Table Of ContentERGEBNISSE
DER BIOLOGIE
HERAUSGEGEBEN VON
H. AUTRUM . E. BaNNING· K. v. FRISCH
E. HADORN . A. KaHN· E. MAYR . A. PIRSON
J. STRAUB . H. STUBBE· W. WEIDEL
REDIGIERT VON
H. AUTRUM
ZWANZIGSTER BAND
MIT 34 ABBILDUNGEN
SPRINGER-VERLAG
BERLIN· GOTTINGEN· HEIDELBERG
1958
ISBN 978-3-540-02262-6 ISBN 978-3-642-51754-9 (eBook)
DOI 10.1007/978-3-642-51754-9
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Briihlsche Universit!itsdruekerei GieSen
Vorwort
Wissenschaft schreitet durch Spezialisierung und deren Dberwindung
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Dberschaubar bedeutet erstens: Beschrankung auf das Gesicherte und
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Gebieten arbeitenden Wissenschaftler. Als im Jahr 1925 die "Ergebnisse
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Literatur.
Die neuen Bande verfolgen ein anderes Ziel: Sie wollen nicht ein
Handbuch erganzen oder ersetzen, sondern vielmehr liber moderne
Probleme berichten und sie libersichtlich zusammenfassen. Die Beitrage
sollen im Umfang begrenzt sein; sie wollen das Erreichte kritisch dar
stellen, offene Fragen herausarbeiten und damit als Grundlage fUr
weitere Forschung und Synthese dienen.
Herausgeber und Verlag hoffen, daB die "Ergebnisse der Biologie"
dem Forscher die Einsicht in Nachbargebiete moglich machen, daB sie
aber auch dem Studenten und dem Lehrer an Hochschule und Schule
helfen werden, sich liber die Ergebnisse der modernen Biologie zu unter
richten und sie unterrichtend weiterzugeben. Die Herausgeber danken
dem Verlag, daB er das Wiedererscheinen der "Ergebnisse der Biologie"
ermoglicht und ihre Arbeit in groBzligiger Weise unterstlitzt.
H. AUTRUM
Wlirzburg, im Januar 1958
Inhaltsverzeichnis
RICHARDS, A. GLENN, Professor, St. PaulfMinnesota(USA). The Cuticle
of Arthropods. With 3 Figures . . . . . . . . . . . . . . . . . .
BURKHARDT, DIETRICH, Dr., Wiirzburg. Die Sinnesorgane des Skelet
muskels und die nervose Steuerung der Muskeltatigkeit. Mit 12 Abbil-
dungen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
ARNOLD, CARL-GEROLD, Dr., Erlangen. Selektive Befruchtung. Mit 1 Ab-
bildung. . . . . . . . . . . . . . . . . . . . . . . . . . . 67
BIER, KARLHEINZ, Priv.-Dozent Dr., Wiirzburg. Die Regulation der
Sexualitat in den Insektenstaaten. Mit 4 Abbildungen . . . . . . . . 97
RENNER, MAX, Dr., Miinchen. Der Zeitsinn der Arthropoden. Mit 1 Ab-
bildung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
KRAUSE, GERHARD, Professor Dr., Tiibingen. Induktionssysteme in der
Embryonalentwicklung von Insekten. Mit 13 Abbildungen . . .. 159
HERAN, HERBERT, Dr. Graz. Die Orientierung der Bienen im Flug 199
Namenverzeichnis. 240
Sachverzeichnis .. 247
The Cuticle of Arthropods*
By A. GLENN RICHARDS, St. Paul/Minn. (USA)
Department of Entomology and Economic Zoology, University of Minnesota
With 3 Figures
Contents
Chitin Chemistry . . . • • • • 2
The Chemistry of Sclerotization • 6
The Chemistry of Other Components. 9
Structure of the Cuticle • . . • • 10
The Multiple Barriers of the Cuticle 14
Diversity within the Arthropods. 15
Ecological Aspects of the Cuticle 16
Literature . . • • • . • • . • 19
The present review is intended to cover the major advances that have
taken place since the appearance of the author's monograph in 1951.
But with the publication of the recent excellent review by WIGGLES
WORTH [170], the treatment has been modified to make the present
review supplementary in so far as possible. Frequent reference will be
made to both of the above.
