups: cohesive, forces which re­
sult from the interaction between the entire chains and also forces be­
tween the end groups. He assumes that in a crystal under ordinary tem­
peratures, these forces are in equilibrium. However, at certain tem­
peratures called by' him, the transitional temperature,. the balance 
between these forces is destroyed. This upset can be due to increased 
amplitude of oscillation of the molecule round its long' axis and by an
!
increase in the dimensions of the lattice perpindicular to the main
1
chain axis. As the temperature is increased, the molecule increases 
its amplitude of vibration and at sufficiently high temperatures the 
molecule might even perform a complete rotation. Thus, the directional *
145
forces vanish at high temperatures and the molecule becomes free to 
turn round its chain axis. The increase in dielectric polarization 
is nothing more therefore, than a manifestation on a macro scale of 
the d)ility of the molecule to orient itself in an electric field, 
with increasing temperature. An increasing polarization also indicates ■ 
tha^ the number of molecules which are free to rotate is increasing 
-'With temperature. . Presumably, at maximum polarization, most of the 
molecules are rotating because all the directional forces have been 
overbalanced by thermal agitation.
Muller (1938).. found evidence to support his belief that this in­
creased freedom of rotation was brought about by an increase in the 
volume of the crystal lattice. When measurements.were performed under 
cohstahh volume, the amount of polarization was greatly decreased. His 
calculations of lattice energies showed.that values comparable to the 
theoritical were obtained only when there was a maximum expansion of 
the lattice.
Figure 25 indicates maximum polarization of beeswax at 5°G.
At this temperature, presumably all the dipoles were rotating round the 
chain axis. This is probably only part of the explanation. Due to the 
complexity of beeswax, the dipolar concentration might contribute mater­
ially to the interaction between the dipoles. Those-that do rotate 
might cause considerable twisting of the chain with the result that the 
polarization is greater than it actually should be. On the other hand, 
it might be argued that the polarization of beeswax at ^0C is not 
what it should be. This„would occur if the rotation of the various •
146
dipoles were in opposite directions, therefore producing a nulling ef­
fect. However, the fact that the polarization decreased after 51.5°C 
indicates a maximum of structural disruption in one form or another.
1^. The Effect. ;of Temperature on Evaporation through. Jilms of Beeswax
and Paraffin.
In an attempt to establish a correlation between the increase in 
polarization of beeswax and increase in evaporation, the rate of evapora- 
. tion through, a film of beeswax into dry air was measured at various tem­
peratures .
A grasshopper was dissected, the fat body was cleaned away and the 
abdominal cuticle cut to a proper size. This membrane was then chloro­
formed and allowed to dry. It was fitted into the ^ evaporating cell .and 
the proper thickness of beeswax from a chloroform solution of known con­
centration was deposited. The cell was then allowed to stand for several 
hours. This was necessary in order to remove the droplets of water 
which accumulated on the undersurface of the'membrane, as a result of the 
rapid evaporation of chloroform off the membrane. Such treatment gave 
consistent results. Otherwise, the rate of evaporation was very erratic. 
The distilled water in the cell was never in contact with the membrane; 
there was always a saturated air. space between the water and the membrane.
In an attempt to first establish the. relationship between thickness 
of wax films and evaporation through such films, various, thicknesses of 
beeswax were deposited, from chloroform solutions-of proper concentra­
tions, on the membrane and 'the rate of evaporation through these'
IkJ
' membranes was jiotdd. The results are presented in Table XXI and Fig­
ure 3 1.
It is seen from this Table and Figure 31 that the'rate of evapora-. 
tion through films thicker than two microns decreases very slowly. A 
film 2u thick suppressed the rate of evaporation by approximately k\.jjo. 
Alexander, Kitchener and Briscoe (19^4) ftiund that it was the initial 
layer of wax that had the greatest waterproofing capacity. Beament 
(1945) reported similar findings. The data presented in Table XXI in­
dicate that the initial O.^u film was responsible for about one-half 
the total suppression in evaporation. This confirms the findings of 
Alexander et al (1944) and Beament (194$).
