Biosynthesis in Insects

Biosynthesis in Insects

Biosynthesis in Insects

E. David Morgan Chemical Ecology Group, Keele University, UK

RSeC advancing the chemical sciences

ISBN 0-85404-691-7

A catalogue record for this book is available from the British Library

0The Royal Society of Chemistry 2004 All rights reserved Apart from any fair dealing for the purpose of research or private study, or criticism or review as permitted under the terms of the UK Copyright, Designs and Patents Act, 1988, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page, Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org Typeset by Refinecatch Ltd, Bungay, Suffolk, UK Printed and bound by TJ International Ltd., Padstow, Cornwall, UK

Preface “It is from the behaviour of simple molecules that we learn our most sign$cant lessons. Sir Frederick Gowland Hopkins, 1913

This book arose out of a course of lectures I was invited to give to young post-graduate students at Universidade Federal de Alagoas, Maceio, Brazil in 1999. Students working on practical aspects of insect pest control through the use of natural pesticides, pheromones and hormones need to know something about the origin of these substances in nature, if they are to use their skills fully in their work. There is not much information available to them in a clear and elementary form in the review literature or books, so I undertook to provide them with an introductory guide to biosynthetic pathways, with special emphasis on insects. I have subsequently expanded the notes prepared for them to make this small book, with the hope that it will prove useful and informative not only to them but to other students in science and technology, and perhaps attract more of them to this interesting and growing area of chemical ecology. Where biosynthetic processes differ between animals and plants or micro-organisms, I have concentrated on animal systems, but I have not confined myself strictly to the Insecta. The close interaction between plants and insects makes it necessary at times to look at plant substances and pathways, and I have, where important or interesting examples present themselves from other areas, strayed to other arthropods, and even man to complete the story. Because this is intended as a didactic work for young students, I have not used the apparatus of references as in a scholarly review. Experience has shown me that faced with many references to research literature, they may consult none of them. I have rather referred in the text to a few especially useful or interesting papers, and added a list of further reading at the end of each chapter. I have also included a few problems so that readers can make a self-assessment of their grasp of what has been discussed.

V

Acknowledgements I am grateful to my friends Neil Oldham, John Brand, Ralph Howard, Athula Attygalle, Graeme Jones and Desire Daloze for reading various parts or drafts and giving me many helpful suggestions, and to Jane Parker for giving me a student view on an early draft. The request for the original lectures came from my former student Ruth do Nascimento, to whom I am much indebted. I thank the British Council office in Recife, and CNPQ for the financial support that enabled me to give the lectures, and Professor Ant6nio Euzebio G. Sant’Ana for his hospitality. I am very grateful to Dr John Shanklin of Brookhaven National Laboratory and Dr Ed Cahoon of the Danforth Plant Sciences Center for kindly providing the diagrams of castor oil desaturase, Prof John Mann and Dr Rishuo Nishida for permission to use figures from their work. I am obliged to Dr Jonathan Banks in Australia and Dr Keith S. Brown in Brazil for helping me concerning the final position of aphinin research. Those of us laboratory-bound do not get to see a great variety of insects in their natural surroundings. I can happily thank various friends for contributing their photographs of (biosynthetically) interesting insects. Stefan0 Turillazzi, Universita di Firenze, for his picture of Polistes wasps, Athulla Attygalle and Maria Eisner for Epilachnis pupae, Steve McWilliam of rECOrd, Chester, for Coccinella septempunctata adults and Jim Klaisch of the Department of Entomology, University of Nebraska - Lincoln for the C. septempunctata larva, Dr Mike Quinn, Texas A&M University, Stephenville, for superb pictures of Hippodamia, Oncopeltus and Danaus, Tom Larsen for Scolopendra, Jens Christian Schou for Arctia caja, Warren E. Savary for Lytta magister, Markku Savela for Harpaphe haydeniana, Dr Hamish Robertson of the South African Museum for Ceroplastes, Dr Paul Choate, Department of Entomology and Nematology, University of Florida for Manduca sexta larva, Socikte Nouvelle des Editions Boubee for permission to reproduce the photo of a female silk moth taken by Jacques Six, Anthony Papadoupolos for the fire bug Pyrrhcorus apterus, the University of Oklahoma Veterinary School for Amblyomma americanum, and our own Terry Bolam for the Myrmica ant trail-following.The cover photo is one vii

...

Vlll

Acknowledgements

of the many beautiful insect photos taken by Ken Preston-Mafham of Premaphotos Wildlife. I have tried to find the owner of the picture of the cheese mite, Tyrophagus putrescentiae without success. I have sought to locate owners of all reproduced material not in my own possession. In a few cases I have been completely unsuccessful, but trust I have not inadvertently infringed any copyrights. Should I have done so I shall of course take appropriate action for any subsequent editions. I would also welcome comments and suggestions on this book. ([email protected])

Contents Chapter 1 Introduction 1.1 The Structures of Natural Products 1.2 Compounds and Function 1.3 Studying Biosynthetic Pathways 1.4 Plant Versus Insect Biosynthesis 1.5 Arthropods and Insects Background and Further Reading Questions Chapter 2 Enzymes and Coenzymes 2.1 The Chemical Reactivity of Enzymes 2.1.1 Lysozyme 2.1.2 Carboxypeptidase 2.1.3 Cytochromes 2.2 Coenzymes 2.2.1 Coenzyme A 2.2.2 Nicotinamide Adenine Dinucleotide 2.2.3 Flavin Adenine Dinucleotide 2.2.4 Thiamine Diphosphate 2.2.5 TetrahydrofolicAcid 2.2.6 S-Adenosylmethionine 2.2.7 Pyridoxal Phosphate 2.2.8 Vitamins 2.2.9 Biosynthesis of Formic Acid in Ants 2.3 Pyruvic Acid 2.4 Chirality 2.4.1 Asymmetric Induction Background and Further Reading Questions

10 10 10 12 13 13 14 15 14 17 19 19 19 21 21 22 23 24 26 26

Chapter 3 Fatty Acids and Derived Compounds 3.1 Fatty Acids 3.1.1 Biosynthesis

28

ix

28 29

Contents

X

3.1.2 Unsaturated Acids and Desaturase Enzymes 3.1.3 Eicosanoids 3.1.4 Branched Fatty Acids 3.2 Cuticular Hydrocarbons 3.2.1 Hydrocarbon Pheromones 3.3 Lepidopteran Sex Pheromones 3.4 Coleoptera 3.4.1 Coccinellines 3.4.2 Epilachnine 3.5 Cockroaches 3.6 Termites 3.7 Honeybees 3.8 Ants 3.9 Spiders 3. I0 Hemiptera 3.10.1 Green Leaf Volatiles 3.1 1 Lactones Background and Further Reading Questions

32 35 36 37 40 41 45 46 47 48 49 50 51 52 53 53 53 55 55

Chapter 4 Polyketides and Acetogenins 4.1 Acetogenins 4.2 Polyketide Derivatives 4.3 Volatile Pheromones 4.3.1 Cyclic Ketals 4.4 Defensive Secretions Background and Further Reading Questions

57

Chapter 5 Experimental Methods 5.1 Tracing Biosynthetic Pathways 5.1.1 Specific Incorporation 5.1.2 Locating the Site of Synthesis 5.2 Radio-isotope Labelling 5.2.1 Examples 5.3 Heavy Isotope Labelling 5.3.1 Examples 5.3.2 Carpophilus Beetle Pheromone 5.3.3 l3C-I3cCoupling 5.4 Isotope Effects 5.4.1 Kinetic Isotope Effects

69 69 70 71 72 73 75 75 80 80 81 81

57 51 61 65 66 67 67

Contents

xi

5.5 Analytical Aspects 5.6 Chirality Background and Further Reading Questions

82 82 83 84

Chapter 6 Terpenes 6.1 Monoterpene Biosynthesis 6.1.1 The Methylerythritol Phosphate Pathway 6.2 Monoterpene Pheromones 6.3 Monoterpene Defensive Compounds 6.3.1 lridoids 6.3.2 Degraded Terpenes 6.4 Sesquiterpenes 6.4.1 Sesquiterpene Pheromones 6.4.2 Cantharidin 6.4.3 Lac Insects 6.5 Homosesquiterpenes 6.6 Juvenile Hormone Background and Further Reading Questions

85

85 87 89 91 91 94 94 96 97 98 99 101 102 103

Chapter 7 Higher Terpenes and Steroids 7.1 Diterpenes 7.1.1 Termites 7.2 Sesterterpenes 7.3 Triterpenes and Steroids 7.3.1 Sterols in Insects 7.3.2 Saponins from Triterpenes 7.4 Insect Moulting Hormone - Ecdysteroids 7.5 Tetraterpenes Background and Further Reading Questions

104 104 105 107 108 111 113 114 116 119 119

Chapter 8 Aromatic Compounds 8.1 Aromatic Compounds in Nature 8.2 The Shikimic Acid Pathway 8.3 Phenyl-C, Compounds 8.3.1 Aromatic Pheromones 8.3.2 Compounds from Chorismic Acid 8.4 Aromatic Amines 8.4.1 Adrenaline Group 8.4.2 Serotonin Group

121 121 121 123 123 125 127 127 128

xii

Contents

8.5 Phenols 8.6 Quinones 8.7 Insect Pigments 8.7.1 Melanin 8.7.2 Quinones 8.7.3 Aphins 8.7.4 Pterins 8.7.5 Tetrapyrroles 8.7.6 Ommochromes and Ommins Background and Further Reading Questions

129 130 132 132 133 134 136 138 139 140 141

Chapter 9 Alkaloids and Substances of Mixed Biosynthetic Origin 9.1 Alkaloids 9.1.1 Alkaloid Precursors 9.1.2 Plant Alkaloid Biosynthesis 9.1.3 Insect Alkaloids 9.1.4 Other Examples 9.1.5 Alkylpyrazines 9.2 Compounds of Mixed Biosynthetic Origin 9.2.1 Luciferin 9.2.2 Volicitin Background and Further Reading Questions

143 143 144 145 145 151 152 154 157 158 159 159

Chapter 10 Plant Substances Stored, Changed or Unchanged, by Insects 10.1 Toxic Plant Substances in Insects 10.1.1 Cardiac Glycosides 10.1.2 Veratrum Alkaloids 10.1.3 Pyrrolizidine Alkaloids 10.1.4 Cyanogenic Glucosides 10.1.5 Glucosinolates 10.1.6 Coniferyl Alcohol 10.1.7 Other Types 10.1.8 A Parting Thought Background and Further Reading Questions The Bonus Question

161 161 162 163 164 166 169 171 171 174 175 175 176

Contents

...