In surveying the literature of the past decade one is impressed with
the number of important advances that have been made, especially in
cuticle chemistry. The work of HACKMAN is particularly important,
partly because he is studying all of the various components found in
cuticle. But these advances also show how extremely complex the story
of cuticle chemistry and structure is going to become. The reports show
that, while the bulk of the cuticle or the ground substance of the cuticle is
composed of chitin plus a protein mixture called arthropodin, the impor
tant properties are due more to compounds present in such small amounts
that they would be classed as trace contaminants until their importance
is discovered. This demonstrates the need for much more research on
* Paper Number 943, Miscellaneous Journal Series, Minnesota Agricultural
Experiment Station, St. Paul, 1, Minnesota, USA.
Ergebnisse der Biologie XX
2 A. GLENN RICHARDS
cuticle components. Fortunately, several competent chemists have
interested themselves in these problems.
Biologists should remember, however, that although some rigorous
chemical determinations have been made, much of the data on arthropod
cuticle are not so soundly based. For much of the work there is no good
alternative to histochemistry. Descriptions of color reactions from
histochemical tests are one matter, but many of the deductions from them
are inconclusive. And histochemical studies sometime lead to the piling
of one logical conclusion upon another, the last interpretation depending
on a prior one which in turn depends on another which is itself uncertain.
For instance, the now commonly stated idea that tyrosine in arthropodin
can be oxidized in situ to produce reactive quinones is, as the cautious
chemist MASON remarks, only an interesting working hypothesis lacking
rigorous documentation. Conclusions today, even many that seem
thoroughly satisfying, are largely tentative.
The increasing complexity is not only apparent in the literature on
chemical components but also in the field of cuticle permeability. Most
authors still attempt to explain penetration in terms of static models
that can be visualized. However, several recent studies [13, 36, 129J
imply that we will soon have to think in terms of dynamic models
expressable by mathematical equations but not satisfactorily shown by
pictures.
What then would I hope to find in the literature a decade hence?
I hope for 1. More comparative analyses, especially treating structures
not usually studied and species from special ecological niches; 2. More
rigorous chemical determinations on components and their reactions by
organic chemists and on the molecular architecture by physical chemists;
and 3. A beginning of the development by biophysicists of dynamic
models expressing penetration phenomena.
Chitin Chemistry
It is generally agreed that chitin is a linear polysaccharide composed
of N -acetylglucosamine residues linked together by p-glycosidic bonds so
that the minimal descriptive unit is chitobiose [124]. Current chemical
terminology would describe chitin as a p-glycosidic linked polymer of
N-acetyl-2-amino, 2-deoxy, D-glucose or even better as a polymer of
2-acetamido, 2-deoxy, IX,D-glucopyranose. These express the over-all
picture but there really is no proof that the all units of the chain are of
this type. There could well be a small percentage of non-acetylated
residues (or even some glucose residues). An occasional non-acetylated
residue would account for the trace of glucosamine found after enzymic
decomposition by HACKMAN [48J and earlier authors. It would also
account for the slight reaction of chitin to the periodic acid-Schiff
The Cuticle of Arthropods 3
reaction (which should be blocked by acetylation), and, of more interest,
would permit the direct linking of chitin chains to tanning quinones [100].
It is, then, highly desirable to know the degree of homogeneity of the
repeating units in a chitin chain but, unfortunately, this is not readily
proven because of the possibility of some deacetylation occurring during
the chemical manipulations. Several authors have t:;ommented on the low
nitrogen values given by KJELDAHL analysis of purified chitin [124] but
this can hardly be interpreted as indicating the presence of some non
amino residues in the chains when such have not been identified following
enzymic decomposition. The chains are reported to have an average
length of 31 residues [69 a].
At the supermolecular level, several types of chitin have been found.
Two crystallographic types appear well documented. The at-chitin
configuration [124] is the common one and the only one known for
arthropods. A second type of chain association, .a-chitin, is found in some
polychaetes and molluscs. In cephalopods, at-chitin is found in the radula,
beak and lining of the gut, but .a-chitin in the skeletal pen '[134J. RUDALL
remarks that .a-chitin is found associated with collagen whereas at-chitin
occurs alone [103] or in association with a non-collagenous protein such
as arthropodin. It seems certain that other types of chitin exist but we do
not yet know whether these are all simply different crystallographic
configurations. Thus, RUDALL reports that Coelenterate chitin gives
a distinct x-ray diffraction pattern,
RICHARDS reports a green instead of
violet chitosan color test from a· Bry
ozoan [124], and KRISHNAN reports an
orange chitosan color test for what he
considers to be chitin in the epicuticle
of a scorpion [84].