When the percent transmission was plotted against thickness, a 
curve was obtained which superficially resembled an exponential curve 
(Figure 32). Plotting the log percent transmission against'thickness, 
should therefore give a straight line.. However, the curve II (repre­
sented by a broken line) shows that such is not the- case (Figure 32).
Curve II also resembles, superficially, the exponential form. From 
this it can be concluded that Curve I is not of the expontential type.
This is exactly what happens in the case of absorption of a hetero­
geneous beam of X-rays by variable thickness of a filter. The data to 
show the latter effect was taken from Stuhlman (1943) and are shown in 
Figure 33« Beament's data were also taken and repELptted in this manner.
The resulting curves are shown in Figure 34 and bear considerable simi- . 
Iarity to those presented in Figures 32 and 3 3. The similarity between the 
Figures taken from the three different sources suggests that the mechanism
Table XXI
I
!
Rate of evaporation through membranes covered with variable thicknesses 
of beeswax. The exposure period to dry air was minutes 
at 30°C. The area of the evaporating Surface = .916 sq. cms.
Thickness Initial IDecrease iii
in microns Wgt0 iWgt./hr. I
0 3.021# ’.0131
12 ll'.99lt3 .0113
I b.9782 . .OlOli
2 li.9662 .0088
3 li..9lili3 .0085
U li.9339 .0080
' hi ■
Rate % Trans-
Log of $ I
mg/cm2/hr. I mission . transmis­
sion I
l6 .lt 100 2.00'
1 2.lt : 73.o 1.87 ;
- 'li.lt 69.3 I.8I1
9.6 .38.3 1.77
9.3 36.7 1.73
9.0 31i.9 ■ l.7li
\
1 2 4-
Thickness of wax
Figure 31. Rate of evaporation of water from an aluminum cell covered by
a cuticular membrane which was coated with films of beeswax of
variable thickness.
P
e
r
c
e
n
t
 
t
r
a
n
s
m
i
s
s
i
o
n
iSo
percent transmission
--- log percent transmission
1006
Thickness
. Decrease in percent transmission and log percent transmission 
of water vapor with increased thickness of beeswax film.
Figure 32
L
o
g
 
p
e
r
c
e
n
t
 
t
r
a
n
s
m
i
s
s
i
o
n
l£l
IOOt
percent transmission
--- log percent trans­
mission
Thickness mm
Figure 33» Decrease in percent transmission and log percent 
transmission of a heterogeneous beam of x-rays with 
increased filter thickness (after Stuhlman).
L
o
g
 
p
e
r
c
e
n
t
 
tr
an
s
m
i
s
s
i
o
n
152
percent transmission
---  log percent transmission
-  1-1
Thickness
Figure 3U. Decrease in percent transmission and log percent 
transmission of water vapor with increased thickness of 
beeswax film, (After Beament)»
L
o
g
 p
e
r
c
e
n
t
 
t
r
a
n
s
m
i
s
s
i
o
n
153
responsible for the decrease in evaporation by increasing thicknesses 
of beeswax is similar to the decrease in transmission of a heterogeneous 
beam of X-rays by variable thicknesses of an aluminum filter.
In the past, the decrease in evaporation by increasing thicknesses 
of beeswax has been attributed to the high orientation of the lowest 
layers of wax. Addition of more wax, which will not be oriented above 
a certain thickness of wax, contributes very little towards increasing 
the impermeability of the organized lower layers. It will be shown here 
that a very simple explanation of this phenomenon can be given by con­
sidering the relationship between films of variable thickness and the 
kinetic energy of the vapor molecules.
The film of wax may be considered as a potential barrier across 
which the penetrating water molecules must pass. The distribution of 
the energies of translation of the water molecules in the vapor phase 
will be given by the well known Maxwell distribution curve.
