Advances in Insect Physiology
Volume 13
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Advances in Insect Physiology
Volume 13
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Advances in Insect Physiology edited by
J E TREHERNE M. J. BERRIDGE and V. 6. WIGGLESWORTH Department ofZoology, The University Cambridge, England
Volume 13
1978
ACADEMIC PRESS LONDON NEW YORK SAN FRANCISCO A Subsidiarv of Harcourr Brace Jovanovich. Publishers
ACADEMIC PRESS INC. (LONDON) LTD 24/28 Oval Road London NW 1
United States Edition published by ACADEMIC PRESS INC. 11 1 Fifth Avenue New York. New York 10003
Copyright @ 1978 by ACADEMIC PRESS INC. (LONDON) LTD
AN Rights Reserved
No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers
Library of Congress Catalog Number: 63-14039 ISBN: 0-12-024213-3
PRINTED IN GREAT BRITAIN AT THE SPOTTISWOODE BALLANTYNE PRESS BY WILLIAM CLOWES AND SONS LIMITED LONDON, COLCHESTER AND BECCLES
Contributors Robert P. Bodnaryk
Canada Agriculture, Research Station, 195 Dafoe Road, Winnipeg, Manitoba R3T2M9, Canada Norbert Elsner
Zoologisches Institut der Universitatzu Koln, 5 Koln-Lindenthal, WeyertalII9,K67n, Germany Bernd Heinrich
Division of Entomology, University of California, Berkeley, California 94720, USA Ann E. Karnrner
Division of Biology, Kansas State University,Manhattan, Kansas 66506, USA Dennis R. Nelson
Metabolism and Radiation Research Laboratory, Agricultural Research Service, US.Department of Agriculture, Fargo, North Dakota 58102, USA Andrej V. Popov
Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad, USSR Richard H. White
Biology Department, University of Massachusetts at Boston, Boston, Massachusetts 02125, USA
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Contents
Contributors
V
Long-Chain Methyl-Branched Hydrocarbons: Occurrence, Biosynthesis, and Function DENNIS R. NELSON
1
Insect Visual Pigments RICHARD H. WHITE
35
Structure and Function of Insect Peptides ROBERT P. BODNARYK
69
Insect Flight Metabolism ANN E. KAMMER AND BERND HEINRICH
133
Neuroethology of Acoustic Communication NORBERT ELSNER AND ANDREJ V. POPOV
229
Subject Index
351
Cumulative List of Authors
373
Cumulative List of Chapter Titles
375
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Long-Chain Methyl-Branched Hydrocarbons: Occurrence, Biosynthesis, and Function Dennis R. Nelson Metabolism and Radiation Research Laboratory, Agricultural Research Service, US.Department of Agriculture. Fargo, North Dakota, USA
1 Introduction 1 2 Occurrence 2 2.1 n-Alkanes and n-alkenes 2 2.2 Cycloalkanes 3 2.3 2- and 3-methylalkanes 3 2.4 Internally branched methylalkanes:Analysis 4 2.5 Monomethylalkanes 6 2.6 Dimethylalkanes 13 2.7 Trimethylalkanes 16 3 Biosynthesis 17 4 Functions 21 References 25
1
Introduction
Surface waxes or lipids of all organisms are responsible for the water-repellent character of their surfaces. For example, the skins of higher animals are kept soft, smooth, and free of cracks by lipids [largely squalene, mono-, di-, and triacyl glycerols, wax esters, and fatty acids in man (Nicolaides, 1974)l. By keeping the skin pliable and continuous, microorganisms are unable to penetrate and cause infections, and the skin surface is prevented from drying out and becoming rough and scaly. Also, the surface lipids of plants (see reviews by Caldicott and Eglinton, 1973 and Kolattukudy, 1975) and insects are important because (1) they allow the uptake of water but prevent excessive water loss when available moisture is low (Beament, 1964, 1967; Browning, 1967); (2) they prevent the penetration of inorganic chemicals (Beament, 1964); (3) they act as a bamer against microorganisms (David, 1967); (4) they affect the absorption of agricultural 1
2
DENNIS R NELSON
chemicals (Ebeling, 1964) [in plants, their formation is inhibited by some herbicides (Still et al., 1970; Kolattukudy and Brown, 1974)l; (5) they may serve as a sex attractant (Evans and Green, 1973); and (6) they may serve as a kairomone for insect parasites and predators (Lewis et al., 1975a,b, 1976). In the present review, I have restricted myself to a consideration of the hydrocarbon components of the surface lipids, particularly to the long-chain internally branched methylalkanes and methylalkenes. These compounds have been extensively investigated since 1970 when di- and trimethylalkanes were identified in an insect (Nelson and Sukkestad, 1970) and the technique of identifying mixtures of the methylalkanes from their mass spectra was elucidated (McCarthy et al., 1968; Nelson and Sukkestad, 1970). The majority of studies of the occurrence and function of the long-chain hydrocarbons has been done with insects. The studies of biosynthesis of alkanes and the origin of the methyl groups have been done largely with plants and microorganisms though some of the more recent investigations have involved insects and other arthropods.
2
2.1
Occurrence n-ALKANES AND n-ALKENES
Although in some insects, the surface lipids are mainly long-chain alcohols (Bowers and Thompson, 1965; Bursell and Clements, 1967), ketoesters (Meinwald et al., 1975), and wax esters (Gilby, 1957a,b; Faurot-Bouchet and Michel, 1964, 1965; Brown, 1975), alkanes are a common component of both insect and plant surface lipids and are ubiquitous hydrocarbons in nature. The hydrocarbons of insects usually occur as mixtures, however only n-alkanes were reported from the hydrocarbon fraction of the lipids from larval cast skins of the beetle, Tenebrio molitor L. (Bursell and Clements, 1967). In addition to the alkanes, alkenes have been reported in the wax of bees, Apis mellifera L. (Streibl et al., 1966), the little house fly, Fannia canicularis (L.) (Uebel et al., 1975a), the house fly, Musca domestica L. (Louloudes et al., 1962; Carlson et al., 1971), the house cricket, Acheta domesticus L. (Hutchins and Martin, 1968), the boll weevil, Anthonomus grandis Boh. (Hedin et al., 1974), the stonefly, Pteronarcys californica Newport (Armold et al., 1969), the cockroaches Periplaneta australasiae (F.) and P. brunnea Burmeister, and P. fuliginosa (Serville) (Jackson, 1970), P. japonica Karny and P. americana L. (Jackson, 1972), the Argentine ant, Zridomyrmex humilis (Mayr) (Cavill and Houghton, 1973), the bull ant, Myrmecia gulosa (F.) (Cavill and Williams, 1967), the fleshfly, Sarcophaga bullata Parker (Jackson et al., 1974), the stable
LONG-CHAIN METHYL-BRANCHED HYDROCARBONS
3
fly, Stomoxys calcitrans (L.) (Uebel et al., 1975b), the face fly, Musca autumnalis De Geer (Uebel et al., 1975~).The pecan weevil, Curculio caryae (Horn), has alkenes and akadienes from 20 to 28 carbons in length (Mody et al., 1975) and volatiles of the confused flour beetle, Tribolium confusum Jacquelin duVal, contained both 1-alkenes and heptadecadiene (Keville and Kannowski, 1975). Tridecene constitutes 90 per cent of the defensive secretion from the prothoracic glands of the lacewing, Chrysopa oculata Say (Blum et al., 1973). The major hydrocarbon component of the surface lipids of the American cockroach, Periplaneta americana L., is cis,cis-6,9-heptacosadiene(Baker er al., 1963; Beatty and Gilby, 1969), whereas all other cockroaches studied have methylalkanes as the major components. This diene is changed by ultraviolet light and oxygen into conjugated unsaturated and oxygenated compounds (Beatty and Gilby, 1969), and antioxidants [polyhydric phenols such as 3,4dihydroxybenzoic acid (protocatechuic acid), which are also involved in tanning] present on the cuticle prevent degradation and the subsequent polymerization (Atkinson and Gilby, 1970; Atkinson et al., 1973). Also, ultraviolet light increases the hydrocarbon content of the cuticular wax (Gingrich, 1975). Alkenes, alkadienes, and alkatrienes and their methyl-branched isomers make up about 90 per cent of the hydrocarbon fraction of the millipede, Graphidostreptus tumuliporus (Karsch) (Oudejans, 1973). n-Alkenes, 2,(m 1)-, 2,(m2)-, and 3,(~-2)-dimethylalkenes,and 2- and 3-methylalkenes have been identified in bacteria (Albro and Dittmer, 1969a; Tornabene and Markey, 1971), and polyolefins have been reported from algae (Youngblood and Blumer, 1973) and mosses (Karunen, 1974). 2.2
CYC L O AL K ANES
Cycloalkanes were reported in M. domestica (Louloudes et al., 1962), wool wax (Mold et al., 1964) and tobacco (Enzell et al., 1969); 1-cyclohexylakanes were found in Nonesuch seep oil (Johns et al., 1966), and 1-cyclopentyl-and 1cyclohexylalkanes, 7- and 9-~yclohexylalkanes,dicyclohexylalkanes and diphenylalkanes were found in paraffin wax (Levy et al., 1961). In G. tumuliporus, cyclopropane alk-1-enes were found only in the female (Oudejans, 1973).