Xlll

Answers to Questions Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 The Bonus Question

177 177 177 178 178 179 180 181 182 183 184 184

Appendix - Common Abbreviations Subject Index

185

187

CHAPTER 1

Introduction The materials in living plants and animals are divided by scientists into two groups: primary and secondary metabolites. Primary metabolites are the substances fundamental to all living matter: simple sugars, aminoacids, nucleotides and fatty acids, etc. Secondary metabolites are substances made by one or a group of species which are not generally vital to the life of the organism. Secondary metabolites may be structural materials, such as bone, chitin or hair, antibacterial or antifungal compounds, they may give protection from predators or foragers, they may be signalling substances (hormones or pheromones) or they may have, as yet, no known function in that organism. The range of secondary metabolites is enormous and presents a never-ending source of research and exploration. What is equally surprising is that this great array of substances are made from relatively few basic building blocks. Figure 1.1 attempts to summarize, very briefly, the way in which all these types of compounds found in nature are made. Notice that the carbon atoms of all substances, from plant or animal, are ultimately derived from carbon dioxide via photosynthesis. The figure shows that many groups of compounds are formed via relatively few biosynthetic paths. Biosynthesis is the building up of chemical compounds through the physiological processes that take place in living animals, plants and micro-organisms. There are by some estimates about one million insect species. They have colonized almost the entire terrestrial world, and are very varied in habitat and behaviour. They share some biochemical characteristics with all living organisms, others with all animals, but others are peculiar to insects alone, or to a few species or even a single caste of a single species. In the words of Jerrold Meinwald and Thomas Eisner, pioneers in insect chemical ecology, “The ability to synthesize or acquire an extremely diverse array of compounds for defence, ofence and communication appears to have contributed significantly to the dominant position that insects and other arthropods have attained. ” The kind of compounds the insects produce are therefore a challenge to our ability to understand their structures 1

2

Chapter 1

and functions. The groups of compounds that are of special interest to us in the study of insects are indicated in boxes in Figure 1.1. The great diversity of secondary metabolites indicated in Figure 1.1 are often spoken of by chemists as natural products. They are varied in their chemical structure, but they are all made by one of these few biosynthetic pathways (in some cases, a combination of more than one of them). By understanding their biosynthetic origins one can make some sense of this great diversity of natural products and group them according to their origin. Moreover, as we come to understand better these biosynthetic mechanisms, we gain greater insight into how we might regulate such reactions in pest species, as well as understanding how these pathways evolved. The general principles are considered first in each case and then their application to insects is discussed. In some cases the principles are discussed first in relation to micro-organisms or plants, because that is where they were first studied or where more is known of them. It should also impress upon the reader the unity and diversity of biosynthetic products.

1.1 THE STRUCTURES OF NATURAL PRODUCTS Knowing the probable biosynthetic origin of a new compound can help to decide what is its likely structure, and what is an improbable structure, and help us to arrive at its structural formula. It can be difficult to rule out a possible structure completely, because nature is full of surprises. This book should help the reader to decide which among some alternative structural possibilities is the more likely. In Figure 1.2, the compounds on the left are insect pheromones where the likely biosynthetic origin can be easily deduced from the structures, while it is very difficult to see how those on the right can be made by the routes we know, but both the structures on the right have been found in at least one insect. When structures like those on the right are proposed, it is particularly important to show that they are correct by synthesizing the structure proposed and comparing it with the natural compound.

1.2 COMPOUNDS AND FUNCTION Many of the compounds from insects considered here are pheromones (Greek, phero = carry or convey), defensive or offensive substances (allomones, Greek, allos = other), or hormones (Greek, hormao = excite or impel). Pheromones can be considered as chemical communication between individuals of the species, while hormones are chemical communication within the individual. In evolutionary terms, it has been

In troduet ion CO2

+

3 hv

H20

GLUCOSE

(plus other 4 , 5 , 6 and 7 carbon sugars)

PHOTOSYNTHESIS Nucleic acids Polysaccharides Glycosides H

S

o Etythrose 4-phosphate Phosphoenol pyruvate

p o H 'OH

\ Shikimic acid

Aromatic amino-acids Aliphatic amino-acids CH~COSCOA

( in plants )

Acetyl coenzyme A

Citric acid cycle

C02

+

H20

+

+ co:!

glyceraldehyde 3-phosphate

ENERGY

+

Fatty acids

\ Acetogenins Hydrocarbons Hormones Pheromones Eicosanoids

CH~COSCOA

Steroids

CH~COCH~COSCOA Acetoacetyl coenzyme A

+ CH~COSCOA

Mevalonic acid

Figure 1.1 A summary of the chief biosynthetic routes to primary and secondary metabolites. Those ofparticular interest here are enclosed in boxes. Notice that glucose, phosphoenol pyruvate and erythrose-4-phosphate are the key intermediates for all these classes of compounds. P indicates a phosphate ester (Adapted from a figure in J. Mann Chemical Aspects of Biosynthesis 1994, by permission of Oxford University Press)

Chapter I

4

rather than

acoocH OH

a trail pheromone of ants

an alarm pheromone of ants

Figure 1.2 On the left are two structures with simple biosynthetic origins, while on the right are two for which a simple biosynthetic route cannot be given

suggested that pheromones were among the first communication chemicals affecting animal behaviour, and the pheromones of primitive singlecell organisms may have evolved into the hormones of multi-cellular animals. On the other hand, the types of compounds used as pheromones and allomones are so varied, they appear to have evolved many times, while the hormones are relatively conserved, and the same hormones serve many or all insects and can be common to many invertebrate classes. Chemicals for communication (semiochemicals, Greek semeion = sign or signal) between species and between plants and animals are called collectively allelochemicals, and are further sub-divided in a system depending upon whether they benefit the sender (allomones, as above), receiver (kairomones), or both (synomones), and other categories. Pheromones are the group of insect compounds that have found greatest application in agriculture and forestry. For example, a large number of lepidopteran species are important agricultural pests. They use sexual pheromones to attract males for mating. The pheromones can be used to aid control of pests in one of several ways. Traps baited with synthetic pheromone can be used to detect the arrival of a pest, or to assess the build-up of the species in a crop, so that insecticides can be used more sparingly and at the correct time. In a few cases, trapping alone can be effective in removing enough of the males to control the pest. Sometimes the pheromone is scattered throughout the crop so that males are unable to locate females (mating disruption). Sometimes a wrong isomer can completely inhibit the response to a pheromone, so a lure containing some of the inhibitor can disrupt mating. Both Coleoptera and Lepidoptera can be pests in forestry, and there too, pheromone traps have been found effective. Pests in stored products are particularly suitable for pheromone trapping, where use of insecticides is undesirable. Sales of pheromones world-wide still represent only a few percent of the total value of sales of insecticides, which are of the order of billions of US dollars, but pheromones sales are steadily growing.

Introduction

5

Insect defensive compounds are usually effective at short distance and their toxicity or repellency is not sufficient for them to have found any industrial application. Venoms can be powerful, but usually require injection. Of the insect hormones, ecdysteroids (Chapter 7) have not yet found practical application, but there are several examples, in special circumstances, of very effective use of juvenile hormone mimics. 1.3 STUDYING BIOSYNTHETIC PATHWAYS When considering the formation of naturally occurring substances, whether simple amino-acids, sugars, or complex proteins, alkaloids, polyketides, terpenes or steroids, it should be remembered that all the reactions involved follow the normal laws of chemistry. One of the fascinating areas of chemistry today is trying to understand how these biosynthetic reactions occur. How it is that reactions we find extremely difficult in the laboratory are accomplished efficiently and quickly at room temperature and near neutral pH inside cells? What kinds of organic chemical reactions can be used in living cells? The immediate answer to these questions is that nature has evolved efficient catalysts called enzymes that lower the energy of activation for these reactions, to make them proceed much more quickly. Enzymes became active catalysts through repeated accidental, evolutionary changes over time. Whatever the apparent “magic” effect of enzymes, the reactions must still obey the laws of thermodynamics, the reaction will still be explicable in terms of electron push and pull, of bond and charge movements, just as in the rest of organic chemistry. Enzymes cannot make reactions go forward if the energetics are unfavourable to formation of the product. To give a fuller explanation it is necessary to consider the nature and function of enzymes and some of the co-enzymes that often function with them. Emil Fischer, at the beginning of the 20th century had two enzymes called invertin and emulsin. Invertin hydrolysed only a-D-glucosides (sucrose is an example) while emulsin hydrolysed P-D-glucosides. From this and his knowledge of sugars he correctly deduced that these enzymes are asymetrically constructed molecules; in modern terms, they are chiral. Biochemical reactions take place on the chiral surface of an enzyme (Chapter 2), which makes an important distinction from solution chemistry. The first enzyme obtained in a pure, crystalline state was urease, in 1926. It soon became clear that it and other enzymes were proteins. The energetics and kinetics of these enzymic reactions are important to the biochemist, but are not essential to our understanding of what kinds of compounds are produced by insects.We should, however, bear in mind that these systems are not static. Schoenhemierand Rittenberg showed in 1936