Now let us assume that only those molecules will pass the mem­
brane which have a kinetic energy greater than the energy of the
154
potential barrier. If such a potential barrier is designated by P, 
then on the basis of our assumption the shaded region under the curve 
to the Ibft of P will represent the number of molecules which have a 
kinetic energy less than the energy of P. A ratio of this area to the 
total area under the curve will give the fraction of the total molecules 
which have an energy of translation less than the critical energy. If 
P is taken .as the barrier of O'.5u .thickness, then the shaded region to 
the left of P would correspond to the percent of molecules which were 
unable to pass through this membrane i.e. 22.5$• This value will be 
called percent suppression. Similarly, if QvR S and T are taken to 
represent potential barriers corresponding to the films of 1 ,2 , 3, and- . 
4 microns thick, then the area to the left of each of these barriers 
would likewise correspond to the !fraction of molecules which had insuf­
ficient kinetic energy to pass through them. A table is given below to 
indicate the fraction of the total area that lies to the left of each 
barrier. The values we re/‘read off the graph.
Membrane Thickness ' # Suppression
0
0.5
1
2
3
4
o
22.5
32.5
40.5 
42.0
42.5
- In:the light of our original assumption these data indicate that 
the increase in the energy of the potential barrier is not linear with 
thickness. Very probably the increase in the .energy of the potential 
barriers with increasing thickness obeys a logarithmic function. Rep-
155
resented diagrammatically, this means that succeeding potential barriers 
must he placed closer together. The energy difference between successive 
barriers would therefore decrease and this will in turn decrease the frac­
tion of molecules which will be screened out as we go from one barrier to 
the next.
Another membrane was prepared and extracted in chloroform in a man­
ner already described. It was fitted in the evaporation cell and the fate
■■ ■
of evaporation through this. membrane into dry air!for thirty minutes was 
determined at various temperatures. Two trials were made at each tempera­
ture .- The data are presented in Table XXII and plotted in Figure 35-1 •
Figure 35-1 indicates a smooth, gradually rising curve. There is 
no indication of a break in any portion of the curve and. in this respect 
it is very dissimilar from the curves already presented, in the discussion 
of critical temperatures a’nd from curves II and III in Figure 35; which . 
relate the rate of evaporation through a membrane coated with films of 
beeswax and paraffin.
The membrane was then coated with two microns of. beeswax. The rate
i
of evaporation through this membrane was determined in. a manner already 
described. The exposure period was thirty minutes and two trials were 
made at each temperature except in the case of temperature '62.5 and 65.
The results are presented in Table XXIII and Figure 35-11.
Curve II indicates the presence of three distinct regions. The 
first region lies between 15-35°C.. Here the increase in evaporation 
appears to be a linear function of increasing temperature. The second 
region lies between 35 -'51.5°C. The rate of evaporation is also a
Eate of evaporation throtigh an.abdominal portion of the' cutical used as 
a membraneo This membrane was treated with chloroform 
to remove the natural wax
156
Table ZXII
Trial I
Temperature Initial Wgt. I Decrease in ( Bate S
0C in gms „ I Wgt./hr. I mg/cm2/hr. J
1 0 .0  - - 5 .0 5 3 0 .0028 ’ 3 . 1 '
2 0 .5 5 .ob?d .007b ' 8 .1
3 0 .0 5 .o b i6 ' .0 1 2 0 13.1
.3 5 ,0 5 .0 2 6b .0170 1 8 .6
k o .o 5 .0 093 .021b 2 3 .b
U5.o b .9 8 7 l .6268 2 9 .3
5 o .o b .9 59b .0386 b 2 .1
5 5 .o . b .9 198 .o5ob 5 5 .0
Trial II ■ - I
Initial- Wgt. Decrease in Rate
I Average Rate J
in gms. ' I Wgt./hr. mg/cm2/hr.