2.3
2-
AND
3-METHYLALKANES
Both 2- and 3-methylalkanes (is0 and anteisoalkanes) have been found in meteorites (Or6 et al., 1968) [no alkanes were found in lunar samples
4
DENNIS R . NELSON
(Meinschein et al., 1970) or in parts of graphitetroilite nodules of iron meteorites not exposed to the earth’s atmosphere (Or6 et al., 1968)1, in petroleum (Hills and Whitehead, 1966), in numerous plants (Eglinton and Hamilton, 1967; Weete, 1972; Kolattukudy and Walton, 1973; Kolattukudy, 1975; and references cited therein), in the land snail, Cepaea nemoralis (L.) (Van der Horst and Oudejans, 1972), and in the millipede, G. tumuliporus (Oudejans, 1972). 2-Methylalkanes were reported from the common house cricket, A. domesticus (Hutchins and Martin, 1968; Blomquist et al., 1976), the female tiger moth, Holomelina opella nigricans (Reakirt) (Roelofs and Carde, 1971), the silkworm, Bombyx mori L. (Shikata et al., 1974), and the crickets, Allonemobius fasciatus (De Geer) and Gryllus pennsylvanicus Burmeister (Blomquist el al., 1976). 3-Methylalkanes were reported from the surface lipids of the big stonefly, P. californica (Armold et al., 1969), the cockroaches, P . australasiae, P. brunnea, and P . fuliginosa (Jackson, 1970), P . japonica and P. americana (Jackson, 1972), L. maderae and B. orientalis (Tartivita and Jackson, 1970), the Mormon cricket, Anabrus simplex Haldeman (Jackson and Blomquist, 1976), the fleshfly, S. bullata (Jackson et al., 1974), and the fire ants, Solenopsis invicta Buren and S. richteri Fore1 (Lok et al., 1975). 3-Ethylhexacosane was reported from the silkworm (Murata et al., 1974), however, their published mass spectrum is more compatible with that of 3-methylheptacosane when compared with spectra of 3-methyl- and 3-ethylalkanes published by the American Petroleum Institute. It should be noted that in plants (Wollrab et al., 1967), Mollusca and Arthropoda, the majority of the 2-methylalkanes has an odd number of carbon atoms, and the majority of the 3-methylalkanes has an even number of carbon atoms, which would be expected if the methyl branch is derived from the amino acids valine and isoleucine, respectively. Also, 2,(w- 1)-dimethylalkanes were reported in the waxes of the horehound, Marrubium vulgare L. (Brieskorn and Feilner, 1968), and dimethylalkenes were reported in bacteria, as noted above (Albro and Dittmer, 1969a; Tornabene and Markey, 1971).
2.4
INTERNALLY B R A N C H E D M E T H Y L A L K A N E S : A N A L Y S I S
Recent reports of the occurrence and structural identification of internally branched mono-, di-, and trimethylalkanes have depended upon the use of molecular sieves (1/16 in. pellets of Linde type 5A) to separate the branched alkanes from the n-alkanes (O’Connor et al., 1962) and the increased use of improved gas-liquid chromatographic and mass spectrometric methods of
LONG-CHAIN M ETHYL-BRANCHED HYDROCARBONS
5
analysis. Monomethylalkanes with the methyl branch located on about carbon 7 to over 18 elute from gas-liquid chromatographic columns such as SE-30, OV-17, and OV-101 with an equivalent chain length (Miwa, 1963) 0.6 to 0.7 carbon atoms less than the n-alkane with the same number of carbon atoms (Mold et al., 1966; Nelson and Sukkestad, 1970, 1975). Additional internal methyl branches have an additive effect. Thus, two internal methyl branches with isoprenoid spacing decrease the equivalent chain length about 1.4 carbon atoms less than the total number of carbon atoms in the molecule, and three methyl branches cause the equivalent chain length to be about 2.2 carbon atoms less (Nelson and Sukkestad, 1970, 1975). If the branch point is closer to the end of the chain, the effect of the branch on the equivalent chain length is less (Mold et al., 1966). However, on polar columns such as cyclohexanedimethanol succinate, iso- and anteisomethyl branches decrease the equivalent chain length 0.65 and 0.75 carbon atoms, respectively, and a centrally located double bond and a terminal double bond decrease it by 0.2 and 0.5, respectively (Albro and Dittmer, 1969a). The equivalent chain length in conjunction with the carbon number determined by mass spectrometry, gives the number of methyl branches, and the position of the methyl branches is then deduced from the mass spectral fragmentation patterns by comparing the relative intensities of significant adjacent even and odd mass peaks. Methylalkanes give relatively simple mass spectra, and some mass spectra have been analyzed by plotting the carbon number of the fragment ion vs. the intensity of the fragment ion (Mold et al., 1966; Hutchins and Martin, 1968; Nishimoto, 1974). However, on the basis of such mass spectra alone, one cannot distinguish between an isomeric mixture of internally branched monomethylalkanes and internally branch di- and trimethylalkanes or isomeric mixtures of di- and trimethylalkanes (McCarthy et al., 1968; Nelson and Sukkestad, 1970, 1975). Biemann (1962) and Hood (1963) noted that internally branched alkanes tended to fragment at the branch point to give a secondary carbonium ion of [C,Hz,+ll+. Formation of the secondary carbonium ion was also accompanied to some degree by the loss of a hydrogen atom to give another secondary carbonium ion of [C,H,,l' (i.e., a doublet appeared in the mass spectrum that corresponded to the odd-mass secondary carbonium ion and to the even-mass secondary carbonium ion, one mass unit less). Of the two competing reactions (cleavage of the carbon-carbon bond on one side or the other of the branch point) for the formation of the two possible [C,,H2,,+,I+ secondary carbonium ions, the preferred cleavage is that which results in the loss of the larger of the alkyl chains (Pomonis et al., 1978). Also, the formation of the primary (straight-chain) carbonium ion is accompanied to some degree by the loss of a hydrogen atom. However, the significance of the loss of the hydrogen atom as
DENNIS R NELSON
6
an aid to the interpretation of mass spectra was not realized until McCarthy et al. (1968) deduced the effects of the size of the straight-chain tail of the secondary carbonium ion and of the presence of other- branch points in the secondary carbonium ion on the intensity of the [C,H,,lt ion (i.e., other branches on the secondary carbonium ion suppressed the formation of the [C,,H,,lt ion). These observations were used to distinguish between the mass spectra expected for 79-dimethylhexadecane and that of a mixture of 7- and 8methylheptadecane (McCarthy et al., 1968) and were later used by Nelson and Sukkestad (1970, 1975), in conjunction with gas chromatographic retention times expressed as equivalent chain lengths, to identify for the first time internally branched di- and trimethylalkanes in insects.
2.5
MONOMETHYLALKANES
Methyl branched alkanes have been identified mainly in arthropods but also in algae, higher plants, and gastropods, in which the methyl branch is located towards the center of the molecule. Alkenes with similar methyl branching have been found in S . calcitrans (Uebel et al., 1975b). Similar methylalkenes were found in the millipede, G. tumuliporus (Oudejans, 1973). The identified monomethylalkanes and their sources are summarized in Table 1. The GLC peak number given there is equal to the number of carbons in the backbone of the molecule, and the letter A designates one internal methyl branch. The shorter chain monomethylalkanes (less than 20 carbon atoms such as 5-methylpentadecane and 7- and 8-methylheptadecanes) are present in meteorites (Or6 et al., 1968), in a number of algae (Gelpi et al., 1970), and blue-green algae (Han et al., 1968; Fehler and Light, 1970). In Hymenoptera, they were identified in the secretions of Dufour’s gland in the ants, Formica nigricans Emery, F. rufa L., and F. polyctena Foerster (Bergstrom and Lofqvist, 1973), Camponotus intrepidus (Brophy et al., 1973), Pogonomyrmex rugosus var. fuscatus Emery and P. barbatus rugosus Emery (Regnier et al., 1973) and in whole body extracts of the Argentine ant, Iridomyrmex humilis (Mayr) (Cavil1 and Houghton, 1973). The 7- and 8-methylheptadecanes were also reported in the lichen, Siphula ceratites (Wg.) Fr. though they may have been from the algal symbiont (Gaskell et al., 1973). Although n-alkanes and 2- and 3-methylalkanes have been identilied in the waxes of a large number of higher plants, the only internally branched monomethylalkanes reported were in the leaf wax of the walnut tree, Juglans regia L. (Stranski et al., 1970), and of wheat (Nishimoto, 1974). A series of methylalkanes from 17 to 34 carbon atoms was present in the walnut leaf wax. In the GLC peaks identified, GLC peak 27-A was a mixture of 7-, 9-, 11-, and 13methylheptacosane, and GLC peak 29-A was a mixture of 11-, 13-, and 15-
7
LONG-CHAIN METHYL-BRANCHED HYDROCARBONS
TABLE 1 Occurence and structure of internally-branched monomethylalkanes GLC peak no.*
Sourcea
Methyl-branched components
Major isomer"
METEORITES
Carbonaceous chondrites P L A N T S (Algae) C. turgidus C. turgidus A. cyanea L . aestuarii NOSIOC sp. C .fritschii N . muscorum P. luridum A . nidulans A . variabilis
I
PLANTS
16-A
6- and 7-methylhexadecane
17-A
7- and 8-methylheptadecane
17-A
4-, 7- and 8-methylhepadecane
17-A
7- and 8methylheptadecane
similar
17-A
7-, and 8-methylheptadecane
similar
27-A 29-A
7-, 9-, 1 I-, and 13-methylheptacosane 1 I-, 13-, and 15-methylnonacosane
13-methyl 1 1 - & 13methyl
23-A 25-A 27-A 28-A 29-A 30-A 3 I-A 32-A 33-A
1 1 -methyltricosane 1 1methylpentacosane
1 I-, and 13-methylheptacosane lo-, and 12-methyloctacosane 1 I - , and 13-methylnonacosane 10, and 12-methyltriacontane 11-, 13-, and 15-methylhentriacontane lo-, and 12-methyldotriacontane 1 1, 13-, and 15-methyltritriacontane
1 I-methyl
35-A
1 I-, 13-, 15-, and 17-methylpentatriacontane
11- & 13-
(Trees)
J. regia PLANTS
5- and 6methylpentadecane 5methylhexadecane
(Lichen)
S. ceratites PLANTS
15-A 16-A
(Wheat)
T. aeskiv um
12-methyl 1 I-methyl
12-methyl 1 I-methyl
12-methyl 11- & 13-
methyl methyl I N S E C T A (Coleoptera)
A. grandis
P. japonica
C. caryae
20-A 22-A 23-A 24-A 25-A 26-A 27-A 28-A 29-A
10-methyleicosane 1 I-methyldocosane 1 I-methyltricosane lo-, 1 I - , and 12-methyltetracosane 1 I - , and 13-methylpentacosane 4-methylhexacosane 5-methylheptacosane 4-methyloctacosane 5-, 11- 13-, and 15-methylnonacosane
12-methyl 1 I-methyl
?
8
DENNIS R. NELSON
TABLE 1 (cont.)
