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Page 1

Springer Series in
Experimental Entomology

Thomas A. Miller. Editor

Page 2

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

Page 151

Insulin-like and Glucagon-like Hormones in Insects 131

C. Development of RIA Systems

Although it is recommended that commercial RIA kits be used for the de-
tection of-insulin-like and glucagon-like peptides, researchers may wish to
use their own system to elicit antibodies. The reader is referred to Berson
and Yalow (1973), Yalow (1976), and Kirkham and Hunter (1971), where
adequate description of all methodology (labeling peptide, producing an-
tibodies, preparing standards, separating complex from peptide, and
validating systems) for total in-lab development of a system is provided.

VI. Immunocytochemistry

Cells that contain insulin-like and glucagon-like peptides can be identified
by the technique of indirect immunofluorescence (Coons et al. 1955;
Sternberger 1977; Orci et al. 1976; Pearse 1969; Baetens et al. 1976;
Sasaki et al. 1975; Sundler et al. 1976; Helmstaedter et al. 1976;
Plisetskaya et al. 1978; and Fritsch et al. 1976). First, a tissue is fixed and
embedded under conditions that maintain the integrity of the cell, particu-
larly the neurosecretory granules (Humason 1972). Second, tissue is sec-
tioned and mixed with a mammalian antibody (I) that complexes with the
insect peptide of interest. Then a second antibody (II) labeled with a
fluorescent compound such as fluorescein and specific for the mammalian
antibody used initially is incubated with the tissue. After the necessary
washings, those cells that bind antibody I and subsequently antibody II
can be localized by using a fluorescence microscope.

Although at the present time a phylogenetic study of the occurrence of
peptide hormone-producing cells has not been extended to insects, I
believe such an advance should be forthcoming soon. 2 Insulin-containing
cells have already been found in gastro-entero-pancreatic cells from a
number of other invertebrates (Plisitskaya et al. 1978; Fritsch et al.
1976). Since reactions with antisera are not absolutely specific and cross-
reactions with other hormones or polypeptides cannot be excluded, im-
munocytochemical evidence should be complemented with chemical and
biological proof that a cell is secretory or endocrine in nature.

VII. Concluding Remarks

The measurement of the potency and concentration of insect peptides in
body fluids and tissues requires more than strictly chemical methods,

"Insulin-like material has been localized in the median neurosecretory cells of C. vumitoria
by the method of immunocytochemical staining. Duve and Thorpe 1979 Cell Tissue Res.
200: 187-91.

Page 152

132 K. J. Kramer

primarily because of the small amounts of material available. Bioassay,
immunoassay, and cytochemical procedures that localize both the pep-
tides and the cells that synthesize them have complementary roles in such
studies. In the past, and certainly in the future, such methodology has and
will broaden our understanding of the nature, physiological role, and
paths of synthesis and metabolism of insect peptides such as those dis-
cussed in this contribution and also be extended to new peptides.


I would like to express my appreciation to Dr. Howard Tager, the University of
Chicago, and Ms. Cynthia Childs, Mr. Roy Speirs, and Mr. Leon Hendricks, U.S.
Grain Marketing Research Laboratory, Manhattan, Kansas, for collaboration in
many aspects of research.


Akanuma Y, Kuzuya T, Hayashi M, Ide T, Kuzuya N (1970) Immunological
reactivity of insulin to sepharose coupled with insulin-antibody. Its use for
the extraction of insulin from serum. Biochem Biophys Res Commun

Assan R (1973) Glucagon antisera. In: Berson SA, Yalow RS (eds) Methods in
investigative and diagnostic endocrinology. North-Holland, Amsterdam

Atkinson DE (1977) Cellular energy metabolism and its regulation. Academic,
New York

Baetens 0, Rufener C, Sri kant BC, Dobbs R, Unger R, Orci L (1976) Identifica-
tion of glucagon-producing cells (A-cells) in dog gastric mucosa. J Cell BioI

Bell RA, Joachim FG (1976) Techniques for rearing laboratory colonies of
tobacco hornworms and pink bollworms. Ann Entomol Soc Am 69:365-73

Berson SA, Yalow RS (1961) Immunochemical distinction between insulin with
identical amino acid sequences Nature 191: 1392-93

Berson SA, Yalow RS (1966) Insulin in blood and insulin antibodies. Am J Med

Berson SA, Yalow RS (1973) Peptide hormone radioimmunoassay. In: Berson
SA, Yalow RS, (eds) Methods in investigative and diagnostic endocrinology.
North-Holland, Amsterdam

Bhakthan NMG, Gilbert LI (1968) Effects of some vertebrate hormones on lipid
solubilization in insect fat body. Gen Comp Endocrinol 11: 186-97

Bowers WS, Friedman S, (1963) Mobilization offat body glycogen by an extract
of corpus cardacium. Nature 198:685

Brown BE (1965) Pharmacologically active constituents of the cockroach corpus
cardiacum: resolution and some characteristics. Gen Comp Endocrinol