5 .0 5 1 6 .0032 3.5 3 .3
5 .0b 53 .007b 8 .1  . • ’ 8„1;-
5 .0 3 5 6 .0138 15.1 lb.I
5 .0 179 .0172 1 8 .8 1 8 .7
Ti.9986 .0230 2 5 .1  ' 2 b .2
' b .9 7 3 7 .0286 3 1 .2  ' 3 0 .2
b.9boi .0b06 bb°3 b 3 .2
If? 7
Table XXIII
Rate of evaporation through a. inembrane of beeswax 2u thick» The 
period of exposure to dry air was 30 minutes»
Trial I .
V  '
Temnerature S Initial Wgt. J Decrease in Rate
OC I in gms. I Wgt./hr. mg/cm2/hr.
15.0 b.9'986 . .00^8 5 .2 b
2 $ .0 L .9912 .0080 8 .7 3
3 0 .0 1 .9 8 6 1 .0102 ll.lb
3 5 .0 li.976L .0120 13.10
3 7 :5 1 .9 6 3 9 .0152 1 6 .5 9
1 2 .5 L .9188 .0182 1 9 .8 7
L 7 .5 1 .9 303 - ' - ' .02^6 2 6 .8 6
5 2 .5 b .9 0 5 5 .0319 3 b .8 l
5 7 .5 L .8719 - .010.0 bb.76
6 o .o U.830lt .0 5 0 2 . - 5b .8 o  '
6 2 .5 li.7 80 li .0559 6 l . l
6 5 .0 1.7131 .071U 7 7 .9
I • Trial II
Initial Wgt. 
in gms,
■ Decrease in 
Wgt/hr.
I Rate.
I mg/cm2/hr, Average Rate-
I
} b .9962 .oobo b .3 7  . b. 8
b .9902 .,0082 8 .9 5 8 .8
! ' b .9810 .0092 10 .0b 1 0 .6
b .970b .0130 lb.19 1 3 .6
i b .9563 .0150 1 6 .3 7 1 6 .b
b .9397 .0188 2 0 .5 2 2 0 .2; b .9180 .0250 2 7 .2 9 2 7 .1
'* b .8869 .0300 . 3 2 .7 5 3 3 .8
! b .8 5 lb .ob.20 b 5 .8 5 b 5 .3
i \ b .8053 .ob98 5b .3 7  . 5b 0 6
\ — - 61.1
I 1; 1 ■ 1■— — 7 7 .9
R
a
t
e
 
of
 
e
v
a
p
o
r
a
t
i
o
n
 
m
g
m
s
/
s
q
.
 
cm
/h
r.
I #
I - uncoated membrane 
II - beeswax-coated membrane 
III - paraffin-coated membrane
6o --
5 io
Temperature 0C
Figure 32. Rate of evaporation of water from an aluminum cell covered 
by a variously coated cuticular membrane.
159 .
'linear function of temperature, ■ but the "slope of the line is much greater. 
It will be recalled that it was in this temperature range that the polari­
zation of beeswax was also an increasing function of temperature. Con­
sequently, as the wax molecules assume more freedom of rotation and in­
ternal distortion, they also become increasingly more permeable. Thus, 
the wax membrane ..loses its impermeability because the forces between 
the wax molecules are weakened, thereby permitting rotation and hence a 
gradual breakdown in structure. The third region begins at 51.5°C. Pre­
sumably, at this temperature, a maximum number of wax molecules are ro­
tating round their chain axis. There is, therefore, a maximum breakdown 
in internal orientation. Under these conditions, the energy of the film"
has perhaps become a constant and the increase in evaporation is due to 
.
the increase in kinetic energy of the penetrating water molecules 
with increasing temperature.