GLC Sourcea
peak
Methyl-branched components
Major isomef
no.b INSECTA (Diptera)
r 25-A
S.bullata (31-A
S. calcitrans
)
5-, 7-, 9-, 1 1-, and 13-methylpentacosane 5-,7-, 9-, 11-, and 13-methylheptacosane 5-, 7-, 9-, 11-, 13-, and 15-methylnonacosane 5-, 7-, 9-, 11-, 13-, and 15-methylhentriacontane 11-, 13-, and 15-methylhentriacontane 13-, and 15-methyltritriacontane 13-, 1 5 , and 17-methylpentatriacontane 13-, and 15-methylheptatriacontane
INSECTA (Hymenoptera) F. nigricans
F.mfa F. polyctena C. intrepidus P. rugosus F. nigricans C . intrepidus P. rugosus P. barbatus F. nigricans F. rufa F. polyctena C. intrepidus P. barbatus P. rugosus P. rugosus P. barbatus F. nigricans C . intrepidus I . humilis I . humilis C. intrepidus I. humilis
1
1
1
}
S.richteri A . mellvera (Beeswax)
5-methylundecane
11-A 12-A 12-A
5-, and 6-methylundecane 4-methyldodecane 5-methyldodecane
12-A
6-methyldodecane
13-A
5-methyltridecane
13-A
5-, and 6-methyltridecane
14-A
6methyltetradecane
15-A
5-methy lpentadecane
16-A 16-A 17-A 23-A 25-A
4-methylhexadecane 5-methylhexadecane 5-methylheptadecane 9-, and 11-methyltricosane 11-, and 13-methylpentacosane 9-, and 11-methylpkntacosane 5-, 7-, 9-, 11-, and 13-methvlhe~tacosane - . 11-, and 13-methylheptacosane 1 1-, and 13-methylheptacosane 9-, 11-, 13-, and 15-methylheptacosane 11-, 13-, and 15-methylnonacosane 9-, 11-, 13-, and 15-methylnonacosane 9-, 11-, 13-, and 15-methylhentriacontane
{ { 25-A 27-A
S.richteri S.invicta M . gulosa A. mell~era M . gulosa
11-A
{
27-A 27-A 27-A 29-A 29-A 31-A
?
11-methyl 1I-methyl 11-methyl 13-methyl 13-methyl 13-methyl ? 15-methyl ? ?
LONG-CHAIN METHYL-BRANCHED HYDROCARBONS
9
TABLE 1 (cont.) I N S E C TA
(Lepidoptera)
H . zea
31-A
7-, 9-, 11-, 13-, and 15-methylhentriacontane 13-methyl ? 9-, 11-, 12-, 13-, and 15-methylhentriacontane lo-, 12-, 13-, 14-, 15-, and 16-methyldotriacontane ? 9-, 11-, 13-, 15-, and 17-methyltritriacontane ? 15-, and 17-methylpentatriacontane 17-methyl 13-, 15-, 17-, and 19methylheptatriacontane 15-methyl 13-, 15-, 17-, and 19-methylnonatriacontane 15-methyl
23-A
26-A
11methyltricosane 12-methyltetracosane 13-methylpentacosane 13-methylhexacosane
27-A
11-, and 13-methylheptacosane
similar
27-A 27-A 27-A 27-A 29-A 29-A 29-A 29-A 30-A 30-A 30-A 3 1-A 3 1-A 3 1-A 32-A 32-A 33-A 33-A 33-A 33-A 33-A
5-, 7-, 9-, 11-, 13-, and 15-methylheptacosane 5-, 7-, 9-, 11-, and 13-methylheptacosane 13-methylheptacosane 5-, 7-, 11-, and 13-methylheptacosane 5-, 7-, 9-, 11-, and 13-methylnonacosane 7-, 9-, 11-, 13-, and 15-methylnonacosane 13-methylnonacosane 5-, 7-, 9-, 1 1-, 13-, and 15-methylnonacosane 8-, 9-, lo-, and 11-methyltriacontane 8-, 9-, lo-, 11-, 12-, and 13-methyltriacontane 13-methyltriacontane 7-, 9-, 11-, 13-, and 15-methylhentriacontane 5-, 7-, 9-, 1 1-, 13-, and 15-methylhentriacontane 5-, 7-, 9-, 11-, 13-, and 15-methylhentriacontane 9-, lo-, 11-, and 12-methyldotriacontane 9-, lo-, 11-, 12-, and 13-methyldotriacontane 11-, and 13methyltritriacontane 11-, 13-, and 15-methyltritriacontane 7-, 9-, 11-, and 13-methyltritriacontane 13-, 15-, and 17methyltritriacontane 11-, 13-, and 15-methyltritriacontane
11-methyl 13-methyl
34-A 34-A 35-A 35-A 35-A 36-A 36-A 37-A 37-A
13-, 15-, and 17-methyltetratriacontane 13methyltetratriacontane 11-, 13-, and 15-methylpentatriacontane 13-, 15-, and 17-methylpentatriacontane 11-, 13-, 15-, and 17-methylpentatriacontane 13-, 15-, and 17-methylhexatriacontane 14-methylhexatriacontane 11-, 13-, 15-, and 17-methylheptatriacontane 13-, 15-, 17-, and 19methylheptatriacontane 12-, and 13-methyloctatriacontane 13-, 15-, 17-, and 19methylnonatriacontane 13-methylhentetracontane
(3i;
H. virescens
M . sexta I N S E C TA
(Orthoptera)
P. australasiae P. brunnea P.$uliginosa L. maderae B. orientalis M . sanguinipes M. packardii P. japonica A . simplex M.sanguinipes M.packardii P. japonica A . simplex M . sanguinipes M . packardii P. japonica M . sanguinipes M . packardii A . simplex M . sanguinipes M.packardii M . sanguinipes M . packardii A . simplex A . domesticus S. vaga
A . domesticus S. vaga A . simplex A . domesticus S . vaga A . domesticus S. vaga A . simplex
S. vaga
1
11-methyl 11-methyl 9-methyl 5-methyl 9-methyl 9-methyl 11-methyl 11-methyl 7-methyl 11-methyl 11-methyl 11-methyl 11-methyl 9-methyl ?
13- & 15methyl ? 11methyl ? 13-methyl ? 13-methyl 13-methyl Similar 13-methyl
10
DENNIS R NELSON
TABLE 1 (cont.)
Source"
GLC peak no.b
Methyl-branched components
Major isomer'
I N S E C T A (Tricoptera)
27-A 29-A
9-methylheneicosane 8-, lo-, snd 12-methyltricosane 9-, lo-, and 12-methylpentacosane 9-, 1 1-, and 13-methylheptacosane 7-, 9-, lo-, 12-, and 14-methylnonacosane
29-A 31-A 33-A 35-A 36-A 37-A 38-A 39-A 40-A 43-A
13-methylnonacosane I I - , 13-, and 15-methylhentriacontane 1 I - , and 13-methyltritriacontane 13methylpentatriacontane 12-, and 14-methylhexatriacontane 1 I-, 13-, 15-, and 17-methylheptatriacontane 12-methyloctatriacontane 1 I -,13-, 15-, and 17-methylnonatriacontane 12-, and 14-methyltetracontane 13-methyltritetracontane
27-A
4-, and 5-methylheptacosane 4-, and 5-methyloctacosane 4-, and 5-methylnonacosane 4.. and 5-methyltriacontane 4-, and 5-methylhentriacontane
P. calfornica
? ? 1 1
CHORDATA
Wool wax
13-methyl Similar Similar 13-methyl 13-methyl 12-methyl
PETROLEUM
Paraffin wax 31-A
4-methyl Similar 4-methyl 4-methyl Similar
a Meteorites: Or0 et a/., 1968, C . turgidus, A. cyanea, L. aestuarii, Nostoc sp: Gelpi el a/., 1970; N . muscorum, A. nidulans, P. luridum, C. fritschii: Han el al., 1968; A . uariabilis: Fehler and Light, 1970; S. ceratiles: Gaskell et a/., 1973; J . regia: Stransky et al., 1970; T. aestivum: Nishimoto, 1974; A . grandis: Hedin et al., 1972; P . japonica: Bennett et al., 1972 and Nelson, D. R. (unpublished); C. caryae: Mody et al., 1975; S . bullata: Jackson et al., 1974; S . calcitrans: Uebel el al., 1975b; F. nigricans, F. rufa, F. polyctena: Bergstrom and Lijfqvist, 1973; C. intrepidus: Brophy el al., 1973; P. rugosus and P . barbatus: Regnier et al., 1973; I . humilis: Cavill and Houghton, 1973; S. invicta and S . richteri: Lok et al., 1975; Beeswax: Stransky et al., 1966, Streibl et al., 1966; M . gulosa: Cavill et al., 1970; H . zea: Jones et a/., 1971; H . virescens: Vinson el al., 1975; M . sexta: Nelson and Sukkestad, 1970, Nelson el a/., 1972; P . australasiae, P. brunnea, and P . fuliginosa: Jackson, 1970; L. maderae and B . orientalis: Tartivita and Jackson, 1970; P. japonica: Jackson, 1972: M . sanguinipes and M . packardii: Soliday et a/., 1974; A . simplex: Jackson and Blomquist, 1976; A. domesticus: Hutchins and Martin, 1968; S. vaga: Nelson and Sukkestad, 1975; P. calfornica: Armold et al., 1969; wool wax: Mold et al., 1966; paraffin wax: Levy et a/., 1961. G L C peaks designated as described herein and in Nelson and Sukkestad, 1970, 1975. The number is equal to the number of carbons in the backbone of the molecule, and the letter A designates one internal methyl branch. The monomethylalkanes eluted with an equivalent chain length 0.6 to 0.7 carbon atoms less than the n-alkane with the same number of carbon atoms (Mold et a/., 1966; Nelson and Sukkestad, 1970; 1975). Determined from the relative intensities of the major characteristic fragmentation peaks in the mass spectra.