6

Chapter I

that when an animal was allowed to drink heavy water (D,O) for a few days, its fatty acids became labelled with deuterium. When normal water then replaced heavy water, the dueterium disappeared from the fatty acids, showing that cells, and whole animals, are in a state of dynamic equilibrium. The kinds of organic chemical reactions that take place in living systems can be divided into five simple types, which are illustrated in Figure 1.3. Enzymes are known which catalyze all these types of reactions, but there may be several ways in which the reaction is catalyzed, particularly so in oxidation-reduction and hydrogenation-dehydrogenation reactions. a. Substitution

R-X

+

b. Addition

R-CH=CH-R H

R

c. Elimination

R++R R

X

d. Rearrangement

R-Y +

Y--

R' R+CH,-OH R

R

+

XY

-

xX R R w R R Y R +HX

- RmR R

R R+CH,-RI

R

OH

or

e. Oxidation-reduction

Figure 1.3 A summary of the types of organic biochemical reactions

1.4 PLANT VERSUS INSECT BIOSYNTHESIS Plants have the ability to make a much greater diversity of compounds than animals can show. Generations of natural product chemists have devoted their skills to solving the structures of plant compounds. For example, there are about 15,000 known terpenes (Chapter 6) made by plants. Above all, plants can use photosynthesis, splitting water in the light reaction (see Figures 1.4 and 5.6) and in the dark reaction creating carbohydrates from carbon dioxide and hydrogen. Plants (and microorganisms) have exclusive access to the shikimic acid pathway (Chapter 8), and the aromatic amino-acids, and to the methylerythritol pathway to terpenes. The case of the polyunsaturated acids (Chapter 3) may be unclear, since at least three insects have been shown able to make linoleic acid, but linolenic acid remains from plants only. The sterols (Chapter 7) can be made by plants and higher animals, but not by insects. The formation of carotenes (Chapter 7) by insects is doubtful. Compounds such as

7

Introduction Light reaction: H20

+

NADP+

ADP

+

H2P04-

hv

NADPH

+

H+

+

i02

ATP

Dark reaction:

Figure 1.4 A summary of the reactions of photosynthesis in green plants

chlorophyll, starch, cellulose, lignin, tannins, anthocyanins, flavones and triterpenes belong only to plants. On the other hand, all the biosynthetic methods, in their broad sense, used by insects, discussed in this book, are available to plants. That is, the formation of fatty acids and their derivatives, such as hydrocarbons; the acetogenins; and especially the terpenes and aromatic compounds are all used by plants. Acetogenins are not as prominent among plant products as the others, except in the formation of anthocyanins and flavones. Only special areas are left to insects alone. It is surprising, as more information accumulates, how insects and plants seem often to have found similar or the same way to biosynthesize certain compounds. Some authors call this parallel evolution. Plants and insects have been evolving together for about 300 million years. In that time plants have produced both physical (hairs, spines and thick waxy surfaces) and chemical (stinging trichomes, alkaloids, toxins and feeding deterrents) defences against insects, while insects have been evolving ways to overcome them. An interesting example of plant counter-attack are the phytoecdysteroids made by plants, which mimic the natural moulting hormone of insects and are stored in the leaves to disrupt normal development of the insect feeding on them (Chapter 7). There are plant anti-juvenile hormone compounds too. Nevertheless, there is probably not a single plant species without at least one insect that has found a way to overcome its defences. 1.5

ARTHROPODS AND INSECTS

The arthropods were the first organisms to emerge from the sea, and insects were the first invertebrates to fly. The arthropods consist of Crustacea (crabs, lobsters, shrimp, barnacles and woodlice), Chelicerata (spiders, ticks, mites, scorpions and others), Hexapoda or Insecta, and Myriapoda (millipedes, centipedes and other minor groups). These classes separated a long time ago, so they have developed quite differently, but it is interesting to discover parallel developments.

8

Chapter I

Spiders and millipedes have sometimes developed chemical defences or communication chemicals similar to those of insects. It is therefore useful occasionally to make comparisons. The insects are the largest single group of animals, with over 800,000 identified species, far more than all the other animals put together. New species are reported at the rate of about 5,000 per year, and total number estimates range from 1 to 10 million. It is estimated there are lo'* individuals alive at any time. They are divided into the Apterygota, primitive wingless insects (springtails and silverfish) which have as yet received little chemical study; and the Pterygota, or winged insects, which form the great majority. The latter in turn are divided into the Exopterygota or Hemimetabola, which hatch from eggs to nymphs which closely resemble their final adult form or imago (grasshoppers, cockroaches, termites, bugs, stick insects, etc.) (Figure 1S); and Endopterygota or Holometabola, which hatch from egg to larvae which may have a very different form and habitat from the adult. They then go into a resting form called the pupa, while the tissues are completely remodelled and from that emerges the adult form (Figure 14. The Holometabola include beetles, butterflies and moths, flies, fleas, bees, wasps and ants. Almost half of all the insect species are beetles. Potentially, the subject of this book is gigantic. The isolation of insect chemicals began slowly. Kermesic acid or venetian red, a pigment from beetles (Chapter 8) has been known and used from ancient times. Wray, in 1670, reported formic acid by distillation of formicine ants. It was not until the 1930s that it began to be recognized that some Lepidoptera males were chemically attracted to females, and

Figure 1.5 Representation of the lije cycles of a hemimetabolous and a holometabolous insect. The symbols J H and M H between stages indicate where the juvenile hormone (Chapter 6 ) and moulting hormone (Chapter 7), which regulate development, are produced Reprinted from ComprehensiveNatural Products Chemistry, Vol. 8, E. D. Morgan and I. D. Wilson. Insect hormones and insect chemical ecology, pp. 263-375. Copyright 1999, with permission from Elsevier.

Introduction

9

only in 1956 was the first sexual attractant (bombykol, from the silk moth Bombyx mori) isolated and identified. From that time onward, with the development of chromatographic and sensitive mass spectrometric techniques, the study of insect natural products has grown to be a major discipline of science.

BACKGROUND AND FURTHER READING

J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993 (Chapters 5 & 8, plants and insects). E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 6, secondary metabolites). H. Oldroyd, Insects and Their World, British Museum of Natural History, London, 1966, pp. 144 (general introduction to insects, out of print) . P. Howse, I. Stevens and 0. Jones, Insect Pheromones and Their Use in Pest Management, Chapman and Hall, London, 1998, pp. 369 (Chapters 1 , 2 & 3, introduction to pheromones). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 7). H. H. Rees, Insect Biochemistry, Chapman and Hall, London, 1977,pp. 64. V. H. Resh and R. T. Card6 (Editors), Encyclopedia of Insects, Academic Press, San Diego, 2003, pp. 1266 (for reference at any point). K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapters 1 & 2) T. D. Wyatt, Pheromones and Animal Behaviour, Cambridge University Press, 2003, pp. 391 (Chapter 1 & 2). A. Zanetti, The World of Insects, Abeville Press, New York, 1985, pp. 256 (general introduction to insects, out of print). QUESTIONS 1. An ant has hydrocarbons on its surface cuticle to repel water and prevent desiccation. The ant feeds largely on honeydew secreted by aphids, which feed on a plant stem. With the aid of Figure 1.1, trace the source of the ant hydrocarbons from carbon dioxide. 2. A bee expends energy in flying from flower to flower collecting nectar. What, according to Figure 1.1 is the ultimate source of that energy? 3. Classify the following substances as primary or secondary metabolites: vitamin A, alanine, camphor, deoxyribose, glucose, penicillin. 4. When a flower produces a perfume to attract pollinating bees, is that an allomone, kairomone or synomone?

CHAPTER 2

Enzymes and Co-enzymes 2.1 THE CHEMICAL REACTIVITY OF ENZYMES An enzyme contains one or more active sites, at which the reaction occurs. The substrate, the substance that is being altered, becomes attached to this site in some way. A co-enzyme, if one is involved, is also attached to, or held close to the active site. “An enzyme first binds its substrate in a particular orientation by using a variety of weak binding forces (hydrogen bonding, electrostatic attraction, dipole-dipole interaction, hydrophobic attraction, and so on), and then uses a variety of strategically placed functional groups and controlled conformational changes to induce reaction between them.” J. W. Cornforth, 1984, see Further Reading

Cornforth has done much of the work in understanding the stereochemistry of many biosynthetic reactions and was awarded the Nobel Prize for Chemistry (with V. Prelog) for this in 1975. For the biochemistry of enzymes the reader is directed to T. Palmer, Understanding enzymes 3rd edtion, 1991, Ellis Horwood, Chichester, and for a detailed treatment of enzyme and co-enzyme reaction mechanisms, T. Bugg, An introduction to enzyme and co-enzyme chemistry 1997, Blackwell Scientific, Oxford. 2.1.1 Lysozyme

Lysozyme has often been chosen as a simple example of how an enzyme works. It is said that Alexander Fleming (who later discovered penicillin) when he had a cold, at one time let the drips from his nose fall onto a bacterial colony on a Petri dish. Rather than throw it away, he kept it to see what would happen. He discovered that his nasal discharge inhibited the growth of the bacterium and this led to the discovery of the mildly antibiotic substance lysozyme in tears. He gave it this name because it is 10

Enzymes and Co-enzymes

11

an enzyme that caused bacterial Zysis. Later lysozyme was found in other body fluids, and elsewhere, but particularly in the white of egg. Lysozyme acts on a group of bacteria that have a cross-linked polysaccharide on their cell surfaces. Lysozyme cuts up the polysaccharide, making the bacterial cell wall very fragile. Lysozyme has a relatively small molecule for a protein, with 129 amino-acid residues linked in a single protein chain, and a molecular mass of 13,930. It was the first enzyme to have its total structure determined by X-ray analysis (1965), and to have its active site discovered. The protein chain of lysozyme is twisted and folded into a shape like a ball with a cleft down one side. The polysaccharide network on the surface of the bacteria fits into the cleft. The structure of the polysaccharide (the substrate) is shown in Figure 2.1 and the schematic structure of the enzyme molecule is shown in Figure 2.2 with the polysaccharide adsorbed on to the active site ready to be cleaved. Six rings of the