The structure and properties of
chitin are said to be most like those of
metastable cellulose III [103]. Some
surprizing discoveries have been made at
this level of association of chitin chains.
DARMON and RUDALL using polarized
infra-red spectroscopy show that the
chains are held together by hydrogen
bonds that presumably occur between
C = 0 and-NRgroupsof the side chains Fig.l. AspatiallydistorteddiagramiIJustra-
ting the two principle type of hydrogen
(and probably also between C = O· .. RO bonds between chitin chains
groups) (Fig. 1). With progressive dea-
cetylation, OR ... OR bonds are formed as in cellulose. But one half of
the acetyl groups are more readily removed by alkali than the other half.
1*
4 A. GLENN RICHARDS
This implies that there are two types of acetyl groups, and it has been
suggested that the two types represent cis-and trans-bonding (Fig. 2), but
the data do not prove this or show which is more readily deace~ylated.
However the chitin chains are held together, they clearly aggregate
into larger units called micelles [124J. With the increasing commonness
of using electron microscopes, the cuticles from a considerable number of
species of various orders have now been examined. Most but not all of the
work has been with thin membranes that
require no elaborate preparation (peritro
phic membranes, gut and tracheal linings,
ecdysial membrane). In general, micro
fibers of about 100 A diameter are found
though a range of 70-200 A is recorded
[38, 39, 57, 90, 99, 101, 126, 127, 171J.
RIEl, noting the commonness of this dia
meter for microfibers of various substan
ces, has suggested that this may well re
present a general average length of cry
, stallinity attainable perpendicular to the
NH CO NH main axis in microfibers which are micellar
~'\//~ aggregates. Working with cellulose, FREY
CO WYSSLING has subdivided microfibers
into aggregates of micelles averaging
cis-bonding trans-bcmding 30 x 70 A; this has the important effect
Fig. 2. Diagram of the probable linkages, of assigning the amorphous or para
related by a screw turn, between chitin crystalline material to a position outside
chains. (after RUDALL, 133)
the micelle yet inside the microfiber. So
much of the data on cellulose is applicable to chitin that this may
well be too.
Apparently we have a hierarchy of chain associations: chitin chains
aggregate into micelles which aggregate into microfibers which, in cuticle,
aggregate into microscopically visible fibers known in the literature as
"Balken". The suggestion by RICHARDS [124J that this is true has now
been well documented by electron microscope and UV microscope
examinations [39,90,117, 126J. ENIGK and PFAFF report seeing micro
fibers in normal Hypoderma, the fibers decreasing in diameter on removal
of the protein; the other authors have had to treat their preparations in
a manner dissociating the chitin-protein complex before seeing micro
fibers. ENIGK and PFAFF interpret their results as indicating a lipo
protein sheath around a chitin core but I see no reason why the fibers
might not equally well be of mixed composition.
Evidence continues to support the idea that chitin is not a naturally
occurring compound but a degenerative chemical product produced in
The Cuticle of Arthropods 5
testtubes [124J. To be sure, K. H. MEYER says that the shield of the
cephalopod Loligo is pure chitin but there are several conceivable explana
tions of this unique case - too little is known about the development of
the structure to warrant speCUlation now. In general, chitin is intimately
associated with protein. In arthropods, chitin is associated with a group
of proteins called arthropodins. Evidence increases that this association
is somehow bonded together but we still have no clear idea of what the
bonds are or even whether they are chemical or physical in nature.
Whatever the nature of the bonds within this glycoprotein, they must be
weak because they are readily disrupted by heat or a moderate change of PH'
Once the unstable chitin-arthropodin bonding is broken, the highly stable
chitin lattice is formed [133]. This is not readily altered. However, the
unstable chitin-arthropodin lattice is rendered stable by the tanning
process called sclerotization (the fragile erythrocyte is also greatly
strengthened by treatment with tannic acid [31J).