LONG-CHAIN METHYL-BRANCHED HYDROCARBONS
11
methylnonacosane. Other internally brdnched monomethylalkanes from 23-A to 35-A were present, including some in which the methyl branch occurred on an even numbered carbon atom (12 or 14). In wheat, the internally branched. methylalkane series was from 21 to 37 carbon atoms and consisted of even carbon numbered 11-, 13-, and 15-methylalkanes and odd carbon numbered 10- and 12-methyl alkanes. Other Hymenoptera and the Coleoptera had longer chain methylalkanes (over 20 carbon atoms). 10-Methyleicosane was the only methylalkane found in the boll weevil, A. grandis (Hedin et al., 1972). The monomethylalkanes 26A, 27-A, 28-A, and 29-A were identified in whole-body extracts of the pecan weevil, C. caryae, (Mody et al., 1975); 27-A, 29-A, and 3 1-A (each GLC peak consisted of a mixture of isomers) were identified in whole-body extracts of the bull ant, Myrmecia gulosa (F.), (Cavil1 et al., 1970), and the monomethylalkanes 25-A, 27-A, and 29-A were identified in beeswax (Strhsky et al., 1966; Streibel et al., 1966); 23-A, 25-A, and 27-A were identified in S . richteri, and 27-A was identified in S . inuicta (Lok et al., 1975) (Table 1). In Diptera, internally branched monomethylalkanes and monomethylalkenes have been reported from the stable fly, Stomoxys calcitrans, (Uebel et al., 1975b), which contained methylalkanes from 32 to 38 carbons in chain length and from the fleshfly, Sarcophaga bullata, (Jackson et al., 1974), which contained methylalkanes from 26 to 32 carbons in chain length (no methylalkanes with an even-numbered carbon backbone were found) (Table 1). One of the major alkane components of the surface lipids of the female tsetse fly, Glossina morsitans Westwood, is 2-methyltriacontane (personal communication, D. A. Carlson, USDA, Insects Affecting Man and Animals Laboratory, Gainesville, Fla.). Among the Orthoptera, the cockroaches, Periplaneta australasiae, P. brunnea, P. fuliginosa (Jackson, 1970), P. japonica (Jackson, 1972), Leucophaea maderae, and Blatta orientalis (Tartivita and Jackson, 1970), have the smallest internally branched monomethylalkanes (between 20 and 3 1 carbon atoms), followed by the grasshoppers, Melanoplus sanguinipes (F.), and M . packardii Scudder (Soliday et al., 1974) (between 28 and 38 carbon atoms). The common house cricket, Acheta domesticus (Hutchins and Martin, 1968), has monomethylalkanes, from 27 to 39 carbon atoms, and the Mormon cricket, A. simplex has monomethylalkanes from 28 to 38 carbon atoms with all the branch points on odd-numbered carbons (Jackson and Blomquist, 1976). The longest chain monomethylalkanes (33 to about 50 carbon atoms) from an orthopteran insect were in the grasshopper, Schistocerca uaga (Scudder) (Nelson and Sukkestad, 1975). Internally branched monomethylalkanes were identified in four Lepidoptera: the tobacco hornworm, Manduca sexta (L.) (Nelson and Sukkestad, 1970; Nelson et al., 1971, 1972), the corn earworm Heliothis zea (Boddie) (Jones et a[., 1971), the tobacco budworm, H. virescens (F.) (Vinson et al., 1975), and
12
DENNIS R. NELSON
the silkworm, B. mori (Murata et al., 1974). M. sexta had monomethylalkanes ranging in chain length from about 20 to 44 carbon atoms, and the major components were GLC peaks 35-A, 37-A, and 39-A. One GLC peak, 3 1-A, of three GLC hydrocarbon peaks of H. zea was identified as a mixture of 7-, 9-, 11-, 13-, and 15-methylhentriacontanes, and the 13-methyl isomer was shown to be a kairomone for the H. zea larval parasite, Microplitis croceipes (Cresson). Murata et al. (1974) reported finding 9-methyltriacontane in B. mori, but their mass spectra leave doubt as to this identification. However, mass spectra that they deduced as coming from 11,12-dimethyloctacosane was completely compatible with the mass spectra expected for a mixture of 11-, 13-, and 15methylnonacosane. The GLC retention time also appeared to be compatible with that expected for a monomethylalkane chromatographed on OV- 1. Similar homologous series of monomethylalkanes were present in both M. sexta and S . vaga. A comparison of GLC peaks 35-A, 37-A, and 39-A showed that the peaks from both insects contained the same mixture of isomers, but the major component of each peak from S. vaga had the methyl branch on carbon 13, and the major component of each peak from M. sexta had the methyl branch on carbon 17 for peak 35-A and on carbon 15 for peaks 37-A and 39A. Whether this difference is of any significance is not known at present. The majority of the internally branched monomethylalkanes has the methyl branch located on an odd-numbered carbon atom, and in plants and insects, this is usually either on carbon 11, 13, or 15. Monomethylalkanes with the methyl branch on an even-numbered carbon atom have been reported in only six insects: the boll weevil, Anthonomus grandis Boheman, with 10methyleicosane (Hedin et al., 1972), the grasshopper, Melanoplus sanguinipes, with GLC peak 30-A a mixture of 8-, 9-, lo-, and 11-methyltriacontanes and GLC peak 32-A a mixture of 9-, lo-, 11-, and 12-methyldotriacontanes (Soliday et al., 1974), the grasshopper, M. packardii, with GLC peak 30-A a mixture of 8-, 9-, lo-, 11-, 12-, and 13-methyltriacontanes and GLC peak 32-A a mixture of 9-, lo-, 11-, 12-, and 13-methyldotricontanes (Soliday et al., 1974), the stonefly, Pteronarcys californica, with GLC peak 23-A being a mixture of 8-, lo-, and 12-methyltricosanes, GLC peak 25-A a mixture of 9-, lo-, and l2-methylpentacosanes, and GLC peak 29-A a mixture of 7-, 9-, lo-, 12-, and 14-methylnonacosanes (Armold et al., 1969a), Popillia japonica, with 12-methyltetracosane (Bennett et al., 1972), and Heliothis virescens, with possibly 9-, 11-, 12-, 13-, and 15-methylhentriacontanesand lo-, 12-, 13-, 14-, 1 5 , and 16-methyldotriacontanes (Vinson et al., 1975). The only report of internally branched monomethylalkanes from a chordate was the finding in wool wax of methylalkanes from 17 to 44 carbon atoms (Mold et al., 1966) (Table 1). The methyl branch occurred mainly at the 13 position for the even-carbon numbered series.
LONG-CHAIN METHYL-BRANCHED HYDROCARBONS
13
Paraffin wax from petroleum contained 4- and 5-methylalkanes (Levy et al., 1961). In meteorites (carbonaceous chondrites) and algae, the internally branched monomethylalkanes occurred as single GLC peaks (in plants and insects, they occurred as homologous series), which were a mixture of two methylalkanes, one with the branch point on an odd-carbon atom and the other with the branch point on an evencarbon atom (Table 1). 2.6
DIMETHYLALKANES
Two homologous series of methylalkanes in addition to the n-alkanes and monomethylalkanes were reported in the tobacco hornworm, Munduca sexta (Nelson and Sukkestad, 1970; Nelson et al., 1971; Nelson et al., 1972) and in the grasshopper, Schistocercu uaga (Nelson and Sukkestad, 1975) in which two (B series) or three (C series) methyl branches, respectively, were located toward the center of the molecule. In both M. sextu and S. vaga, the major methylalkanes in the A-series were 35-A; in the B-series, 35-B; and in the Cseries, 35-C. The homologous B-series (dimethylalkanes) ranged from about 35 to 55 carbons in S . uagu and from about 21 to 47 carbons in M. sextu. As previously noted, for the monomethylalkanes, M. sexta had the methyl branching located farther down the chain than did S. vaga, and this was also true for the dimethylalkanes. The major component of 33-B in S . ougu was 13,17-dimethyltritriacontane,but in M. sexta, the only two isomers present (13,17- and 15,19-dimethyltritriacontane)were present in about equal amounts (Table 2). Likewise, the major component of 35-B in S. uugu was 13,17dimethylpentatriacontane and in M. sexta, it was 15,19-dimethylpentatriacontane; the major components of 37-B were 13,17-, 15,19-, and 17,21dimethylheptatriacontane in S. ouga and 15,19-dimethylheptatriacontane in M . sextu; the major component of 39-B was 13,17-dimethylnonatriacontane in S. vaga, but in M . sextu the three isomers 13,17-, 15,19-, and 17,21dimethylnonatriacontane were present in about equal amounts. Similar homologous series have recently been reported to be present in the stable fly, Stomoxys culcitrans (Uebel et al., 1975b), in the Mormon cricket, A. simplex (Jackson and Blomquist, 1976), and in the grasshoppers, Melanoplus sunguinipes and M. puckardii (Soliday et ul., 1974). The major isomers in M. sunguinipes were the 11,15-dimethyl isomers, but in M. packardii, the 13,17dimethyl isomer was the major isomer in three of five GLC fractions identified. The female tsetse fly, G. morsituns, was reported to have a homologous series of dimethylalkanes, one of which was a mixture of 15,19- and 17,21-dimethylheptatriacontane (personal communication, D. A. Carlson). Bennett et al. (1972) established that the dimethylalkanes 9,13dimethyltricosane and 11,15-dimethylpentacosane were present in the
TABLE 2
a
P
Occurrence and structure of internally branched dimethylalkanes
Sourcea
GLC peak no!