HOOC

HOOC

Figure 2.1

The polysaccharide molecule found in the walls of certain bacterial cells is the substrate broken by the lysozyme molecule. The polysaccharide consists of alternating residues of two kinds of amino sugar N-acetylglucosamine and N-acetylmuramic acid. In the portion of polysaccharide chain shown here A, C and E are N-acetylglucosamine residues; B, D and F are N-acetylmuramic acid residues. Ring D is distorted when adsorbed on the enzyme. The position attacked is indicated by the arrows

Figure 2.2

The amino-acid chain of lysozyme ( a ) is folded so it roughly forms a ball, with a cleft down one side, into which the polysaccharide chain of the bacterium fits. In ( b ) is shown how the aspartic acid 52 and glutamic acid 35 work together to break the sugar chain

12

Chapter 2

polysaccharide fit into the cleft of the lysozyme molecule, and are held firmly in position by hydrogen bonds and other interactions (see the quotation from Cornforth, above). Ring D is held in a flattened conformation, so the bond to ring E is strained and prepared for reaction. Adjacent to it are two carboxylic acid groups; that of aspartic acid 52 is in polar surroundings and is in the ionized form; that of glutamic acid 35 is in non-polar surroundings and is therefore in the un-ionized form. These two groups and a water molecule complete the reaction as shown in Figure 2.2.

2.1.2 Carboxypeptidase In the example of lysozyme, the catalytic effect is entirely due to the protein. In many enzymes there is a prosthetic group that often contains a metal ion bound to the protein, as in the example of carboxypeptidase, which contains a zinc atom in the active site (Figure 2.3), that takes part in the reaction. There are at least four carboxypeptidases which differ in the particular amino-acid linkages they are able

__--_

Figure 2.3 The carboxylic acid end of a protein sitting in the active site, a pocket in the enzyme carboxypeptidase, showing how it is held in place by bonding to the Zn2+atom and various amino acids. The probable mechanism, based on the hydrolysis of a known peptide, is shown, with the water molecule used for the hydrolysis shown in bold type. Once the amino-acid is cleaved, it can difuse away and the protein moves up into the pocket for the next aminoacid to be cleaved in the same way

Enzymes and Co-enzymes

13

to hydrolyze. Carboxypeptidase hydrolyzes amino-acids from the carboxyl end of proteins, one by one. Some 400 enzymes are known that contain zinc atoms. Other common metals in enzymes are Fe, Mg, Ca and Mn. A more advanced picture of enzyme action in biosynthesis is given in Chapter 3 when discussing desaturase enzymes.

2.1.3 Cytochromes There is in all living cells a family of enzymes known as cytochrome P,,, oxygenases. They contain a haem group (Figure 2.4) attached covalently, and have an absorption maximum at 450 nm in the violet region of the spectrum, and are coloured yellow, hence their names. They are important in the oxidizing of alkanes to alcohols, alkenes to epoxides, in the transformation of sterols (see Chapter 7) and for introducing an OH group into aromatic rings. They are also important for the detoxifying of many ingested substances, whether they be from plants or animals, or are synthetic substances, like pesticides or environmental pollutants. Cytochromes activate molecular oxygen, which attacks the substrate of whatever kind, as in Figure 2.5. Notice that the Fe3+atom must be reoxidized to Fe4+before the enzyme can be used again. It accepts another molecule of 02, splits off OH- and is restored to Fe4+-O-for re-use. Other substances that contain haem are haemoglobin, other cytochromes and catalase.

HOOC

Figure 2.4

-COOH

The structure of haem. The tetrapyrrole without the iron atom is known as protoporphyrin I X (see also Chapter 8 )

2.2 COENZYMES Working with the enzyme is frequently a co-factor, called a co-enzyme, a relatively small (compared with the enzyme) organic molecule which may itself be reversibly changed during the reaction. Remember that there are only five basic types of chemical reaction (see Figure 1.3) and all these types of reaction are encountered with enzymes and coenzymes.

Chapter 2

14 .Yenzyme

Efenzyrne

I

0’)

RLH

The overall reaction is: R-H

+ O2 i-2H+ +

-

2e-

R-0-H

R-OH

+

H20

Figure 2.5 Schematic drawing of the centre of a haem group of a cytochrome enzyme catalysing an oxidation of an organic molecule. This is a free radical reaction, as indicated by the arrows with a single barb

2.2.1 Coenzyme A Coenzyme A can be described as a “handle” for carboxylic acid groups. It picks up and drops acetyl groups, or acyl groups in general. It is particularly important in the degradation of fatty acids and in the first stages of terpene synthesis (Chapter 6). Its structure is shown in Figure 2.6. The essential part of the molecule is the thiol group, so it is usually represented as CoA-SH. The thiol reacts with, e.g., acetic acid, to give a thioester, coenzyme A thioacetate, or briefly CH,COS-CoA or AcS-CoA. Why a thioester? Esters are much more common in organic chemistry, but esters are less reactive than thioesters. Aldehydes and ketones have relatively reactive carbonyl groups, with reactivity slowed chiefly by bulky R groups. In esters, reactivity is decreased by the OR group through orbital overlap. There is no orbital overlap with the larger sulphur atom, so that reactivity of the thioester is more like that of a ketone. Notice the large increase in the acidity constant for the removal of an a-proton from a thioacetate compared with an acetate in Figure 2.7. The importance of this will become clear when considering the biosynthesis of fatty acids (Chapter 3) and terpenes (Chapter 6). pantothenic acid A

mercaptoethylamine

adenine

p-alanine

diphosphate

0I OH 0-7-0’

o’

ribose 3-phosphate

acetyl-CoA R-C, IP 0-H

Figure 2.6

+

H-S-COA

+

synthetase

~

R-c/t

+

H20

S-COA

Coenzyme A , showing its constituent parts, and the reaction between coenzyme A and an acid

Enzymes and Co-enzymes

15

2.2.2 Nicotinamide Adenine Dinucleotide Nicotinamide adenine dinucleotide (NAD' and NADP') are the reagents for alcohol to aldehyde, ketone or carboxylic acid oxidation-reductions. The equivalent reagents in the laboratory would be KMnO, or Na,Cr,O, for oxidations and NaBH, or LiAlH, for reductions. The complexity of the molecule should not hide its essential reactive part, the nicotinamide portion (Figure 2.8). The rest of the molecule is a polar handle to orient it correctly in the enzyme active site.

H3C-t C 0-R H&-6:

0

Z

@

+

HzC-C, 0 /p 0-R

@

+

HzC-C,

0 /P

K, = 10-'O

S-R

S-R

Figure 2.7

3x

K,

There is a large increase in the ease of dissociation of an a-proton in a thioester compared with a normal ester, which is important for biosynthetic condensation reactions

QcoNH22

?W

-

0+P,-O' 0

Ho

H

H

CONH2

e-

H+ NADH or NADPH

O-(H or phosphate)

Figure 2.8 Nicotinamide adenine dinucleotide NAD' (or N A D P with an extra phosphate on C-2 of ribose) oxidized and reducedforms. The sphere in the right-hand structure represents the remainder of the molecule

The reaction of a general alcohol with NAD' is illustrated in Figure 2.9. Note that when the alcohol is held on the enzyme surface it is possible to distinguish between the pro-chiral hydrogen atoms (for an explanation see the section on chirality at the end of this chapter). We know that the pro-R hydrogen atom is removed, and becomes attached to the upper side (as drawn) of the reduced nicotinamide molecule. When NADH is used for reduction, the same hydrogen, from the upper side of the molecule is transferred to the back of the carbonyl group (as drawn in Figure 2.9).

Chapter 2

16

The overall reaction is: RCH20H

Figure 2.9

+

-

alcohol dehydrogenase enzyme R-CHO

NAD+ Cf

+

NADH

+

H+ CI-

Oxidation of an alcohol with NAD+, and its reverse reaction, reduction of an aldehyde or ketone. B: represents some basic group on the enzyme. The subscripts R and S are a means of distinguishing between the two hydrogen atoms of the methylene group, see the section on chirality at the end of this chapter

This has been established by replacing either the pro-S or the pro-R hydrogen by deuterium and seeing whether the deuterium is retained by acetaldehyde or is taken up by NADH. NAD’ is used in metabolism or catabolism, for example, turning sugars into energy, NADP’ is used for anabolism or building up chemicals. Nature, in this way, keeps the two kinds of process separated. 2.2.3 Flavin Adenine Dinucleotide Flavin adenine dinucleotide (FAD) is also an oxidation-reduction reagent more specifically confined to reduction of C=C bonds and removing 2H from adjacent carbon atoms. The two hydrogen atoms added to the coenzyme and removed from the substrate are circled (Figure 2.10). FAD also oxidizes oxy-acids, amines and some aminoacids. The reduced form of the coenzyme has to be re-oxidized by molecular oxygen to FAD for use again. 0

FH2

YH‘OH

FAD

CH-OH

H2C-0,

,o\

FADH2

y.42

CH-OH

(ICJ

,o.

o ’ p \ o ~ o ~ ~ ~ oc w

OH

HO

-&A-I I

H H

The overall reaction is:

+

FAD

enzyme

‘c=c/ /

+ FADH~

\

Figure 2.10 Flavin adenine dinucleotide, FAD, oxidized and reducedforms. The two circled hydrogen atoms in FADH, are those removedfrom carbons

17

Enzyrn es and Co-enzyrn es

2.2.4 Thiamine Diphosphate Thiamine diphosphate is a particularly interesting substance because it illustrates a chemical evolution from lactic acid bacteria to yeasts and then to higher animals. It converts pyruvic acid, which is an a-keto-acid, into the equivalent of a 9-keto-acid (with C=N' instead of C=O), which can then lose CO, by decarboxylation. We know that the hydrogen atom next to nitrogen in the thiazole ring is acidic and easily removed because if we shake thiamine with D,O, we get rapid exchange of that atom (Figure 2.1 1). Some simple bacteria, such as those that produce yoghurt, reduce the pyruvic acid to lactic acid, and the reaction stops there, without much of

@ = phosphate

thiamine diphosphate

Figure 2.11 Thiamine diphosphate exchanges one labile hydrogen atom with D,O, indicating the reactive centre

0

+ OH

NADH

+

H+

YH C

H&'/,'9

0

+

NAD+

OH lactic acid

Figure 2.12 The reduction of pyruvic acid to lactic acid with the consumption of NADH formed earlier in the degradation of glucose

the energy of glucose being released (Figure 2.12). For each molecule of glucose that is broken down in metabolism, two molecules of NAD' are required (see Figure 2.20). Eventually two molecules of pyruvic acid are produced together with two molecules of NADH + H'. The NADH produced in catabolism of glucose is used by the lactic acid bacteria to reduce the pyruvic acid.