Supporting evidence for the ability of chitin to combine with proteins
has been provided by HACKMAN [49J who first showed that N-acetyl
glucosamine can react with arthropodin, peptides and amino acids,
especially tyrosine. The union is unstable at acid PH but reasonably
stable at higher PH'S. Then, second, HACKMAN [50J reported that purified
chitin can adsorb up to 8 % of its weight in protein. The bonding is not
affected by reasonable temperatures but is effected by salts and by PH'
falling off rapidly above the isoelectric point of chitin and reaching zero
at PH 9. He concludes that there is a weak chitin-arthropodin bond of
some sort; its easy rupture by a mild PH change he interprets as implying
that the bond is neither covalent nor H bonding. The fact that he
succeeded in obtaining a maximum of only 8 % adsorbed is not inter
pretable. Perhaps it only means that the micelles of his purified chitin
were too tight for penetration and that hence adsorption was limited to
their surfaces. Certainly the amount of protein adsorbed was far below
that found in arthropod cuticle.
Chitinases have now been found in a wide variety of organisms. Their
properties have been studied in preparations from snails and their
intestinal flora [48,70,74, 75J, fungi [158J, and the exuvial fluid of various
insects [71, 72, 76, 112J. Destruction of chitin implying the presence of
chitinase has been reported for various soil bacteria [11, 165J, various
fungi, both free-living [40, 78, 143J and parasitic species that penetrate
insect cuticles [64,93, 131, 150J, and, among animals, eelworms, earth
worms and soil amebae [155-157J. The recorded PH optima are usually
in the range 4.8-5.5 [48, 72, 75, 158J, and the products are acetyl
glucosamine plus a trace of glucosamine for crustacean chitin [48J but
only acetylglucosamine for Sepia chitin [158J. Thechitinasesfrominsect
exuvial fluid have similar PH and temperature optima to the microbial
6 A. GLENN RICHARDS
enzymes [71, 72, 76J. The activity of fungal chitinase is said to be
augmented by the presence of extra protein [158J. It is also an adaptive
enzyme [122 AJ. The use of chitinases is now under sufficient control
that TRACEY [159J recommends their use for chitin detection.
On the other hand, the museum pest Anthrenus eats insects, pulveri
zing the chitin but not digesting it [73J. And the exuvial fluid of maggots
which seems not to digest any en do cuticle at molting is reported to lack
chitinase [173J.
No important advances appear to have been made on the inter
mediary metabolism of chitin or cuticle. However it does appear that the
synthesis and assemblying of the components is under the control of the
animal and independent of environmental conditions except as these
affect the development of the whole animal. TSAO and RICHARDS report
for Blatta, Tribolium, Tenebrio, Galleria and Phormia that the quantity of
chitin and cuticle was little if any altered by variations in temperature,
humidity, or nutrition when feeding was ad libitum (previous reports of
an effect of feeding on quantity of cuticle relate to quantity of food; this
study dealt with composition of the diet). In Tribolium and Phormia the
amounts were not altered by diets lacking free carbohydrate but the
casein used contained some glucosamine which may well have been
utilized as chitin precursor. Within a single stage of a single species the
amounts of chitin remained surprizingly constant when values are
expressed as per cent of body weight - individual variations from the
average seldom exceded 10%.
However, as is well-known, the thickness and weight of cuticle does
vary considerably from one region of the body to another. Additional
quantitative determinations have been published for Periplaneta [25,
117J, Blatta [163J, and Bombyx [153J.
The Chemistry of Sclerotization
As indicated by TRIM'S work [124J, the proteins are heterogenous even
in a single species. Electrophoretic separations of the water-soluble
proteins (arthropodin) by HACKMAN [47J show that they are all fairly
similar in physical and chemical properties in the seven species studied
(Orthop., Coleopt., Hemipt.). Free oc-amino groups were found in four
but free carboxyl groups in only one. The same amino acids that have free
amino groups in the water-soluble fraction, also have free amino groups
in the water-insoluble fraction. There are some differences in amino acid
composition but we have yet no idea what this signifies. Thus the water
insoluble, alkali-soluble (N NaOH at 50 fractions lack proline and
0)
hydroxyproline; the alkali-insoluble fractions lack serine, threonine and
lysine. The water-insoluble fraction also contains 3.3% carbohydrate.