Methyl-branched components
Major isomef
INSECTA (Coleoptera)
P. japonica
23-B
9,13-dimethyltricosane 11,15-dimethyIpentacosane
31-B
11,15- and 13,17-dimethylhentriacontane 11,15-, 13,17- and 15,19dimethyltritriacontane 11,15-, 13,17- and 15,19dimethylpentatriacontane 11,15-, 13,17- and 15,19-dimethylheptatriacontane 15,19- and 17,21dimethylheptatriacontane
{ 25-B
INSECTA (Diptera)
S . calcitrans
G. morsitans INSECTA (Hymenoptera) P. rugosus P. barbatus I N s EC TA (Lepidoptera)
37-B
3,5-dimethyldodecane
1
M.sexta
{ 3,4-dimethyltridecane 27-B 33-B 35-B 37-B 39-B 41-B
9,13-dimethylheptacosane 13,17- and 15,19-dimethyltritriacontane 13,17- and 15,19-dimethylpentatriacontane 13,17-, 15,19- and 17-21dimethylheptatriacontane 13,17-, 15,19- and 17,21dimethylnonatriacontane 13,17- and 15,19-dimethylhentetracontane
Similar 15,19-methyl 15J9-methyl Similar Similar
33-B
9,13-, 11-15-, 13,17-, and 15,19dimethyltritriacontane
llJ5-methyl
33-B 33-B 34-B 35-B
11,15-, and 13,17-dimethyltritriacontane 9,13-, llJ5-, and 13,17-dimethyltritriacontane 12,16-, 13,17-, and 14,18dimethyltetratriacontane 11,15-, 13,17-, and 15,19-dimethylpentatriacontane
13,17-methyl 13~7-methyI SimiiU 11,15-methyl
INSECTA (Orthoptera)
M . packardii M . sanguinipes A . simplex S . vaga S. vaga M . packardii
1
zz z
v)
P Z rn r v)
0
z
*
6 Z
c
? 0 M . sanguinipes A . simplex S . vaga S . vaga M . packardii M . sanguinipes A . simplex S . vaga .S. vaga M . packardii S . vaga M . packardii M . sanguinipes
1
~
_
37-B 37-B 38-B 39-B 39-B 41-B 41-B 41-B 43-B 45-B 47-B 49-B 51-B _
~
e
z
5
14,18-dimethylhexatriacontane
37-B
S . vaga
I
9,13-, 11,15-, 13,17-, and 15,19-dimethylpentatriacontane 11,15-, 13,17-, and 15,19-dimethylpentatriacontane 9.13-, 1 1,15-, 13,17-, and 15,19-dimethylpentatnacontane
35-B 35-B 35-B 36-B
11,15-, 13,17-, 15,19-, and 17,21dimethylheptatriacontane
13,17-methyl
11,15-, 13,17-, 15,19-, and 17,2 1-dimethylheptatriacontane
15,19-methyl 13,17- & 15,19-methyl Similar 13,17-methyl 13,17-methyl 13,17-methyl 11,15-methyl 13,17-methyl 13,17-methyl
11,15-, 13,17-, 15,19-, and 17,21dimethylheptatriacontane 1 I,l5-, and 14,18-dimethyloctatriacontane 1 1,15-, 13,17-, 15,19-, and 17,21-dimethylnonatriacontane 13,17-, 15,19-, and 17,21-dimethylnonatriacontane 11,15-, 13,17-, 15,19-, 17,21-, and 19,23-dimethylhentetacontane 11,15-, 13,17-, 15,19-, 17,21-, and 19,23-dimethylhentetracontane 13,17-, and 19,23-dimethylhentetracontane 13,17-, and 19,23-dimethyltritetracontane 13,17-dimethylpentatetracontane 13,17-dimethylheptatetracontane 13,17-dimethylnonatetracontane 13,17-dimethylhenpentacontane -
-I
I
_ppp._____p-p
“P.japonica: Bennett el al., 1972; S.calcirrans: Uebel er al., 1975b; G. morsitans: Carlson (personal communication); P. rugosus and P. barbarus: Regnier el al., 1973; M . sexfa: Nelson and Sukkestad, 1970, Nelson er al., 1972; M. packardii and M . sanguinipes: Soliday et al., 1974; S . vaga: Nelson and Sukkestad. 1975; and A . simplex: Jackson and Blomquist, 1976. bGLC peaks designated as described herein and in Nelson and Sukkestad, 1970, 1975. The number is equal to the number of carbons in the backbone of the molecule, and the letter A designates one internal methyl branch. The dimethylalkanes with isoprenoid spacing eluted with an equivalent chain length about 1.4 carbon atoms less than the n-alkane with the same number of carbon atoms (Nelson and Sukkestad, 1970, 1975). Determined from the relative intensities of the major characteristic fragmentation peaks in the mass spectra.
< 7 W a D
Z 0 I
rn 0
I
re-promelittin ’* (or protomelittin) as a translation product of promelittin mRNA. As seen in the preceding section, the venom gland of honey bees is a highly specialized tissue synthesizing mainly one peptide, melittin, via its precursor promelittin (Kreil and Bachmayer, 1971, 1973). Consequently, the messenger for promelittin is likely to be one of the most abundant species of mRNA in the gland. Kreil and co-workers have turned their attention to the translation of the mRNA of promelittin in frog (Xenopus) oocytes and in mammalian cell-free system. Their work has given insight into the fundamental nature of the translation process and the molecular biology of secretory polypeptides. Unfractionated RNA prepared from young queen bee venom glands and injected into Xenopus oocytes directs the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced: the 17 amino acid residues identified correspond exactly with the sequences found in promelittin from the venom gland (Kindas-Mugge et al., 1974). The authors draw the r‘ollowing conclusions: (a) the informational content of the codons for a variety of amino acids is the same in cells from different phyla; (b) the results yield final proof that at least part of an injected messenger RNA can be translated with great fidelity and without translational error; (c) the translation of a gland cell insect messenger in Xenopus oocytes demonstrates that at least some of the translational systems of the frog cells are neither phylum nor cell-type specific. Some of the post-translational modifications of promelittin that occur in the venom gland were not observed in the oocytes. Conversion of promelittin to melittin was not detected and the conversion of the carboxyl terminal amino acid of promelittin to the amide form probably does not occur in the oocyte (Kindas-Miigge et al., 1974). Since vertebrates do not synthesize melittin, it is hardly surprising to find that these post-translational mechanisms are lacking in Xenopus. However, the oocyte-derived promelittin, like venom gland promelittin, was heterogeneous at the amino end, suggesting that either the oocyte contains proteases of the correct specificity which can catalyse the activation of the precursor of promelittin, or that the total RNA preparation used in the study contained messengers coding for these proteases. Kreil’s group turned to a cell-free, protein-synthesizing system for further answers, arguing that post-translational modification might be absent or much reduced in vitro. The ability of total RNA preparations from the venom glands of young queen bees to serve as a template in a cell-free system prepared from mammalian sources was investigated by Suchanek et al. (1975). The cell-free system consisted of purified ribosome subunits, rat liver pH-5 fraction and partially purified initiation factors from rabbit reticulocytes and was made
110
ROBERT P BODNARYK
optimal for the translation of rabbit globin mRNA. The heterologous system was found to translate the venom gland mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized by this in uitro system that had amino acid sequences characteristic of promelittin and which liberated a melittin-like peptide after digestion with a bacterial protease. The polypeptide was larger than promelittin and probably contained the whole of the promelittin sequence, plus a number of additional amino acids at the amino end. Suchanek et al. (1975) note that synthesis of a larger, “prepromelittin” (or protomelittin) by a cell-free system has certain analogies with other observations on the translation of messenger RNAs in heterologous systems. These are: (1) the synthesis of a murine immunoglobin “light” chain containing about 20 additional amino acid residues at the amino end in frog oocytes and a cell-free system from reticulocytes (Stevens and Williamson, 1972; Milstein et al., 1972; Mach et al., 1972; Schechter, 1973) and (2) the synthesis of a polypeptide larger than proparathyroid hormone (“preproparathyroid hormone”) in a cell-free system from wheat germ (Kemper et al., 1974). It can be postulated that in all three cases the large polypeptide products represent precursors, too short-lived to be detected in intact cells but relatively stable in cell-free systems. Suchanek et al. (1975) suggest the intriguing possibility that secretory polypeptides, including melittin, may generally start with some “leading amino acid sequence” which acts as a signal in one of the complex steps involved in secretion. Patterns of promelittin and melittin biosynthesis during bee maturation. Bee stings are used in the defence of the colony against both vertebrate and invertebrate animals [melittin, for example, is highly toxic to Drosophila larvae (Mitchell et al., 1971) and probably to most other invertebrates]. Worker bees undertake a variety of tasks in the hive, followed by a transition from house-bee activity to field-bee activity (Lindauer, 1952). Aging of the worker bee is accompanied by structural changes in the venom apparatus and increased production of venom which reaches its full capacity within about two weeks after emergence. The poison sac is full of venom by this time and the bee can serve as a fearsome guard (Autrum and Kneitz, 1959; da Cruz Landim and Kitajima, 1966). The pattern of synthesis of promelittin and melittin during the maturation process has been followed by Bachmayer el al. (1972). In worker bees, production of promelittin increases slowly from the time of emergence to reach a maximum eight to ten days later, and then declines again. Conversion of promelittin to melittin does not occur during the first two days after emergence, but by the ninth day is proceeding at a maximal rate. Evidently, synthesis of the precursor and its conversion to product are independently controlled. The situation in queen bees is quite different. Both synthesis of promelittin and its conversion to melittin operate close to full capacity in the newly emerged
STRUCTURE A N D FUNCTION
OF INSECT PEPTIDES
111
queen. Bachmayer et al. (1972) point out that full production of melittin, the major toxin of venom, may be needed for imminent duels with other newly emerging queen bees in the colony. Although melittin is the major component of bee venom, its synthesis in worker bees cannot be said to mirror the production of the complete venom. The histamine content of venom has been studied by Owen and Braidwood (1974) and was found to be relatively low (approx. 100 ng/venom extract) in 1week-old worker bees. It continues to increase, reaching approx. 400 ng in 2week-old bees and 1600ng (the maximum) in 5-week-old bees. Owen and Braidwood (1974) conclude that the rise in the histamine content of worker bees is concomitant with the transition from house-bee activity to field-bee activity, and suggest that the venom is only fully completed at the time at which bees start to leave the hive. c Phylogenetic relationships between honeybees as deduced from melittin sequence data There are four real species of Apis: A . mellifera; the Indian bee, A . cerana; and the free-nesting forms A . dorsata and A.jlorea. The amino acid sequences of melittins isolated from the venom apparatus of these species has been obtained by Kreil (1973, 1975) (Fig. 8). All four melittins are composed of 26 amino acid residues; all four begin with the sequence Gly-IleGly-Ala and the carboxyl terminus of each is in the amide form. Differences exist among the species only at positions 5, 10, 15, 22 and 26, the remaining being identical for each species. Kreil (1972, 1975) has used the sequence data to deduce phylogenetic relationships between the various species; these have proven to be consistent with morphological, immunological and other biological criteria. Thus, the sequences of melittin from A . mellifera and A . cerana are identical (Fig. 8a,b) and these two species are very closely related. Cross-fertilization between them is possible, but embryonic development stops at an early stage (Rutner and Maul, 1969). Apparently, in the evolution of honey bees, A . mellifera and A . cerana diverged only recently and it is perhaps not surprising that their melittins are identical. On the other hand, melittin from A . dorsata differs by five residues from that of A.florea, but only by three residues from those of the A. melliferalA. cerana pair. At the nucleic acid level the remote relationship of A.florea to the other three species is even more evident: no fewer than six base changes would be required to convert thejlorea to either the dorsata or the mellueralcerana sequence, using the genetic code of E. coli (Kreil, 1975). A . jlorea is a more distant relative, having only half the number of chromosomes (N = 8) of the A . mellfera1A. cerana pair, is much smaller than the other bees and has many other distinctive features (Kreil, 1973). Based on the structure of melittin, Kreil (1975) proposes a phylogenetic tree for the genus Apis, where
ROBERT P. BODNARYK
112
the line of descent for A.jlorea branches off first from the trunk representing the ancestor common to all honey bees. An identical picture of the evolutionary history of honey bees has been obtained from electrophoretic and immunological cross-reactivity studies of the blood proteins of the different species by Engels et al. (1973). 8.1.2
Mast cell degranulating peptide (MCD-peptide)from bee venom
Mast cells (basophil leucocytes) of mammals are a prominent source of histamine, and in many animals there is a good correlation between the histamine content of a tissue and the number of mast cells (see Bell et al., 1965). Mast cell destruction is the earliest visible process resulting from the sub-cutaneous injection of bee venom in mice (Habermann, 1972). Both melittin and lysolecithin are mastocytolytic agents (lysolecithin is the reaction product of phospholipase A, a key component of bee venom, Habermann,
7 s s ’
Ile Lys Cys Asn Cys Lys Arg His Val Ile Lys Pro His Ile Cys Arg Lys Ile
Cys Gly Lys Asn NH,
1
s
-
s
-
Fig. 9. The structure of MCD-peptide, a mast cell degranulating peptide from bee venom (Haux, 1969; Vernon el al., 1969).