Carroll NY, Longley RW, RoeJH (1956) The determination of glycogen in liver
and muscle by use of anthrone reagent. J BioI Chern 220:583-93

Page 301

Pepsin 265 - 6
Peptide digestion 12
Peptide linkage 237
Periodate oxidation 267
Periplaneta americana (American

cockroach) I, 2, 41,92, 104, III,
117, 138, 144, 148, 165,249

Phenoloxidase 126, 140
Phenylmethylsulfonyl fluoride 118
Phenylthiourea 126, 164
Phormia regina 156
Physiological solution (See saline

Picrotoxin 162
Pieris brassicae 92, 148, 246
PIF (puparium immobilization factor)

156, 167, 169, 170-1
Plasticizing activity 189, 192
Plasticizing factor 81, 199

chemistry of 190 - I
pharmacology of 191
receptor for 193

Plodia interpunctella (Indian meal
moth) 117, 120-1

Polypeptide sequencing III
Polyvoltine 216-17, 221, 223

bioassay of proctolin on 3
Proctolin 1, 10, 12

extraction of 13
hydrolysis of 22
isolation of 14
synthesis of 27
u v spectrum of 22

Programmed cell death 141
Prohormone 101
Pronase 265 - 6
Prothoracic glands 216, 244, 254 - 5
PSF (puparium stimulating factor) 156,

PTF (puparium tanning factor) 155,

165, 169-170, 175
pronase digestion of 170

PITH (prothoracicotropic hormone)

(Also called brain hormone) 220
partial purification of 258
preparation of crude 257 - 8

Index 281

preparation of highly purified 260 - I
synonymy of 244

Pupal assay (Bombyx mori) 252
(Hyalophora cecropia) 253

Pupal ovaries 218 - 19
Pupariation 154, 158
Pupariation factors (See also ARF,

PDF, PIF, PSF, PTF) 155-6
purification of 171 - 2

Pupariation formation
sequence of 158
period of contraction of 160

Puromycin 141
Pyrrhocoris apterus 198

Radioimmunoassay (RIA) 127,

Rearing blowflies 156-7
Rectal longitudinal muscle 4
Rectum equivalents 13 - 14, 15
Red pigment-concentrating hormone

(RPCH) 33, 41, 267
Red-spiracle larvae 175, 158
Renin 128
Retrocerebral tissue 118
Rhodnius prolixus 81-3, 85, 88-9,

108, III, 139, 180-8, 191,244,

RIA (See radioimmunoassay)
Ring gland 154
Ringer, Ephrussi and Beadle 203
Ringer's solution (See saline solution)
Royal jelly 125
RPCH (See red pigment-concentrating

Ryanodine 162

Saline solution 6, 8, 34, 82, 94-5,
96-97,103,108,160-1, 184-5,

Samia cynthia ricini 26, 250-2,

S amia unit 251, 261, 264, 271
Sarcophaga argyrostoma 156
Sarcophaga bullata 138, 141, 146,

170-1, 173-4

Page 302

282 Index

Sarcophaga crassipalpis 156
Sarcophaga scoparia 156
Satumiid silk moth (See Antheraea

pernyi and Hyalophora cecropia)
Schistocerca gregaria 31,38,46,49,

54,56-7,92, 138-9, 146
Schistocerca nitens (formerly S. vaga)

Sclerotization 139
Serum 122
SG (See sUboesophageal ganglion)
SG-brain complex

(suboesophageal-brain complex)

Silkworm (See Bornbyx rnori)
Sodium dodecyl sulfate 123
Somatostatin 116, 128
Somatotropin 116
Soybean trypsin inhibitor 118
Spectrofluorometric method 149
Spiracle apolysis 255
Storage lobe (of corpora cardiaca) 92
Subcellular fractions, electron

microscopy of 56
Suboesophageal ganglion (SG)

Swallowed air 142
Synchronous emergence 145

Tanning 160, 170, 179
Tanning assay 145
Tanning, inhibition of 162
Tanning score 146
Target organ (pupal ovaries) 218

TCA (See trichloracetic acid)
Tenebrio rnolitor 138, 143,250
T enebrio test 147
Tetrodotoxin 162
Theophylline 143, 170
Thermolysin 266
Transducer, isotonic 8, 10
Transducer, linear variable

displacement 186
Trasylol 118
Trehalase 218
Trehalose 126
Trehalosemic assay 126
Trichloracetic acid (TCA) 149, 170-1
Trypsin 109- 10, 238-9, 265-6,

Tungsten needle 93

electrolytically sharpened 88
Turbo comutus 267
Tyrosinase 140, 144
Tyrosine 139
Tyrosine depletion assay 148
Tyrosine uptake 148

Univoltine 216-17
Urea 123

Vermiform larvae 146-47
Vermiform S chistocerca larva assay


White puparium 160, 162
Wing assay 201, 205, 212
Wing hypodermal cells assay 149

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