Table Of ContentSpringer Series in
Experimental Entomology
Thomas A. Miller. Editor
Neurohormonal
Techniques in Insects
Edited by
Thomas A. Miller
With a Foreword by Gottfried S. Fraenkel
With Contributions by
R. J. Aston . T. Goto . L. Hughes· H. Ishizaki
M. Isobe . K. J. Kramer' S. H. P. Maddrell
W. Mordue . S. E. Reynolds· I. M. Seligman
A. N. Starratt . R. W. Steele· J. V. Stone· A. Suzuki
J. W. Truman' J. zditrek
Springer-Verlag [$]
New York Heidelberg Berlin
Thomas A. Miller
Department of Entomology
University of California
Riverside, California 92521
With 90 Figures
Library of Congress Cataloging in Publication Data
Main entry under title:
Neurohormonal techniques in insects
(Springer series in experimental entomology)
Bibliography: p.
Includes index.
I. Insect hormones. 2. Neurosecretion. I. Miller,
Thomas A. II. Series.
QL495.N48 595.7'01'88 79-27343
All rights reserved.
No part of this book may be translated or reproduced in any form without written
permission from Springer-Verlag.
The use of general descriptive names, trade names, trademarks, etc. in this
publication, even if the former are not especially identified, is not to be taken as a
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Act, may accordingly be used freely by anyone.
© 1980 by Springer-Verlag New York Inc.
Softcover reprint of the hardcover 18t edition 1980
987654321
ISBN-13: 978-1-4612-6041-7 e-ISBN-13: 978-1-4612-6039-4
001: 10.1007/978-1-4612-6039-4
Series Preface
Insects as a group occupy a middle ground in the biosphere between bac-
teria and viruses at one extreme, amphibians and mammals at the other.
The size and general nature of insects present special problems to the
student of entomology. For example, many commercially available in-
struments are geared to measure in grams, while the forces commonly en-
countered in studying insects are in the milligram range. Therefore, tech-
niques developed in the study of insects or in those fields concerned with
the control of insect pests are often unique.
Methods for measuring things are common to all sciences. Advances
sometimes depend more on how something was done than on what was
measured; indeed a given field often progresses from one technique to
another as new methods are discovered, developed, and modified. Just as
often, some of these techniques find their way into the classroom when
the problems involved have been sufficiently ironed out to permit students
to master the manipulations in a few laboratory periods.
Many specialized techniques are confined to one specific research labo-
ratory. Although methods may be considered commonplace where they
are used, in another context even the simplest procedures may save con-
siderable time. It is the purpose of this series (1) to report new develop-
ments in methodology, (2) to reveal sources of groups who have dealt
with and solved particular entomological problems, and (3) to describe ex-
periments which might be applicable for use in biology laboratory
courses.
THOMAS A. MILLER, Series Editor
Call to Authors
Springer Series in Experimental Entomology will be published in future
volumes as contributed chapters. Subjects will be gathered in specific
areas to keep volumes cohesive.
Correspondence concerning contributions to the series should be com-
municated to:
Thomas A. MiIIer, Editor
Springer Series in Experimental Entomology
Department of Entomology
University of California
Riverside, California 92521
USA
Foreword and Overview
It should be emphasized from the outset what this book is meant and what
it is not meant to be. It brings together the very considerable and diffuse
information about neurohormones in insects largely from the point of
view of the hard facts-evidence for their existence, their chemical na-
ture, and the techniques used in obtaining this information. In this re-
spect, it is invaluable to everyone entering this field and despairing how to
pick the right insect and method out of a seemingly infinite variety of
choices. The book does not give an integrated picture ofthe interaction of
these hormones, and omits to tell the often strange and exciting stories of
the devious ways by which these hormones were discovered.
What gives this volume a certain distinction and authority, different
from similar ventures, is the fact that most chapters were written by the
very person or group that made the original discoveries, worked out the
original methods, and are still active in the field.
Classification of Insect Neurohormones
This book deals with an almost bewildering variety of neurohormonal
manifestations, which makes the reader wonder about how to view them
in an orderly scheme. A classification has recently been devised by Seh-
naP and is given here in somewhat abbreviated form (translated from the
German):
1 Sehnal F. (1979). Neuroendokrine. Regulation der Entwicklung der Lepidop-
teren. In, Probleme der Korrelation neuraler und endokriner Regulation bei Ever-
tebraten. Ed. H. Penzlin. Wissenschaftliche Beitrage der Friedrich-Schiller-
Universitat, Jena, 154-175.