1968, 1972). However, early experimental work indicated that the amount of melittin and phospholipase A in venom did not account entirely for the histamine released in various test tissues. A histamine-releasing fraction was obtained from bee venom (Fredholm, 1966; Fredholm and Hagermark, 1967, 1969) and a peptide (MCD-peptide) with specific histamine-releasing properties was purified by Breithaupt and Habermann (1968). The primary sequence of MCD-peptide was reported by Haux (1969) and confirmed by Vernon et al. (1969) who also determined the position of the two disulphide bridges in the peptide (Fig. 9). MCD-peptide resembles apamin (Fig. 10, section 8.1.3) in that both have two disulphide bridges and both are extremely basic, the MCD-peptide having five lysine and two arginine residues in a total of 22 amino acid residues. Positions three to six in MCD-peptide correspond to positions one to four in apamin, and the carboxyl terminus of both is in the amide form. The MCD-peptide differs from melittin in its specific activity as a histamine releaser, the MCD-peptide being 10-100 times more active. Since bee venom consists of about 50 per cent of melittin and only 1-2 per cent of MCD-
STRUCTURE A N D FUNCTION OF INSECT PEPTIDES
113
peptide, both factors contribute significantly to mast cell destruction caused by whole venom (Breithaupt and Habermann, 1968). MCD-peptide has no hemolytic activity and is practically nontoxic when given by intravenous injection, in marked contrast to the modes of action of melittin and apamin (Habermann, 1972). In the rat, degranulation of mast cells results in the release of histamine and 5-hydroxytryptamine. The MCD-peptide therefore is strongly inflammatory in this species. Recent interest in the MCD-peptide has centred about its potent anti-inflammatory (sic) properties and its ability to suppress the development of adjuvant arthritis and reduce the severity of primary and secondary lesions in established adjuvant arthritis in the rat (Billingham et al., 1973; Hanson et al., 1974). Whether these two apparently opposite biological activities are related is unclear at present (Gauldie et al., 1976). Nevertheless, these intriguing observations, in addition to their obvious potential practical
H Cys Asn Cys Lys M a Pro Glu Thr Ala Leu Cys Ala Arg Arg Cys Gln Gln His NH, I
I
s
-
s
I
I
Fig. 10. The structure of apamin, a neurotoxin from bee venom (Haux et al., 1967; Shipolini et
al., 1967; Callewaert et al., 1968).
significance, may relate to an ancient and apocryphal belief that the venom of honey bees is beneficial in certain arthritic and rheumatoid conditions (Beck, 1935; Broadman, 1963). 8.1.3
Apamin, a neurotoxic peptide from bee venom
Habermann’s group, while screening the peptide fractions of whole bee venom by gel filtration, obtained a potent neurotoxin that was further purified on carboxymethylcellulose and subjected to amino acid analysis. The neurotoxin-a relatively small peptide-was called apamin (Habermann and Reiz, 1964,1965aYb;Habermann, 1972). a Structure The amino acid sequence of apamin (Fig. 10) has been established by two independent groups (Haux et al., 1967; Shipolini et al., 1967). The peptide consists of 18 amino acids, four of them being half-cystines. One disulfide bridge connects a half-cystine in position 1 and a half-cystine in position 11. The second bridge connects a half-cystine in position 3 and a halfcystine in position 15 (Callewaert et al., 1968). Apamin is a basic peptide containing one lysine, one histidine and two arginine residues. The apamin octadecapeptide is the smallest neurotoxic peptide known. Its basicity and cross-linking by disulfide bridges are features common to the snake toxins
114
ROBERT P. BODNARYK
which are generally classified as short neurotoxins (60-62 residues; four disulphide bridges) and long neurotoxins (7 1-74 residues; 5 disulfide bridges) (rev. Yang, 1974). b Synthesis Synthesis of apamin has been achieved recently by van Rietschoten et al. (1975) using a solid-phase procedure. Synthetic apamin reported by the Marseille group represents the first synthesis of neurotoxin with full toxic activity. It is also the first laboratory-synthesized neurotoxin for which the chemical purity and identity with the natural peptide have been demonstrated. By comparison, Aoyagi et al. (1972) reported the synthesis of a peptide with cobrotoxin activity; this synthetic peptide had only about 20 per cent of the activity of the natural snake toxin. van Rietschoten et al. (1975) attribute their remarkably successful synthesis to the use of solid-phase methodology (Merrifield, 1969) with high yields of incorporation of each amino acid (99.3 per cent on average over 15 steps), to the use of the fluorescamine test to check completeness of coupling (Felix and Jimenez, 1973) and to the high quality of the solid support resin (Tregear, 1972). c Structurefunction relationships Specific chemical modifications of natural apamin have been used to study the residues involved in its toxic action (Vincent et al., 1975). Their work has shown that the alpha-amino group of Cys 1, the epsilon-amino group of Lys 4, the carboxylate side chain of Glu 17 and the imidazole group of His 18 are not essential for the toxic activity of apamin. A synergistic effect was observed when several functions were modified. Thus, apamin in which the alpha- and epsilon-amino groups of Cys 1 and Lys 4 have been acetylated and the imidazole of His 18 has been carbethoxylated is devoid of activity. Complete loss of toxicity also results from reduction and alkylation of both disulphide bridges, a finding recently confirmed by Gauldie et al. (1976). Evidently, the disulphide bridges are necessary for biological activity. According to Vincent et al. (1975), the most important part of the apamin sequence for neurotoxic activity appears to be the C-terminal region containing the two arginine residues. Chemical modification of Arg 13 and Arg 14 eliminated toxicity, as did removal of Arg 14 of acetylated apamin by digestion with trypsin. Further progress on structure-function relationships can be expected from work on synthetic structural analogs of apamin (van Rietschoten et al., 1975). d Pharmacological activity and site of action A lethal dose (LD50 = 4 mg/kg) (Habermann, 1972; Gauldie et al., 1976) of apamin given to mice by intravenous injection induces uncoordinated hypermotility within
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15 min, culminating in generalized convulsions followed by respiratory distress and death. Sublethal doses cause extreme hyperexcitability which can last for up to 60 h. The site of action is localized in the spinal cord as judged from neurophysiological studies on the effect of apamin on spinal reflexes (Wellhoner, 1969). Radioactive apamin is found predominantly in the spinal cord after its injection into mice (Vincent et al., 1975). The effect of apamin is to augment polysynaptic reflexes and render excitatory polysynaptic pathways more effective than inhibitory polysynaptic mechanisms (Wellhoner, 1969). Current research interest is directed towards understanding the molecular mechanisms of toxic action. Little is anything is known about the in vivo biosynthesis of apamin in the venom gland of the bee. 8.1.4
Melittin F, tertiapin and secapin
A large-scale fractionation of 700 g (sic)of crude bee venom has resulted in the isolation of three new peptides (Gauldie et al., 1976). Melittin F, a 19-amino acid residue peptide, is evidently a fragment of melittin consisting of residues 8-26. Tertiapin is a 20-residue basic peptide. Both peptides are present in venom in very small amounts. Their pharmacological properties have not yet been explored. The third newly discovered peptide, secapin, comprises about 1 per cent of lyophilized venom and apparently has been overlooked by other workers. It is a 24-residue basic peptide containing a large proportion of proline and one disulphide bridge. Secapin has a very low mammalian toxicity. At high doses in mice it produces marked hypothermia and signs of sedation. 8.1.5
Other bee venom peptides
Nelson and O’Connor (1968) have reported two small peptides from bee venom with histamine at the C-terminus. They have been characterized as alagly-pro-gln-histamine and ala-gly-gln-gly-histamine (procamine) by Peck and O’Connor (1974). Synthetic procamine is said to have the same chromatographic properties as the natural peptide by the authors. The presence of histamine peptides in bee venom could not be confirmed by Gauldie et al. (1976) in their large-scale fractionation of venom (section 8.1.4), and no explanation of this discrepancy has been offered. Minimine, a basic peptide reported by Lowy et al. (1971) was not found by Gauldie et al. (1976). Probably, minimine activity as described by Lowy et al. (197 1) was due to phospholipase A activity (Habermann, 1972). The total number of bona Jide bee venom peptides discovered to date, including variants of apamin and the MCD-peptide, appears to be eight (Gauldie et al., 1976).
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1
2
3 4
5
6
7
8 9
Arg Pro Pro Gly Phe Ser P r o Phe Arg Bradykinin Arg P r o Pro Gly Phe Ser P r o Phe Arg Lysylbradykinin (kallidin) Met Lys
Arg Pro Pro Gly Phe Ser Pro Phe Arg Methionylly iylbrady kinin
pGlu Thr Asn Lys Lys Lys Leu Arg Gly
Arg Pro Pro Gly Phe Ser Pro Phe Arg Polisteskinin
c1 c2 I 1 Thr Ala Thr Thr Arg Arg Arg Gly
Arg Pro Pro Gly Phe Ser Pro Phe Arg Vespulakinin 1
c 1 c2
I
I
Thr Thr Arg Arg Arg Gly
Arg Pro Pro Gly Phe Ser Pro Phe Arg Vespulakinin 2 Arg Pro Pro Gly Phe Thr P r o Phe Arg Thr6-bradykinin
Ala Arg
Arg Pro Pro Gly Phe Thr P r o Phe Arg
Alanylargininyl-Thr6-bradykinin Fig. 11. A comparison of the structures of bradykinin and bradykinin derivatives with bradykinin-like peptides from the venom of wasps. Polisteskinin is from the venom of wasps of the genus Polistes (Nakajima el al., 1967; Pisano, 1968). Vespulakinins 1 and 2 are from the venom of the yellow jacket Vespula maculifons (Yoshida et al., 1976). The carbohydrate prosthetic group of the vespulakinins has not been fully characterized to date. C : N. Ac. 2, galactose 1; C2: N. Ac. Galactosamine 2 3, galactose 2. Thr6Galactosamine 1 bradykinin and alanylargininyl-Thr6-bradykininare from the Japanese wasp P. rothneyi iwatai (Watanabe et al., 1975).