18

Chapter 2

+

c02

H+ H,

enzyme

H+

The overall reaction is: CH3COCOOH

-

CH3CHO + NADH

+

CH3CHO

H+-

+

C02 CH3CH20H

+ NAD+

Figure 2.13 The reaction of thiamine diphosphate with pyruvic acid in yeasts to release carbon dioxide and give acetaldehyde, and then ethanol. The structure marked A is used again in Figure 2.14

More advanced organisms, like yeasts, can use thiamine diphosphate with different enzymes to oxidize glucose to ethanol and carbon dioxide (Figure 2.13). The NADH produced in the glucose break-down is then used to reduce acetaldehyde to ethanol. Yeast is therefore used by the brewer for the alcohol produced and by the baker for the CO, to aerate the bread. Higher organisms have evolved a system to oxidize the pyruvic acid further, with a substance called lipoic acid as an intermediate, to acetic acid (as CoA thioester) and CO, (continuing the reaction from stage A in Figure 2.13). Approximately half the CO, we exhale is produced by decarboxylation with thiamine. The lipoic acid inserts itself after the decarboxylation step (Figure 2.14). The acetyl coenzyme A produced in the last step is either ultimately oxidized to CO, (through the citric acid or Krebs cycle) or it is the vital starting material for the biosynthesis of fatty acids, acetogenins and terpenes (Figure 1.1).

A

part of lipoic acid

tl

Figure 2.14 The$nal steps in the oxidation of pyruvic acid with thiamine diphosphate and lipoic acid to give acetyl coenzyme A

Enzymes and Co-enzymes

19

2.2.5 Tetrahydrofolic Acid The ultimate source of a one-carbon fragment is tetrahydrofolic acid, which obtains one carbon atom from formic acid, formaldehyde, or the amino-acids serine or glycine. The carbon atom from one of these sources becomes attached to N-5 of the folic acid and is reduced to methyl with the now familiar NADH (Figure 2.15).

tetrahydrofolic acid

Figure 2.15 The reduction of a one-carbon fragment attached to tetrahydrofolic acid to give the source of a methyl group. "C" represents one of several sources of a single carbon atom. This sequence of reactions can run in either direction, to give a methyl group from a formyl group, or to produce a formyl group from a methyl group as required

2.2.6 S-Adenosylmethionine The methyl group attached to N-5 of tetrahydrofolic acid becomes transferred to S-adenosylhomocysteine and the S-adenosylmethionine thus formed is the compound that transfers methyl groups (for methyl esters and ethers, and N-methyl groups) in nature (Figure 2.16).

S-adenosylhornocysteine

S-adenosylmethionine

S-adenosylhornocysteine

Figure 2.16 Reaction between 5-methyltetrahydrofolic acid and S-adenosylhomocysteine gives S-adenosylmethionine which can react with an alcohol, phenol, or carboxylic acid to give a methyl ether, a phenolic methyl ether or a methyl ester respectively, regenerating S-adenosylhomocysteine

2.2.7 Pyridoxal Phosphate Pyridoxal phosphate is the coenzyme that removes amine groups in metabolizing amino-acids (Figure 2.17). This is achieved by a series of reactions while the pyridoxal phosphate is bound to a transaminase

Chapter 2

20

The overall reaction is:

H

R-c-CoO-

+

pyridoxal

9

R-C-COOH

+

pyridoxamine

NH3+

Figure 2.17 The removal of ammonia from an amino-acid by pyridoxalphosphate ( P L P ) and u transaminuse enzyme. The PLP is held tightly to the enzyme by a lysine and ionic bonding of the phosphate group. B indicates some general base

enzyme by several interactions. Pyridoxal forms an imine with the amino-acid, and that is converted to an imine of pyridoxamine and a keto-acid. Hydrolysis of the imine gives an a-keto-acid and pyridoxamine which must be converted back to pyridoxal for re-use. In the metabolism of proteins the protein is first broken down to the individual aminoacids, which are de-aminated in this way. The carbon skeleton of the amino-acid (now as an a-keto-acid) is passed to the citric acid cycle (Figure 1.1) to be converted to energy. In fish the very toxic ammonia is usually excreted directly. In higher animals it is converted into harmless products, in insects uric acid is the important one. In mammals the amine group is transferred to the urea cycle and is excreted as urea. Pyridoxal is also used to move amino groups between amino-acids and, with different enzymes takes part in a number of other reactions involving amino-acids. An example of the steps by which an amino-acid

Enzymes and Co-enzy m es

21

Y

f

+ co2 l p

R-C N

H+

,

H+

0'

A

A

pyiiboxal

Figure 2.18 The sequence of steps by which an amino-acid attached to pyridoxal and a decarboxylating enzyme is converted to an amine and CO,

is decarboxylated are shown in Figure 2.18. Amines derived from aminoacids are discussed in Chapter 9.

2.2.8 Vitamins It should be noted in passing that higher animals have lost the ability to synthesize some of the coenzymes or parts of them. By definition, a vitamin is an essential substance that the body cannot make for itself and must acquire through its food. Humans cannot make pantothenic acid (vitamin B5)needed for coenzyme A, nicotinamide (vitamin B3) for NAD+,riboflavin (vitamin B2) for FAD, thiamine (vitamin BJ, folk acid (vitamin Bc or M) or pyridoxal (vitamin B6). We possess the ability to phosphorylate or otherwise convert the vitamins into active coenzymes. Some of these substances are also vitamins for insects, plus some others which higher animals can make themselves.

2.2.9 Biosynthesis of Formic Acid in Ants All ants of the subfamily Formicinae have lost the ability to sting but can spray a concentrated solution of formic acid (up to 65% in some species) from their venom glands. Blum and Hefetz (Science, 1978, 201, 545) studied the biosynthesis and showed by using radio-labelled compounds that the formic acid can be formed from serine (CH,OHCHNH,COOH) or glycine (CH2NH,COOH) with the help of tetrahydro folic acid (Figure 2.19). Apparently any compound capable of contributing a C , fragment can be a potential source of formic acid. The four enzymes necessary for these steps were all shown to be present in the venom gland. Other insects, including the larvae of the lepidopteran Schizura concinna and many beetles of the family Carabidae and some other arthropods also make and use formic acid in defensive or offensive secretions.

Chapter 2

22 H,

..-.C.

labelled serine

SOOH

M

M

' f E Y

I

ti

part of folate molecule

k

formic acid

Figure 2.19 Formation of formic acid in ants according to Blum and Hefetz. The black dot on carbon indicates the labelled atom which shows which atom of serine is used for formic acid

2.3 PYRUVIC ACID Pyruvic acid and its derivative phosphoenol pyruvate have already appeared in Figure 1.1, and pyruvic acid was required in discussion of the action of thiamine diphosphate (Figures 2.12 to 2.14). They are important intermediates and will appear again. It is worth looking briefly at the origins of these compounds now. When glucose is broken down in metabolism, the first steps are to convert glucose to glucose &phosphate, which is isomerized to fructose &phosphate and then converted to fructose 1,6-bisphosphate (Figure 2.20). This is cleaved by an aldol reaction running in reverse, and catalyzed by the enzyme aldolase. The products are dihydroxyacetone 1-phosphate and glyceraldehyde 3-phosphate, which are really equivalent compounds because they are interconverted through a common enol form. Glyceraldehyde 3-phosphate is phosphorylated again and oxidized to glyceric acid 1,3-bisphosphate, linked to the conversion of one molecule of NAD' to NADH. Glyceric acid bisphosphate loses one phosphate to ADP, forming ATP while the 3-phosphate is isomerized to 2-phosphate. Loss of water from this compound gives phosphoenol pyruvate and transfer of the phosphate to ADP gives another molecule of ATP, and conversion of enolpyruvate to the keto-form gives pyruvic acid. The summary in Figure 2.20 does not give all the stages, nor considers the mechanisms or the energetics of this important process. For that the reader is referred to a standard textbook of biochemistry. Under anaerobic conditions, the NADH produced during the oxidation of glyceraldehyde to glyceric acid is re-oxidized in the reduction of pyruvic acid to lactic acid (in bacteria), or in the reduction of acetaldehyde to ethanol (in yeast); and, under aerobic conditions, is oxidized (via

23

Enzymes and Co-enzymes

"

\

HO-CH2 Dihydroxyacetone phosphate

glucose 6-phosphate

H$-OH CH

fructose 1,6-bisphosphate phosphate

@ = phosphate

Q H,C-C-C,

,p

OH pyruvic acid

glyceric acid bisphosphate

-H20

c--

/ H H27-C-


H

.