X Foreword and Overview
a) Glandotropic neurohormones guide the activity of endocrine
glands, viz. prothoracicotropic hormone (Chap. II), allatotropic
hormone.
b) Morphogenetic neurohormones guide the speed and direction of
ontogenesis, i.e., shape, structure, color, viz. bursicon (Chap. 5),
pupariation hormones (Chap. 7), diapause hormone (Chap. II).
c) Myotropic neurohormones affect the kinetics of the heart, intes-
tine, the Malpighian tubules, the oviducts, ovaries and other inter-
nal organs, viz. proctolin (Chap. I).
d) Metabolic neurohormones influence metabolism, viz. adipokinet-
ic hormone (Chap. 2), insulin-like hormones (Chap. 5), diuretic
hormone (Chaps. 3 and 4).
e) C hromotropic hormones affect rapid color change by migration of
pigment (rather rare in insects, not dealt with in this book).
f) Ethotopic neurohormones act on the nervous system, viz.
eclosion hormone (Chap. 9), the pupariation factors (Chap. 7).
Historical Background
Unlike vertebrate endocrinology, which has developed largely through
the study of the control of individual growth and metabolic processes, in-
sect endocrinology developed almost exclusively from the study of
complicated morphogenetic events, such as molting and metamorphosis.
The latter turned out to be controlled largely by the two master glands,
the corpus allatum and the prothoracic gland, with an overall control by
neurohormones. In this sense, vertebrate endocrinology was from the
beginning biochemically oriented, while insect endocrinology largely
stemmed from a study of morphology and developmental physiology.
This preoccupation with the hormonal control of developmental events
so dominated insect endocrinology, including neuroendocrinology, that
the study of the control of the more metabolic functions in insects has
lagged behind by several decades. I t was really only in the past ten years
that metabolic hormones in insects, which all turned out to be neurohor-
mones, were seriously studied, and the real success stories from the point
of view of the endocrinologist, the isolation, identification, and synthesis
of such neurohormones, have broken within the past five years.
We have learned very recently that insects also possess insulin- or
glucagon-like hormones (Chap. 5). However, in retrospect, the existence
of specific metabolic neurohormones should have been expected in inver-
tebrates with no less certainty than is now known for vertebrates. This
only shows that the dogma, still ripe when I did my first endocrinological
studies with insects, that hormones were something special for ver-
Foreword and Overview XI
tebrates and developed very late in animal evolution, took a long time to
die.
The general concept of neurosecretion and neurohormones hardly goes
back 40 years and was crystallized largely in the work of the Scharrers.2
But this was foreshadowed in the early 1920's by Kopec's discovery of
the brain function in the development of Lepidoptera, which took almost
30 years to be recognized as the driving force in insect development. Al-
though the role ofthe "brain" hormone, as it was first called, was well es-
tablished in the early 1950's and investigations and speculations on the
nature of what is now most often called the prothoracicotropic hormone
(Chap. 11) followed each other in an unending stream, we are now, 30
years later, still very largely in the dark about the identity ofthis hormone,
as the last chapter in this book surprisingly reveals.
The ways in which scientific concepts develop are often strange and
devious, and nothing illustrates this better than the topic of the hormonal
control oftanning in insects, a subject I have been connected with, on and
off, for over 45 years and which came to play also a dominant role in the
development of our concepts in insect neuroendocrinology. The hormone
now known as ecdysone, was originally discovered as the factor that
brings about tanning of the fly puparium. Although the wider implication
of ecdysone in molting and metamorphosis was soon recognized. it took
over 25 years to recognize that in pupariation, ecdysone controlled not
only tanning but also other morphogenetic events that bring about
pupariation, though only indirectly as it turned out later. The all impor-
tant role of ecdysone as the tanning hormone was generally assumed for
30 years, when another hormone, bursicon, a product of neurosecretion
(Chap. 6), was recognized as the tanning hormone for the adult fly. It then
turned out that the role of ecdysone in tanning of the pupariation was an
exception, a freak, as it were, among insects, and that possibly all conven-
tional tanning after a molt is generally controlled by bursicon. Surpris-
ingly now, even the concept of ecdysone as the tanning hormone in
pupariation no longer seems to be true, as follows from the discovery of
the pupariation factors (Chap. 7), neurohormones which are set in motion
by ecdysone, one of which (PTF) seems specifically to have the function
of controlling tanning.
Bursicon, which originally was found just to effect tanning is now seen
also to control many other events during the consolidation of the cuticle
after a molt, plasticization during general, and specifically wing expan-
sion, deposition of the endocuticle, cell death between the lamina of the
wings, and possible formation of the apodemes. Fortunately, this does
2 Scharrer, E., Scharrer B. (1963). Neuroendocrinology. New York. Columbia
University Press. 289 pp.
XII Foreword and Overview
not invalidate the propriety of the term, which was originally derived from
the Greek bursicos-pertaining to tanning-because this is derived from
the word bursa-skin. So bursicon now stands appropriately as a term
for a hormone that gives the insect cuticle its peculiar properties after a
molt.