-
8.2
Y
K I N I N S FROM W A S P S A N D HORNETS
The venoms from wasps and hornets contain bradykinin-like peptides but apparently lack melittin, apamin and MCD-peptide. Bradykinin and the structurally related kallidin (lysylbradykinin) and methionyllysylbradykinin
STRUCTURE A N D FUNCTION OF INSECT PEPTIDES
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(Fig. 11) are smooth muscle active, hypotensive agents. Bradykinin is among the most potent pain-producing agents (revs. Erdos, 1966; Ryan et al., 1970). Venom kinins have pharmacological actions similar to bradykinin and kallidin. Vasoactive peptides in animal venoms are concisely reviewed by Pisano (1968) whose bibliography provides reference to many general works. Insect peptides similar to bradykinin were first found in venom of the European wasp Vespa vulgaris (Jaques and Schachter, 1954; Schachter and Thain, 1954; Holdstock et al., 1957). Crude venom from V. vulgaris was resolved into three peaks of kinin activity (a major and two minor peaks) by ion-exchange chromatography (Mathias and Schachter, 1958). The activity of the wasp kinins was much reduced by trypsin (bradykinin is resistant to trypsin digestion) indicating that structural differences exist between these two types of kinins. The hornet Vespa crabo contains a single, trypsin-resistant kinin that is clearly different from the wasp kinins by chromatographic and enzymic tests and is about one-tenth as active as bradykinin in contracting guinea pig ileum (Bhoola el al., 1961). The structure of insect venom kinins is known in three cases: polisteskinin from wasps of the genus Polistes, P-11-1 and P-111 from the Japanese wasp, P. rothneyi iwatai, and vespulakinin from the yellow jacket Vespula maculfrons.
8.2.1 Polisteskinin
Polisteskinin was purified from extracts of the terminal three abdominal segments from 6000 wasps of mixed species, Polistes annularis, P. fuscatus and P. exclamans (Prado et al., 1966; Pisano, 1968) and its structure determined (Nakajima et al., 1967; Pisano, 1968). The octadecapeptide contains the nonapeptide bradykinin at its carboxy-terminal end (Fig. 11). Polisteskinin is a strongly basic peptide containing three arginine and three lysine residues out of a total of 18 amino acid residues. Laboratory synthesis has not been reported to date, although this would seem straightforward if synthesis was started from bradykinin. Polisteskinin has pharmacological properties that distinguish it from mammalian bradykinin, kallidin and methionyllysylbradykinin. Unlike these kinins, polisteskinin is not inactivated by passage through the rat pulmonary vascular bed (Ryan et al., 1970), it has a longer acting hypotensive effect and it is the most potent naturally occurring releaser of histamine from rat mast cells (Johnson and Erdos, 1973). Very little is known about the biosynthesis of polisteskinin in the venom gland. In mammals, bradykinin is formed from an inactive protein precursor in blood plasma (bradykininogen) by the action of specific proteases at the site of its action (Erdos, 1966). No kininogen has been found for polisteskinin either
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in venom or whole wasp extracts, at least not in the preliminary experiments of Prado et al. (1966), thus leaving open the question of insect kinin biosynthesis. 8.2.2 Thr6-bradykininand ala-arg-thr6-bradykinin
Two new bradykinin analogues have been reported recently in the venom of the Japanese wasp P. rothneyi iwatai by Watanabe et al. (1975). These are Thr6bradykinin and Ala-Arg-Thr6-bradykinin.The discovery of these bradykinin analogues in Polistes indicates that active peptides in wasp venom may be different from species to species. 8.2.3
Vespulakinins I and 2
Vespulakinins are newly discovered carbohydrate-containing bradykinin derivatives from the venom sac of the yellow jacket Vespula maculifons (Yoshida et al., 1976). They are the first reported naturally occurring glycopeptide derivatives of bradykinin and the first reported vasoactive glycopeptides. Vespulakinins are similar in structure to polisteskinin (Fig. l l ) in that bradykinin is at the carboxy-terminal end. The heptadecapeptide vespulakinin 1 and pentadecapeptide vespulakinin 2 are also highly basic peptides containing no fewer than five arginine residues per 17 and 15 amino acid residues, respectively. Their most distinctive feature is the carbohydrate prosthetic group (which has not yet been fully characterized). The extent to which the carbohydrate moiety contributes to biological activity is not known at present. Vespulakinin 1 is at least twice as potent as bradykinin (on a weight basis) in lowering rat blood pressure, but the duration of the response is not significantly longer (Yoshida et al., 1976). Further pharmacological testing of the potency of the vespulakinins in releasing histamine from mast cells and leucocytes and their ability to produce pain will be of interest.
9
Concluding remarks
The list of peptides found in insects undoubtedly will continue to grow as our knowledge of peptide-mediated processes continue to increase. It is evident that enormous experimental benefit accrues to the individual or group of individuals who, early in the course of their work with peptides, devote their attention to establishing primary structure. Synthesis, structure-function relationships, isotope studies and precise, definitive physiological experimentation are then all possible, and soon follow with the psychological advantage of dealing with a known substance.
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Acknowledgements
I wish to thank Dr Leo Levenbook for generous and unfailing advice throughout the writing of the review and Dr P. S. Chen for valued guidance on the peptides from Drosophila. I am indebted to Drs H. Baumann, B. E. Brown and A. N. Starratt who provided me with copies of their manuscripts prior to publication. Special thanks are due to Mr. K. D. Oliver and library staff for locating reference material. This chapter is Contribution No. 762, Canada Agriculture. References Adams, T. S. and Nelson, D. R. (1968). Bioassay of crude extracts for the factor that prevents second mating in female Musca domestica. Ann. ent. SOC.Am. 61, 112-1 16. Autrum, H. and Kneitz, H. (1959). Die Giftsekretion in der GiRdriise der Honigbienen in Abhangigkeit vom Lebensalter. Biol. Zentralbl. 18,595-602. Aoyagi, H., Yonazawa, H., Takahasi, N., Kato, T., Izumiya, N. and Yang, C. C. (1972). Synthesis of a peptide with cobrotoxin activity. Biochim. Biophys. Acta, 263,823-826. Bachmayer, H., Kreil, G. and Suchanek, G. (1972). Synthesis of promelittin and melittin in the venom gland of queen and worker bees: patterns observed during maturation. J. Insect. Physiol. 18, 1515-1521. Balabaskaran, S. and Smith, J. N. (1970). The inhibition of l,l,l-trichloro-2,2-bis-(pchlorophenyl) ethane (DDT) dehydrochlorinase and glutathione S-aryltransferase in grassgrub and housefly preparation. Biochem. J. 111,989-996. Balogun, R. A. (1974). A sex-specific ninhydrin-positive component detected in the accessory glands of adult male tsetse flies (Diptera, Glossinidae). Nigerian J. Ent. 1, 13-16. Bargmann, W., Lindner, E. and Andres, K. H. (1967). ~ e Synapsen r an endokrinen Epithelzellen und die Definition sekretorischer Neurone. Untersuchungen am Zwischenlappen der Katzenhypophyse. Z . Zellforsch. 11,282-298. Baumann, H. (1974a). The isolation, partial characterization, and biosynthesis of the paragonial substances, PS-I and PS-2, of Drosophilafunebris. J. Insect Physiol. 20,2181-2194. Baumann, H. (1974b). Biological effects of paragonial substances PS-1 and PS-2, in females of Drosophilafunebris. J. Insect Physiol. 20,2347-2362. Baumann, H. and Chen, P. S. (1 973). Geschlechtsspezifische Ninhydrin-positive Substanzen in Adultmannchen von Drosophila funebris. Rev. Suisse 2001.80,685-690. Baumann, E. and Gersch, M. (1973). Untersuchungen zur Stabilitat des Neurohormons D. Zool. Jb. Physiol. 71, 153-160. Baumann, E. and Gersch, M. (1974). Versuche zur Markierung von Neurohormon D aus Periplaneta americana mit Hilfe von Dansylchlorid. Zool. Jb. Physiol. 18,533-54 1. Baumann, H., Wilson, K. J., Chen, P. S. and Humbel, R. E. (1975). The amino acid sequence of a peptide (PS-1) from Drosophila funebris: A paragonial peptide from males which reduces the receptivity of the female. Eur. J. Biochem. 5 5 521-529. Beard, R. L. (1963). Insect toxins and venoms. Ann. Rev. Ent. 8, 1-18. Beck, B. F. (1935). “Bee venom therapy.” D’Appleton-Century Co., Inc., New York. Bell, G. H., Davidson, J. N. and Scarborough, H. (1965). “Textbook of Physiology and Biochemistry.” 6th Edn. E. & S. Livingstone Ltd., Edinburgh and London. Belton, P. and Brown, B. E. (1969). The electrical activity of cockroach visceral muscle fibres. Comp. Biochem. Physiol. 28,853-863.