To determine the nature of evaporation through a paraffin film and 
compare it with that through a beeswax film, two microns of paraffin were 
deposited from a chloroform solution of proper concentration.upon a mem­
brane from the abdominal portion of the cuticle. The rate of evaporation 
through this film was determined in exactly the same manner as in the pre­
ceding case. The data are presented tabularly in Table XXIV and .graphi­
cally in Figure 35-III.
It is seen from a comparison of Curves II and III that.the mechan­
ism involved in-.the evaporation of water through a paraffin film id dif­
ferent from that through a beeswax filib.. This difference is undoubtedly 
determined at least partially by the non-polarity of paraffin. Muller-
160
Bate■of evaporation at.various temperatures through a film of paraffin 
two microns thick„ The m.p. of the paraffin was £0“£2°Cl
Table XXIV
Trial I
Temperature I Initial Wgt. I Decrease in I Rate
OC I in gms. j Wgt./hr. mg/cm2/hro
22.0 U.92U7 .00U3 Uo 69
30.2 U.9199 .0060 6.22
32.0 U.9071 .0070 -7.6U '
Uo.o U.9033 .009U 10.26
U6.0 . U.89U3 .0118 12.88
U8.0 U.8783 .0129 1U.08
22.0 U.8606 .0172 18.78
27.o U.8U91 .0312 3U.39
62.0 U.8281 .0UU8 U8.91
Trial II
Initial Wgt . 
in gms.
Decrease in 
Wgt/hr.
Rate
rng/cm2/hr. ■
Average Rate •
I
U.9218 .0038 Uol2 UoU
U.9169 .0022 ' 2.68 6.1
— — 7.6
U.8986 .0086 9.39 9.8
U.886U - .0116 12 .6 6 . 12 .8 .
U.8697 .0136 1U.82 ■ 1U.2
—  M M 18.8
—  —  UM 3).uU
■  — BM —  ™ U8.9
l6i
(1937) using a paraffin of melting point 44 - 46°C found no increase in 
dielectric polarization with increasing temperature. Consequently, the 
rapid rise in evaporation beginning at approximately 52°C cannot be at­
tributed to an increase in dielectric polarization. It is assumed to 
be caused by a change in phase of the paraffin at Its melting point. , The 
gradual increase in evaporation through this film up to its melting, 
point is attributed to a gradual softening of paraffin as the tempera­
ture was increased, and/or the ,gradual increase in the kinetic energy 
of the water molecules.
It is therefore concluded that the phenomena of increased evapo­
ration at certain temperatures called critical temperatures could be 
caused by two mechanisms depending,,.; upon..the nature of the potential bar­
rier. If the potential barrier is polar then"the increased evaporation 
at the citical temperature will be caused by the breakdown of internal 
orientation resulting in an increased dielectric polarization. This oc­
curs long before the true melting, poiht. 1$ reached, At the melting
,
point, i.e. change in phase of such a compound, there is.no change" in 
evaporation. On the other hand, if the potential barrier is nonrpolar, 
then the increase in evaporation at the critical temperature coincides 
with the melting-point of the compound.' In other words, it appears to 
be caused by a change in phase of the non-polar material.
X. Nature of the Transitional point.
In the light of the previous discussion, it is now possible to - 
propose a very simple mechanism which would account for the increase 
in the■rate of evaporation at.the critical temperature as observed in.
162
the intact cuticles.
Muller (1932) found by means.of X-ray analysis that crystalline 
changes occur in n-paraffins a few degrees below their true melting .
.point. The coefficient of lattice increase in the "a" direction was 
3-4 times that in."b" direction. There was very little increase in the 
"c" direction. This indicates two things. First,, an increase in volume 
of the crystal lattice occurs a few degrees below the true melting point 
and secondly, that the forces holding the chains together, were much 
weaker than those holding the atoms within the chain. In 1937; he 
found that an increase in dielectric polarization of certain ketones 
increased at a temperature 10 - 20 degrees below the true melting-point. 