The history of insect endocrinology, and particularly neuroen-
docrinology, is replete with surprising discoveries that uncovered the ex-
istence of unique processes or adaptations. These discoveries could only
have been made originally by observers familiar with good, "old-
fashioned" natural history. It is as if "nature" had contrived to reveal its
secrets to the observer in certain rare and striking phenomena. Let us
consider a few notable examples.
The adipokinetic hormone (Chap. 2). Locusts use fat as energy for
flight, in contrast to many other insects which use carbohydrates. The fat
is stored in the fatbody and released into the hemolymph within a few
minutes of beginning of flight.
Bursicon and plasticization hormone in flies (Chaps. 6 and 8). The
adult fly emerges from the puparium in the soil and has to dig its way out
before it expands body and wings and tans the body. These processes, to
be effective, must be delayed (inhibited) until the fly is free from the soil.
Then they are initiated by bursicon, which plasticizes the cuticle to make
it inflatable, then tans the body, and subsequently controls a number of
other processes. Similar processes are operating in other insects, but it
was the particular ease with which they can be demonstrated and tested in
flies which at first led to these discoveries.
Plasticization and diuretic hormones in Rhodnius (Chap. 8). At the
very beginning of insect endocrinology stands the discovery that Rhod-
nius, a then obscure South American large blood sucking bug, takes only
one blood meal in each instar. This blood meal can be 12 times the vol-
ume of the body, and this is only made possible by the secretion of the
plasticization hormone which makes the cuticle expandable. Sub-
sequently, the diuretic hormone is released which controls the rapid
excretion of the excess water in the blood. Similar events probably occur
in other blood-sucking insects.
The Pupariation factors (Chap. 7). Puparium formation in flies
(pupariation) is a unique morphogenetic event among insects and has
proved of enormous heuristic value in insect endocrinology. In this pro-
cess, a soft, colorless larva contracts into a rigid dark puparium under the
influence of what is now recognized as a series of hormonal events. One
of the beauties of these reactions is that they take place within one hour.
It started with the discovery of the hormone now known as ecdysone.
Thirty-five years later the pupariation factors (Chap. 7) were discovered,
neurohormones set in motion by ecdysone that control a variety of
Foreword and Overview XIII
manifestations during pupariation, anterior retraction (ART), immobiliza-
tion (PIF), possibly a stimulation factor (PSF), and ultimately tanning
(PFT). It is still not known whether neurohormones like the pupariation
factors are unique in this process, or are elicited by ecdysone in also other
contexts.
The eclosion hormone (Chap. 9). Recognition that eclosion from a
pupa is controlled by a specific hormone is of very recent date, and still
confined to a few species of moths. This hormone triggers typical
eclosion behavior even in an isolated abdomen!
Diapause hormone in Bombyx mori (Chap. 10). Diapause (arrest of
development) occurs in many insects in a great variety of manifestations,
and is often caused by a lack of ecdysone. But the recognition of a specif-
ic diapause hormone in the common silkworm is so far unique. This was
the outcome of an enormous and prolonged effort to breed different races
of silkworms in Japan.
Making good use of the specific reactions that led to the discovery of
the various insect neurohormones, the following, mostly rapid and specif-
ic tests were developed:
Proctolin: Motility of the isolated cockroach hindgut (proctodeum).
Adipokinetic hormone: Mobilization of lipids from the locust fatbody,
in vivo and vitro.
Diuretic hormone: Elimination of fluid from isolated Malpighian
tubules of Rhodnius.
Bursicon: Neck ligation in a fly immediately after emergence, tested for
tanning. Other tests proved less specific and convenient.
Pupariation factors: Acceleration of pupariation and tanning in Sar-
cophaga larvae selected several hours before pupariation (early red-
spiracle larvae).
Cuticle pLasticizing factors: Stretchability of cuticle in neckligated flies
immediately after emergence (as in bursicon test), or stretchability of
Rhodnius cuticle immediately after a blood meal.
Eclosion hormone: Precocious eclosion of the pharate adults of
Antheraea pernyi; or induction of eclosion behavior in ligated abdomens
of HyaLophora cecropia several hours before natural eclosion.
Diapause hormone: Injection of brain-suboesophageal ganglion ex-
tracts into pharate adults of non-diapausing strains of Bombyx mori. An
important feature of this test is the fact that diapausing eggs are colored.
Prothoracicotropic hormone: The brains of the Satumiid Samia
cynthia ricini were removed early in the pupa. The test consisted of in-
ducing adult development, and proved superior to, and more reliable than,
previous attempts with Bombyx mori and H. cecropia pupal assays, or a
larval assay with Manduca sexta.