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Insect Flight Metabolism Ann E. Kammer' and Bernd Heinrich2
' Division of Biology, Kansas State University, Manhartan. Kansas, USA
Division of Enromology. University of California, Berkeley, California, USA
1 Introduction 134 2 The metabolic rate during flight 134 2.1 Ambient temperature 137 2.2 Wing-loading and body mass 139 2.3 Flightspeed 143 2.4 Cost of transport 146 2.5 Ecology and evolution of high metabolic rates 146 3 Neural control of power output 147 3.1 Insects with neurogenic rhythms 147 3.2 Insects with myogenic rhythms 151 3.3 Novel aerodynamic mechanisms 154 3.4 Some comparisons and conclusions 155 4 Supplying the energy demanded: Control of flight metabolism 156 4.1 Insect flight muscles 156 4.2 Oxygen supply 157 161 4.3 Biochemical processes in flight muscle 4.4 Mobilization of stored fuels 169 4.5 Methods for studying flight muscle metabolism 171 4.6 Hormonal control mechanisms 173 4.7 Hemolymph circulation 178 4.8 Substrate availability and flight speed 179 5 Interrelations of flight muscle temperature and metabolic rate 180 5.1 Effects of temperature on the flight motor 181 5.2 Pre-flight warm-up 184 5.3 Stabilization of thoracic temperature during flight 190 5.4 Shivering and nonshivering thennogenesis 19 1 5.5 Why require a high muscle temperature? 195 6 Development and senescence 197 6.1 Hemimetabolous insects 198 6.2 Lepidoptera 200 6.3 Holometabolous insects with fibrillar muscles 203 6.4 Adult diapause, regeneration and polymorphism 206 6.5 Maturation related to use and disuse 208
133
ANN E. KAMMER AND BERND HEINRICH
134
7
1
6.6 Hormonal control 209 6.7 Age and flight metabolism Conclusions 211 Acknowledgements 21 1 References 2 12
210
Introduction
Actively flying insects achieve the highest metabolic rates known, and they do so in the fraction of a second required to shift from quiescence to flight. The various adaptations that make possible the high metabolic rates necessary for flight constitute the subject of this review. Flight depends on the biochemical and mechanical work done by the flight muscles, which must be continually supplied with oxygen and fuel. The work of the muscles is under neural control and therefore the metabolic rate is also under neural control. Hormones participate as part of the biochemical mechanisms by which the neural commands are executed and also as part of the internal milieu supportive of flight. In larger insects, high metabolic rates and the associated heat production result in elevated body temperatures; temperature effects and temperature regulation are thus closely related to flight and they are considered extensively in the following discussion. Much has been written about the flight of insects. Recent reviews have already covered the biochemistry of the flight fuels (Bailey, 1975) and their utilization (Sacktor, 1970, 1975; Crabtree and Newsholme, 1975), nervous coordination (Wilson, 1968), ventilation (Miller, 1966, 1974; Kammer, 1976) and temperature control of the flight muscles (Heinrich, 1974a). Other reviews have considered aerodynamic problems (Lighthill, 1975; Weis-Fogh, 1975), the comparative physiology-anatomy of flight mechanisms (Pringle, 1957, 1968, 1974), and migration (Johnson, 1969,1974; Rainey, 1976). We shall not attempt to provide another review of the above-mentioned aspects of insect flight, but shall draw on these areas insofar as they concern intensity and regulation of flight metabolism.
2
The metabolic rate during flight
Energy expended in flight appears partly as aerodynamic work and partly as heat. The work output must first of all be sufficient to counteract the force of gravity, and secondly, the work done must provide forward thrust. The energy input for flight, on the other hand, must be much greater than the work output of the wings on the air, because of biochemical and mechanical inefficiencies. Typically the muscles are no more than 20 per cent biochemically efficient (i.e.
135
INSECT FLIGHT METABOLISM
TABLE 1 Rates of oxygen consumption of insects at rest and in flight* Metabolic rate
ml O,/g body wt/h
Species
At rest
W * N-' in flight
Reference
In flight
DICTYOPTERA
Periplaneta americana
0.36
36
21
Polacek and Kubista (1960)
ORTHOPTERA
Schistocerca gregaria
10-30 (45) 6-18 (27)
Krogh and Weis-Fogh (1951);Weis-Fogh (1952)
-
17-24
10-14
Weis-Fogh (1967)
0.55 0.7 0.75 0.73 -
54 92 56 51 40-90 64 55 59 82 (105) 29 43 7
32 55 33 30 24-53 38 33 35 49 (62) 17 25 4
Zebe (1954) Zebe (1954) Zebe (1954) Heinrich (1971) Casey (1976a) Zebe (1954) Heinrich and Casey (1973) Heinrich and Casey (1973) Zebe (1954) Zebe (1954) Zebe (1954) Nayar and Van Handel (197lb)
57 (1 11) 36 (61) 12 (18)
0.63
ODONATA
Aeschna grandis LEPIWPTERA
Vanessa w Metopsilus procellus Mimas tiliae Manduca sexta Hyles lineata Hyles euphorbia Hyles euphorbia Deilephila elpenor Saturnia pavonia Antheraea pernyi Triphaenapronuba Spodopterafmgiperda
1
~
DIPTERA
Lucilia sericata Musca vomitoria Drosophila repleta
-
1.8
96 (188) 60 (103) 21 (30)
Drosophila melanogaster Drosophila melanogaster Drosophila gibberosa
5 .O -
12 (22) 33 (40) 19
7 (13) 20 (24) 11
Davis and Fraenkel(l940) Axenfeld (1911) Chadwick and Gilmour ( 1940) Wigglesworth (1949) Hocking (1953) Sotavalta and Laulajainen
Drosophila hydei
-
20
12
Sotavalta and Laulajainen
Drosophilafunebris
-
10
6
Sotavalta and Laulajainen
Tabanus aflnis Tabanus septentrionalis A edesflavescens Aedes nearcticus Simulium venustum
0.8 1.1 3.8 5.6 4.4
22 (63) 56 (68) 22 21 (58) 27 (37)
(1961) (1961) (1961)
13 (37) 33 (40) 13 12 (34) 16 (22)
Hocking (1953) Hocking (1953) Hocking (1953) Hocking (1953) Hocking (1953)
136
ANN E. KAMMER AND BERND HEINRICH
TABLE l-continued Metabolic rate Species
ml O,/g body wtlh At rest
In flight
W * N-' in flight
Simulium vittatum Eristalis tenax
4.8 -
50 (53) 23
30 (3 1) 14
Fannia caninrlaris
-
23
14
Glossina morsitans
-
90
53
Apis meififera
2.0
87 (100)
52 (59)
Apis mellifera Apis mellifera Apis mellifera Apis mellifera Bombus uosnesenskii
3.2 0.5 1.3
60 (98) 312 70 60 55-66
36 (58) 185 42 36 33-39
Bombus spp. Vespa crabro
-
80 ( 110) 17-24
47 (65) 10-14
Reference
Hocking (1953) Sotavalta and Laulajainen (1961) Sotavalta and Laulajainen (1961) Hargrove (1976)
HYMENOPTERA
Jongbloed and Wiersma (1934) Hocking (1953) Kosmin el al. (1932) Bastian and Esch (1970) Sotavalta (1954b) Kammer and Heinrich (1974) Heinrich (1975) Weis-Fogh (1967)
In most cases oxygen consumption was measured; in some cases it was calculated from other data [see Hocking (1953) for discussion of the older work]. We have converted these values into SI units, using 1 L 0, = 5 kcal for all substrates, and 1 cal-h-'-gf" = 0.1186 W-N-'. Flight values are means and, in parentheses, maxima. In this table we have not distinguished among free, tethered on a flight mill, or fixed flight; the oxygen consumption in free flight can be double that in fixed flight.
the ratio of mechanical work done to metabolic cost is approximately 0.2, although values can range from 0.35 to -1.2 (Hill,1939; Weis-Fogh, 1972; Tucker, 1973, 1975)). Of the mechanical work performed by the muscles, only a fraction is aerodynamically useful. The efficiency varies among different kinds of flight, such as fast forward flight, gliding, or hovering. For the latter, WeisFogh (1972) calculated an aerodynamic efficiency (momentum imparted to the aidtotal aerodynamic power) of 50 per cent for a hummingbird and 30 per cent for Drosophilu. Thus for a hovering Drosophilu, for example, of every 100 calories expended, only about 6 calorites (100 x 0.2 x 0.3) result in useful work. The work input, or total energy expended during flight, is reflected in the metabolic rate. Measured and calculated metabolic rates of flying insects vary
INSECT FLIGHT METABOLISM
137
-
over a large continuum. In general, most values fall between about 12 to 60 W N-I (Le., 100 to 500 cal g-' * h-l) (Table 1). These metabolic rates are among the highest known. They represent 50- to 100-fold increases over the resting rate. In comparison small mammals running at maximal speed have metabolic rates of 1-4 W. N-', only 7 times greater than resting rates, and flying birds show a similar 7- to 14-fold increase (Schmidt-Nielsen, 1972). The metabolic rate of a hovering hummingbird, 24 W-N-' (Lasiewski, 1963), is comparable to that of many insects, however. Hovering flight places heavy energetic demands on small birds and bats as well as on insects (Weis-Fogh, 1972), and it is this mode of locomotion, not the systematic position of the animal, that demands high metabolic rates. The large range in the measured metabolic rate reflects intrinsic differences between species as well as different conditions of measurement. The full range of possible rates is probably not yet known. For example, very low values of metabolic rate possible during gliding flight such as that of some butterflies (Nachtigall, 1967) and some dragonflies (May, 1976) have not been measured in the laboratory, where flight metabolism has largely been analysed in relatively small respirometers under conditions such that gliding flight is not possible. Free hovering flight, which is most expensive, has been measured, however, and these results probably represent maximal values for these species. Metabolic rate during flight may be influenced by ambient temperature, load, and flight speed. These factors will be discussed in the following sections.
2.1
AMBIENT TEMPERATURE
The maximum rate at which a muscle can transform chemical energy into mechanical work depends on muscle temperature. If ambient temperature determines the thoracic temperature, then the ambient temperature will strongly influence the metabolic rate. If an insect can regulate its temperature behaviorally or physiologically, then its metabolic rate will be relatively independent of ambient temperature. In a number of species of sphinx moths (Heinrich, 1971a; Heinrich and Casey, 1973; Casey, 1976a), bumblebees (Kammer and Heinrich, 1974; Heinrich, 1975) and syrphid flies (Heinrich and Pantle, 1975), muscle temperature is stabilized during flight over a relatively wide range of ambient temperature, and metabolic rate remains constant (Fig. 1). In these large insects, then, metabolic rate during flight is independent of ambient temperature, but energy is expended prior to flight to raise the thoracic temperature to a level at which flight is possible (section 5). The locust Schistocercu greguriu does not regulate its temperature during flight, at least during tethered flight (Weis-Fogh, 1956a). One might therefore expect that in this insect metabolic rate would be influenced by ambient
138
ANN E. KAMMER AND BERND HEINRICH
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