O

-

0

0

CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)1BCOOH CH3 (CH,),CH=CH(CH2),COOH

palmytoylgroup

c-~ stearoyl group

-

Myristic acid Palmitic acid Stearic acid Oleic acid

oleyl group

C14:o C,6:o cj8:O c18:I

Figure 3.1 An example of a triglyceride or glycerol triester, (the structure shown is 1-palmitoyl-2-stearoyI-3-olein) and the formulae of the most common fatty acids, their common names and abbreviations

Fatty acids are indeed synthesized by head-to-tail condensation of two-carbon units, but the reality is a little more complex. In building up fatty acids, nature uses the Claisen condensation.to make the new carbonto-carbon bonds. In the laboratory we use a strong base (ethoxide ions) and anhydrous conditions to make this difficult reaction work between two molecules of ethyl acetate. Nature can perform the same in an 28

Fatty Acids and Derived Compounds

29

aqueous system and at neutral pH, but in order to make an acetate group sufficiently reactive, two activating effects are applied. We have already seen in Chapter 2 (Figure 2.7) that using a thioester increases reactivity. The second effect is converting the acetate into a malonate (Figure 3.2). The acidity value for the CH, group in a malonic thioester is not readily available, but it is evidently very high. In biosynthetic reactions the combined effects of thioester and additional carboxylate are used to activate the CH, portion of acetic acid for condensations.

@

4?

H3C-C,

0

+

0 /p

H2C-C,

OR

0

K, = 3~

OR K, = 1 x10-13

0 H3C-
o

3-isoxazolin-5-one UDP-glUCOSe

H O O C Y

COOH

c02

HOOCbNo2

t

NO2

Figure 9.19 Theformation of the defensive secretion of Chrysomela tremulaefrom labelled aspartic acid, Both the isoxazolinone part and the nitropropionic acid part are derivedfrom aspartic acid. The 6'-nitropropionic ester as well as the 2',6'-bis-(nitropropionic) ester shown, are present in the secretion

0

HF-7

Figure 9.20

The guanosine derivative designated HF-7 from venom of Hololena curta, a funnel-web spider, and the amide of oxalic acid with agmatine (decarboxylated arginine) from a hunting spider Plectreurys tristis

adapting derivatives of primary metabolites (compare section on spiders in Chapter 3, p. 52). a-Tocopherol acetate (Figure 9.21) is present in the secretion of the pupae of the squash beetle Epilachna borealis. It is made from chorismic acid via p-hydroxybenzoic acid with isoprene units added. Tocopherol contains a completely reduced diterpene chain fused onto a trimethylhydroquinone. The extra methyl groups on the quinone part come from S-adenosylmethionine. Tocopherol is an antioxidant and is vitamin E in humans, so this application of the compound is curious. It is related to the ubiquinones, also known as coenzyme Q, they are primary metabolites, found in all cells, with a function in electron transport.

Alkaloids and Compounds of Mixed Biosynthetic Origin

157

YOOH + P O P O y y - )

Q OH

n

+

P O P O + q q H 4

Figure 9.21 Outline of the formation of a-tocopherol acetate from p-hydroxybenzoic acid, a reduced terpene chain and additional methyl groups, and, for comparison, the formation of ubiquinones

9.2.1 Luciferin Bioluminescence, the ability of living organisms to emit light, is widely distributed throughout more primitive orders. It occurs in insects (fireflies and glow-worms) among the Collembola, Hemiptera, Diptera, Lepidoptera and Coleoptera. It is found particularly in three Coleoptera families: Lampydidae, Elateridae and Phengodidae. All the insects examined share the same system of luciferin and luciferase to produce light. Luciferin is synthesized by the fusion of quinone and cysteine to give first 6-hydroxybenzothiazine-2-carboxylicacid. This condenses with a second molecule of cysteine before oxidation and re-arrangment in an unexplained way to give luciferin (Figure 9.22). Luciferin is oxidized enzymically to oxyluciferin, which is formed in two excited states (Figure 9.23). To reach ground state, one form emits red light, the other green, the resultant effect is close to white light. One third of the molecules that undergo oxidation emit light (White, Miano and Umbreit, Journal of the American Chemical Society, 1975, 97, 198). Insects emitting light make an easy target for prey. It is not surprising that lampyrid beetles also employ chemicals, lucibufagins (Chapter lo), to make themselves unpalatable.

HS)

Figure 9.22

The biosynthesis of luciferin. The carboxyl group of cysteine was labelled with 14C

158

Chapter 9

aN\HNrcyH luciferase + ATP

HO

s

Mg2+_

[ luciferin - luciferase - ATP] + pyrophosphate

s

\red

hv

green hv

Figure 9.23 The light-emitting reaction of luciferin. When "0,gas was used, labelling was found equally in CO, and oxyluciferin. The asterisks indicate the excited electronic states

9.2.2 Volicitin Some lepidopteran larvae and grasshoppers have been shown to induce plants on which they are feeding to release volatiles (mostly terpenes) that attract predators and parasitoids of the plant-eating insects, by the saliva or regurgitated juices from the mouths of the larvae. The cause in at least one insect, Spodopteru exiguu, feeding on corn seedlings, is called volicitin, an elicitor of plant volatiles. Volicitin has been identified as N-( 17S-hydroxylinolenyl)-~-glutamine (Figure 9.24). The linolenic acid, taken from the plant, is hydroxylated by the insect and conjugated with glutamine. The saliva also contains 17-hydroxylinolenic acid, 17-hydroxylinoleic acid, linolenyl-glutamine and linoleyl-glutamine, but none of these show activity comparable to volicitin. There does not seem to be any obvious benefit to the Spodopteru, nor is it clear how different insect species affect the bouquet of plant volatiles differently, so that parasitic wasps, specific to that insect, are attracted. Probably other compounds, with action similar to volicitin, will be isolated. The subject is new and more will be learned as research progresses.

*YNH2 volicitin

Figure 9.24

Volicitin, a derivative of linolenic acid from a plant and glutamine from the insect, which stimulates the plant to release volatile compounds that attract predators of the insect

Alkaloids and Compounds of Mixed Biosynthetic Origin

159

BACKGROUND AND FURTHER READING M. S. Blum, Biosynthesis of arthropod exocrine compounds, Annual Review of Entomology, 1987,32,381-413. M. S. Blum, Biochemical defenses of insects, in M. Rockstein, editor, Biochemistry of Insects, Academic Press, New York and London, 1978, pp. 465-513. D. Daloze, J. C. Braekman and J. M. Pasteels, Ladybird defensive alkaloids: structural, chemotaxonomic, and biosynthetic aspects (Col.: Coccinellidae), Chemoecology, 1995,516, 173-1 83. E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 3, alkaloids). J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993, pp. 3 18 (Chapter 7). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 6, alkaloids). K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 8).

QUESTIONS 1. Note that reserpine (Figure 9.1) has a mixed biosynthetic origin. The centre part is more difficult, but what are the precursors of the leftand right-hand parts? 2. Note that both portions of the molecule of exochomine (Figure 9.9) have 13 carbon atoms. Suggest a biosynthetic scheme for the formation of the dimethylaza-acenaphthalenoneportion. 3. The biosynthesis of solenopsin (cis and trans) was studied by feeding sodium [ 1-14C]acetate(activity 60 mCi mmol-') to several thousand ants of 5'. geminata over 60 h. The solenopsins were then isolated and found to contain 6.1 x mCi mmol-'. Calculate the YOincorporation of acetate into solenopsins. 4. (a) In the experiment above with sodium [1-l4C]acetate,the methyl group on the pyrimidine ring of the solenopsins was selectively removed (for details of the method, see Leclercq, Braekman, Daloze, Pasteels and Vander Meer, Naturwissenschaften, 1996,83,222). What fraction of the total radioactivity would be expected in this carbon atom? (b) In a repeat of the experiment with [2-14C]acetate,used in exactly the same way, how much of the radioactivity would be expected in this same carbon atom? 5. The pupae of the ladybird Subcoccinella vigintiquatuorpunctata are

160

Chapter 9

protected by hairs which contain oily droplets. HPLC-mass spectrometry showed the secretion consisted mainly of three compounds that have molecular masses of 530, 532, and 534. They are derived from the compounds below (compare Figure 3.29). Suggest molecular structures for the three compounds. The three compounds all contain free NH groups but are not acidic. 0

T -

OH

HN\/-OH

mol. mass 283

0

OH HN-OH

mol. mass 285

6. In a study of luciferin biosynthesis in the beetle Pyrearinus termitilZuminans uniformly labelled cysteine (''C(U)cysteine, 30 nCi per insect) was injected into five insects and after two days the luciferin was separated by thin layer chromatography and counted. In the total extract of five insects, 480 dps were found. What is the specific incorporation of cysteine into luciferin?

CHAPTER 10

Plant Substances Stored, Changed or Unchanged, by Insects 10.1 TOXIC PLANT SUBSTANCES IN INSECTS In their constant evolutionary struggle against insects that attack them, plants have evolved many kinds of toxins to defend themselves. While most plant-eating insects have to avoid these toxins, some insects, in turn, have evolved the ability to overcome them, and some even to store them in their bodies as their own defence against predators (other insects, arthropods or higher animals). Butterflies and moths, followed by beetles, stand out prominently as insect sequesterers of plant chemicals. Day-flying, brightly coloured butterflies and moths would otherwise be particularly attractive prey. The plant substances may be stored unchanged, or changed slightly or metabolized so much that their connection with the plant may not be immediately apparent. Insects use a variety of enzymes to degrade or modify the plant products, including oxidases, reductases, hydrolases, esterases and transferases. The insect may be able to absorb selectively certain compounds and reject others or control the level of toxin they store. The ingestion of toxic compounds for protection is sometimes called pharmacophagy, and the insects are called pharmacophagous species. The earlier chapters of this book have been organized by fundamental biosynthetic route. No system can be totally logical. It might be argued that the section on sterols and the carotenes in Chapter 7 and the whole of Chapter 8 on aromatic compounds should be included here, but all insects modify dietary sterols to make moulting hormones and all animals modify the aromatic amino-acids to make important compounds. Here we are considering small groups of insects and essentially compounds normally toxic to insects. It is, moreover, a compilation, and cannot be expected to cover all known examples.