This increase in dielectric polarization was correlated hy him in 1938 
with the increased 'yolume of the.lattice. It has already been pointed 
out that a similar increase in dielectric polarization of beeswax was 
first observed about 28 degrees below its melting point.
Beament (1945) states that Chibnall (1944) communicated to him the 
information that insect waxes consist of esters, acids and paraffins. 
This means that they are polar. Consequently, it is highly probable 
that they would exhibit a similar increase in polarization at the cri­
tical temperature.
The wax layer covering the surface of the cuticle may be considered 
as a potential barrier. Let Tjj, be a temperature just below the critical' 
temperature T, and Iq just above T., Then P]_ and Vq will be the value of 
the energy of the potential barrier at temperatures Tq and Tq respec­
tively. 1 As the temperature is increased from Tq to Tq the dielectric
163
polarization of the wax increases. This internal reorientation de­
creases the value of the energy of the potential barrier from to 
P2 . At the same tune the distribution of the velocities of the water 
molecules as given by Maxwell's distribution curve is shifted to the 
right, i.e. their kinetic energy is increased. Consequently, this de­
crease in the energy of the potential barrier in the region 
•coupled with the increase in the kinetic energy of the water molecules 
as the temperature is increased from T1 to T2 produces a sudden increase 
in the rate of evaporation through the wax film at the critical tem­
perature.
Coupled with the decrease of the energy of the potential barrier 
as a result of polarization, there might be and probably is an increase 
in the size of the intermolecular pores. This increase in the volume 
of the crystal lattice would permit an easier penetration of the wax
Energy ->
layer by the water molecules.
SUMMARY AED COECLUSIOES
The literature .on the arthropod cuticle is reviewed, discussed 
and criticized in detail. The important contributions, to our know­
ledge of structure and permeability of the cuticle, by some of the in­
vestigators in this field are brought to the forefront. An attempt 
was made at a unification and synthesis of their findings.
The apparatus and methods employed in this study are described 
and discussed in detail.
A formula for the determination of the surface area of Melanoplus 
bivittatus was fitted to the data obtained by dissecting the insect ' 
body into various geometrical forms and then measuring their areas by. 
means of a microprojector and a planimeter. The relationship between 
surface area and weight was found to be best expressed by S = 10.27 
W,. ‘7^. The form of this formula compared favorably with that obtained 
for other insects. •
The epicuticle of Melanoplus bivittatus was found to consist' of 
at least two layers. From within outward they are (a) cuticulin layer, 
and (b) wax layer. The cuticulin layer is presumably a lipoprotein. It 
is resistant to prolonged periods of chloroform extraction. In the pre­
sence of the fumes of an aqueous solution of osmic acid it assumes a 
black color. The wax layer overlies the cuticulin layer. It is the 
main barrier to evaporation through the cuticle. If.jthe cuticle.■> is ex- 
. tracted in _ch.lbroform,for a very short period of time, or abraded, it 
•ldses its property of impermeability because of the removal of.the wax 
layer. Evidence has been obtained that these layers are not discrete;
165
rather, they fuse into each other (Chefurka, unpublished data).
The wax layer is completely deposited on the new cuticle before 
the old cuticle is shed. New cuticle extracted in cold chloroform be­
comes highly permeable as compared with untreated cuticle. Measure­
ments of contact angles of distilled water on the new cuticle during 
the moulting process, and on cuticle several days after moulting> gave 
values of 105.9° and. 105.0° respectively. When the new cuticle was 
stained in.ammoniacal silver hydorxide, no evidence of staining was pre­
sent .
I
The similarity in the values of contact angles given above indi­
cates that the interfacial reactions which might occur-between the 
polar groupings of the protein and those of the wax do not affect the 
orientation of the wax molecules at the wax-air interface.