161

162

Chapter 10

10.1.1 Cardiac Glycosides The first example of the collection of toxic plant compounds to protect insects was demonstrated by Miriam Rothschild. She showed that the larvae of the Monarch butterfly Danaus plexippus (Plate 12) feed on milkweed, Asclepias curassavica which contains cardiac glycosides like calotropin (used as an arrow poison) (Figure 10.1). The compounds are stored unchanged in the larva and through metamorphosis, making the adult butterfly unpalatable to predators like birds. Calotropin is highly toxic to vertebrates but evidently has no ill effects on the insect. In addition to calotropin, the butterfly also stores three volatile alkylmethoxypyrazines (Figure 10.1) from the plant. The warning odour of these volatile compounds, associated with calotropin, is enough to deter a bird on close approach from trying to eat a butterfly charged with calotropin. It does not follow that because an insect feeds on a toxic plant that it will sequester the toxins. Another danaid butterfly Danaus chrysippus does not seem to sequester calotropin from milkweed.

0

calotropin

Figure 10.1 The structures of calotropin and the three volatile pyrazines sequestered by the Monarch butterfly

The popular flowering tropical shrub oleander (Nerium oleander) contains toxic cardenolides. The principal one is oleandrin (Figure 10.2), but the bug Aspidiotus nerii feeding on it sequesters only a minor component, adynerin. The ladybird Coccinella undecempunctata preying on Aspidiotus nerii sequesters the adynerin from its prey while another ladybird, C. septempunctata feeding on the same bugs does not. The aphid Aphis nerii (see Chapter 8, and Plate 7) on the same plant collects and stores three of its cardiac glycosides. The formation of cardiac glycosides from non-toxic plant sterols (Figure 7.12) and of related saponins from common triterpenes (Figure 7.13) have already been described.

Plant Substances Stored, Changed or Unchanged, by Insects

EQG0 H3CO

163

H

oleandrin

Figure 10.2 Two cardiac glycosides from oleander used by insects. Oleandrin is not sequestered by the aphid C. undecempunctata, but only the minor product adynerin. The sugar in oleandrin is oleandrose, in adynerin it is digitalose

10.1.2 Veratrum Alkaloids The veratrum alkaloids are a group of particularly toxic steroidal alkaloids. A study of the specialist sawfly Rhadinoceraea nodicornis has demonstrated how they store in their haemolymph ceveratrum alkaloids from the host plant, false, or white hellebore Veratrum album. The alkaloids in sawflies can either be directly sequestered, partly metabolized and sequestered, excreted intact or destroyed. The principal alkaloid stored in the haemolymph is 3-acetylzygadenine (Figure 10.3). 3Angeloylzygadenine of the plant is probably hydrolyzed in the gut to zygadenine and then acetylated. At the same time protoveratrines A and B (Figure 10.3) are degraded. Another sawfly, Aglaostigma sp., fed on false hellebore leaves neither had alkaloids in their haemolymph nor excreted any (Schaffner, Boeve, Gfeller and Schlunegger, Journal of Chemical Ecology, 1994,20, 3233-3250).

A

R=

do.. HO '

protoveratrine A

R= or

R=

OH

HO

zygadenine esters

9.. HO

COCH~

*

protoveratrine 6

Figure 10.3 Alkaloids from Veratrum plants. The angeloyl ester of zygadenine is present in the plant, the acetyl ester in the insect. Protoveratrines are, at the same time, metabolized completely by the sawfly R. nodicornis

164

Chapter 10

10.1.3 Pyrrolizidine Alkaloids The pyrrolizidine alkaloids such as seneciphylline from ragwort, Senecio jacobaea, and monocrotaline from Crotolaria species (Figure 10.4) are well-studied examples of metabolized plant substances converted to insect use. The subject of pyrrolizidine alkaloids and their presence in insects is too detailed to consider fully here. Taxonomically unrelated insect groups feed on unrelated plants, chiefly in the Asteraceae and Boraginaceae which contain these alkaloids and store the compounds in their bodies. The alkaloids consist of two parts, the basic pyrrolizidine (the necine part), and various hydroxy and branched acids esterified to it (the necic acid part). A feature of pyrrolizidine alkaloids is that they exist in two interchangeable forms, the non-toxic N-oxides, and the pro-toxic free bases. They only become toxic when the free bases are metabolized to highly reactive pyrroles by cytochrome P450oxidases. In most plant taxa the alkaloids are stored as the N-oxides. On ingestion by many insects they seem to be reduced in the gut to free bases and then re-oxidized for storage. The origin in the plant of the nitrogen-containing necine part is putrescine, which by exchange with spermidine gives first homospermidine (Figure 10.4), while the carboxylic acid portion of seneciphylline comes from amino acids, with many changes. In monocrotaline, the dicarboxylic acid comes from two molecules of 2-methylbutanoic acid.

spermidine homospermidine

OH HO

CHzOH hydrolysis of ester

retronecine

- 2H 0 monocrotaline N-oxide

hydroxydanaidal

by insect

+

danaidone male sex attractant

0 seneciphylline N-oxide

Figure 10.4 Some examples of pyrrolizidine alkaloids and the sex attractants made from them by danaid butterflies and ornate moths. The biosynthesis in plants of the retronecine part of the pyrrolizidine alkaloids begins with reaction between putrescine (from ornithine, Chapter 9 ) and spermidine

Plant Substances Stored, Changed or Unchanged, by Insects

165

Some adapted insects can metabolize the alkaloids and use the metabolites as pheromones. For example, male danaid butterflies collect pyrrolizidine alkaloids from Senecio plants, part of the alkaloid they store, and part degrade to danaidone (Figure 10.4) to use as a sex attractant. The danaidone encourages the females to copulate with them. It shows the females how rich in alkaloids the males are. The danaidone is presented on organs called hairpencils. Female butterflies are able to detect which males have more toxin by the amount of danaidone they secrete, and choose males with most alkaloid. The males pass on the alkaloids with their sperm, and the females invest their eggs with the alkaloids in turn. The ornate moth Utethesia ornatrix produces (R)-(-)-hydroxydanaidal (Figure 10.4) from pyrrolizidine alkaloids like monocrotaline N-oxide, and uses it as a sex attractant for females as the danaid butterflies do. The aphid Aphis jacobaeae also feeds on Senecio plants and stores these alkaloids. The ladybird Coccinella septempunctata (Plate 3) feeding on the aphids accumulates the pyrrolizidine alkaloids in turn, but does not accumulate cardiac glycosides (see earlier). Two groups of chrysomelid beetles have evolved different ways of dealing with these alkaloids. The genus Oreina feed on Senecio and Adenostyles plants. They make their own cardiac glycosides (see Figure 7.12), and accumulate pyrrolizidines as N-oxides, in their haemolymph and glands. One species has only the pyrrolizidines for its defence. Generally the insects do not alter the alkaloids, but two examples, one of hydrolysis and one of epoxidation are known. A typical Oreina alkaloid and these two metabolized products are shown in Figure 10.5. The mechanism for handling alkaloids in the genus Phtyphora is different. Only open-chain pyrrolizidines are accumulated, along with the saponins these insects make themselves (Figure 7.13). The alkaloids are stored as the tertiary amines, not the N-oxides, and only in the haemolymph of larvae and defensive glands of adults. Some alteration of their structures is possible by the insects as shown in Figure 10.6. These species

t

0

t

0

t

0

Figure 10.5 Small changes made in the pyrrolizidine alkaloid acetylseneciphylline N-oxide by Oreina species. The epoxide example is not found in plants

166

Chapter 10

rinderine

lycopsamine

interrnedine

0

OH

retronecine

+i$ OH

L%

OH

OH

N

Figure 10.6 Changes made in open-chainpyrrolizidine alkaloid by Platyphora boucardi by epimerization, and new alkaloids made by this species from retronecine

were also shown to be able to take ingested retronecine and esterify it with propionic, lactic and a-hydroxyisovaleric acids to make new alkaloids. It has been suggested that the toxic effect of pyrrolizidine alkaloids does not really protect insects, because the toxicity only comes into effect when the compound is metabolized, which is well separated from the eating or tasting of the insect, rather it is only the bitterness of the taste of the compounds that offers protection.