No evidence was found for the presence of a cement layer. Cuticles 
extracted in chloroform at 25°C and 40°C for 30 minutes showed the same 
degree of permeability. Cuticles .extracted, in chloroform at 25°C for 
variable periods of time also showed the same degree of permeability, 
indicating that, once the superficial layer of wax is removed, con­
tinued extraction does not alter the permeability. This is further e v i ­
dence for the absence of a cement layer. Chloroform vapors do not pro­
duce any appreciable disruption,of the wax layer. In order that it be 
disrupted, it must be in contact with the chloroform.
When the cuticle is heated to a certain "temperature, it suddenly 
becomes more permeable to water. The temperature at which- the rate of 
change of rate of evaporation is a maximum is here called-the "critical
f 166
temperature" of the wax. For M. hivittatus this temperature was found 
to be W 3C. It remained', constant for all the instars. The method by 
which the, critical temperature was determined' is outlined.
On the basis of analogous experiments with beeswax, it is con­
cluded that the increase in the rate/of evaporation at the critical 
temperature is caused by an increase, in the dielectric polarization of 
the wax at this temperature. A theory for the explanation of the ‘cri­
tical point is therefore offered. It considers the wax layer as a po­
tential barrier. At the critical temperature, the value of the energy
of the potential barrier is decreased due to the weakening of the dipole
1
forces. This means' :that more water molecules have a kinetic energy 
greater than the energy of the potential barrier at this temperature. 
This combined effect is responsible for the sudden rise in evaporation.
There is a progressive decrease in the permeability of the cuticle 
with increasing time after moulting. This is attributed to:
1. Progressive increase in the thickness of the cuticle with 
time after moulting.
2 .  ' Gradual hardening of the cuticle caused by a tanning of the
protein by quinones. One of the polyphenols,1:2 dihydroxy- 
benzene, was found to be present in the cuticle of M. 
hivittatus. It is proposed that the process of protein tan­
ning is completed by 6 hours after moulting. The cuticle is 
presumably formed by approximately 50 hours after moulting.
The percent, loss of body moisture into dry air was found to be' 
approximately 1$, 2$, and 3$ at 25°C, 30°C,.and 40°C respectively.
The rate of loss of body moisture into dry air-, per hour was found.to be 
approximately constant at any particular temperature, irrespective of 
the time of exposure..
The rate of evaporation into dry air at a series of temperatures 
was found to be slightly higher from grasshoppers whose spiracles were 
hot closed.
The variation of the dielectric polarization.of beeswax with tem­
perature was measured. It was found that the polarization increases 
from 35°C to 51.5°C. It is assumed that this is caused by a gradual 
weakening of" the elastic forces holding the dipoles in a stationary- 
condition . As the directional forces are weakened, the dipoles and 
molecules increase the. amplitude of oscillation, and ultimately perform 
a complete rotation around their chain axis. In the-.case of beeswax 
this maximum rotation is assumed to occur at 51 • 5°C
When the rate of evaporation into dry air was measured through a 
2 micron film of beeswax, Sb. was found that an increase in the rate of 
evaporation occurred in the region 31-51.5°C. Consequently the wax 
molecules become more permeable to water as a result of the gradual in­
crease in polarization. BTo break in the evaporation vs. temperature 
curve of beeswax- was noted■at the melting point of this wax. When the 
rate of evaporation .was measured through paraffin films.of the same 
thickness, a sudden increase in the rate of evaporation occurred at its 
melting point. It is concluded, therefore, that, in the case of polar 
compounds, the'increase in the rate of evaporation at the critical tem­
perature is caused by a gradual weakening of the dipolar forces'. In
168
non-polar compounds the increase in the rate of evaporation at the cri­
tical .temperature is due mainly to. a change in phase of the substance 
at its melting point.
The rate of evaporation into.dry air,through increasing thicknes­
ses of beeswax films., decreased gradually with increasing thickness of 
the film. If the film is considered as a potential barrier then this 
gradual decrease in evaporation is attributed to the non-linearity be­
tween increasing thickness and: increasing energy of tbepotential barrier
\
i
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MONTANA STATE
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