10.1.4 Cyanogenic Glucosides Many plants store cyanogenic glucosides, which on removal of the glucose can decompose to release hydrogen cyanide. As HCN is a powerful toxicant to all haem groups containing complexed iron (present in both plants and insects), it is remarkable how such compounds can be safely sequestered by plants or insects. More than 40 cyanogenic glucosides are known in plants, mandelonitrile glucoside, prunasin (Figure 10.7), and its epimer sambunigrin, are typical examples. A glucosidase cleaves the prunasin when the plant is damaged to give glucose and mandelonitrile. The released mandelonitrile is either cleaved spontaneously, at acid pH, or by oxynitrilase in alkaline medium, giving benzaldehyde and HCN. Among animals, this type of defence is practised by millipedes, centipedes and insects only. The larvae of the Australian beetles of Puropsis and Chrysophtarta, feeding on Eucalyptus leaves produce mandelonitrile and prunasin. When freeze-dried insects were treated with pglucosidase and nitrilase, HCN was released (Figure 10.7). The larvae

Plant Substances Stored, Changed or Unchanged, by Insects

w:sRH HHo

Q+I

+

HCN

or oxynitrilase

H

mandelonitrile P-glucoside or prunasin

Figure 10.7

uncatalysed

167

mandelonitrile

benzaldehyde

Conversion of plant-producedprunasin to HCN in Australian beetles

have special glands that secrete the HCN, but eggs, pupae and adults also release some HCN but without specific glands. In beetles and millipedes, which do not live on plants that produce cyanogenic glycosides, the compound is almost certainly made by the insect or arthropod itself. In millipedes mandelonitrile is stored in an inner chamber of their defensive glands, which is separated from the outer chamber which holds enzymes that catalyze the breakdown of mandelonitrile to benzaldehyde and HCN. A muscle controls the valve between the two chambers. The millipede Oxidus gracilis converted racemic [2-14C]phenylalanine to HCN, but did not make HCN from [2-'4C]tyrosine.The steps from phenylalanine to mandelonitrile have been studied in the millipede Harpaphe haydeniana (Plate 13) by feeding a wide range of potential precursors, from which the biosynthetic scheme shown in Figure 10.8 was constructed. Phenylalanine was much less well incorporated into mandelonitrile than N-hydroxyphenylalanine, but that may be because there are many competing uses for phenylalanine. Phenylpyruvic acid oxime was also incorporated, but not as well as phenylacetaldehyde oxime, which leaves the two possible routes open in the middle of the sequence. Cyanogenic species are much more common among Lepidoptera, and many of them are able to synthesize the cyanogens. The bright red-andblack burnet moth Zygaena trifolii obtains the cyanogenic glycosides linamirin and lotaustralin from Lotus corniculatus. This species is also

rnandelonitrile

Figure 10.8 The investigated route to mandelonitrile in the millipede Harpaphe haydeniana. The actual route was notJirmly established, but with small possible variation, it is the same as the route in plants

168

Chapter 10 H3C, CH-CH I ,COOH H3C'

.hH

Fe2+ * 2 0 2 NADPH

H3C\~

-co2

II

CH-CH H3C'

____c

.L

H3C,, H3C' CH-? .\I

02

NH-OH

valine

H

N-OH

I

linamarin

- -

H2 H3C-C, ,COOH CH-CH H3C' L NH2 isoleucine

H2 H3C-C, ,O-glucose

4

/c,

H3C

CSN

lotaustralin

Figure 10.9 The biosynthesis of the cyanogenic glycosides linamarin and lotaustralinfrom amino-acids in the burnet moth. The dot on nitrogen and the prime and double prime on carbon represent 15N and I3C respectively. Labelling showed that these atoms were retained in place during the synthesis

able to synthesize more linamirin and lotaustralin from valine and isoleucine (Figure 10.9). This may be a case of co-evolution according to the suggestions of J. M. Pasteels. Other cyanogenic species do not feed on cyanogenic plants at all. The ability to make and store these compounds seems general in the heliconiine tribe of butterflies. About one third is stored in the haemolymph and two thirds in the integument. Biosynthetic studies in both Zygaena and Heliconius species showed that larvae fed with uniformly labelled 13C-valineor isoleucine were able to synthesize linamarin and lotaustralin respectively. Studies showed that the carbon skeleton was retained intact except for the carboxyl group. For release of HCN, the glucose is enzymically cleaved by linamarinase and the cyanohydrin spontaneously decomposes (Figure 10.10). The cyanide once released can be detoxified as in plants, by conversion to cyanoalanine and asparagine (Figure lO.lO), or by the enzyme rhodanese, which converts cyanide into the relatively innocuous thiocyanate. The formation and decomposition of HCN in millipedes and insects is therefore a remarkable example of parallel evolution, where insects and plants both produce HCN and benzaldehyde in similar ways and avoid the toxic effect of HCN by converting it to the same cyanoalanine. It is not easy to perform tests for the presence of rhodanese and so it is uncertain how widely it occurs in insects. It is the view of at least one author, L. Kassarov, that the cyanogenic effect has no influence on rejection of butterflies and moths by birds and animals. The release of HCN is too slow to protect the insect. Not even the bitterness of the compounds is responsible, since the compounds are

Plant Substances Stored, Changed or Unchanged, by Insects

COOH CN-

1

-

/!F H

-

NH2 cysteine

N-c--\

,COOH CH

H20

A

NH2

*

169

H2N7fYcooH 0 NH2 asparagine

cyanoalanine

pyridoxal phosphate

R-CEN

rhodanese

*

R-S-EN

Figure 10.10 The release of cyanide from linamarin and the detoxiJication of cyanide either by conversion of cysteine to asparagine or by oxidation to thiocyanate

internal and water soluble, not on the cuticular surface, which is hydrophobic. He suggests the whole concept needs to be re-thought. Some of the primitive cycad plants produce the highly toxic glycoside cycasin (Figure 10.11). Yet some butterflies and moths can feed on the plants and sequester the cycasin. The hairstreak butterfly Eumaeus atala florida absorbs the toxic glucoside, so that larvae contain 0.02% and adults 1.O to 1.8%cycasin by weight. When the moth Seirarctia echo was fed on the aglycone, methylazoxymethanol, they stored cycasin. They probably hydrolyze cycasin in their gut and then re-glycosylate it again to store it. Cycasin and methylazoxymethanol are carcinogenic, hepatotoxic, mutagenic and radiomimetic, all of which can be summarized by saying that they are active alkylating agents. OH

cycasin

methylazoxymethanol

Figure 10.11 Cycasin and its aglycone methylazoxymethanol

10.1.5 Glucosinolates

Glucosinolates, or mustard oil glycosides, are like the cyanogenic glycosides, non-toxic substances which give toxins after the enzymic cleavage of the glycoside. About 100 of them are known, and some have been found in all species of Cruciferae examined. Their synthesis in the plant begins with N-hydroxylation of an amino-acid or modified amino-acid and decarboxylation. Further steps are not known with certainty, but probably proceed as shown in Figure 10.12.

170

Chapter 10

HN-OH

-cS,-

0-D-glucose

II

N

4

-

‘o-so3

glucose POSO3

Na+

SH

R-c:,

N-OH

a t hiohyd roximic acid

a glucosinolate

Figure 10.12 The partly speculative route by which glucosinolates are formed in plants. POS03- represents a phosphoric-sulphuric anhydride which donates sulphate

On decomposition they release isothiocyanates or mustard oils, but can also give thiocyanates, and at low pH can give cyanides (Figure 10.13). They generally act as feeding deterrents but some insects, including the Cabbage white butterflies (Pieris sp.) have adapted to feed only on Brassica plants and use the glucosinolates to attract them to the plants for egg-laying and larval feeding. As well, they sequester one of these glucosinolates, sinigrin (Figure 10.13) and its corresponding allyl isothiocyanate. The latter is both toxic and repellent to most insects. There is in the foregut of the Diamondback moth PZuteZZa xyZusteZZa a sulphatase which converts the glucosinolate to a non-toxic oxime thioglucoside (Figure 10.13) which is excreted in the faeces. Glucosinolates are toxic to the thyroid and liver of domestic animals and humans, but many humans are attracted by their pungent effect in cabbage, horseradish and cress.

HzC=HC-CH

-?,S-P-D-glucose

sinigrin N. o-sO3-

H&=HC-CH2-s-C-N allyl thiocyanate

N

+

11

H2C=HC-CH2-C~~ allyl cyanide

,+c/S-~-D-g~ucose “

mvrosinase* glucose

~af

-

%-so3

Naf

sulphatase H20

@-H

‘NLO-SOJ

N i

+

NaHS04

allyl isothiocyanate

*

R-

c, S-P-D-g lucose II

N . ~ - ~

non-toxic

Figure 10.13 The spontaneous release of allyl isothiocyanate and lesser amounts of other compounds from sinigrin in damaged Brassica leaves after the cleavage of the thioglycoside by myrosinase. Sinigrin can also be detoxified by some insects by selective cleavage of the sulphate group

Plant Substances Stored, Changed or Unchanged, by Insects

171

10.1.6 Coniferyl Alcohol A small example, similar to the conversion of pyrrolizidine alkaloids to male copulating pheromones by some Lepidoptera, is the conversion of flower compounds to attractants by males of the oriental fruit fly Bactrocera dorsalis. These adult males feed on the fragrant flowers of Fagraea berteriana which contain (E)-3,4-dimethoxycinnamylalcohol and smaller amounts of (E)-3,4-dimethoxycinnamylacetate (Figure 10.14). The male flies convert these to (E)-coniferyl alcohol (Figure 10.15) and store it in rectal glands. The coniferyl alcohol is very attractive to females, although they do not appear to derive any benefits from the compound. A neat use of the simple thin layer chromatography plate to locate the position of the attractive compounds on the plate with the fruit flies is illustrated in Figure 10.16. 10.1.7 Other Types Harborne (Ecological Biochemistry, pp. 98-1 00, see Further Reading) lists 12 types of plant toxin sequestered by insects, including the cardiac glycosides, veratrine and pyrrolizidine alkaloids, cyanogenic glycosides, and glucosinolates already considered. The others are aristolocic acids, quinolizidine alkaloids, iridoids (Chapter 7), and phenols. Nishida (Annual Review of Entomology, see Further Reading) lists 17 types for Lepidoptera alone. Even so, insects have only harnessed a small fraction of the hundreds of types of plant toxins that they can encounter. Another example of partial modification in the insect is found with the beetle Diabrotica speciosa. It feeds normally on plants of the Cucurbitaceae (cucumbers) which contain bitter triterpenoids (Chapter 7) called cucurbitacins. D. speciosa when fed on ['4C]-cucurbitacinB converts it by removing the C-25 acetate and reduces the side-chain double bond to give dihydrocucurbitacin D (Figure 10.17). Other species raised on diets free of cucurbitacins, when fed cucurbitacin D, were able to glycosylate it (at the 2-OH), hydrogenate, desaturate and acetylate it. The water beetles of the Dytiscidae family convert dietary sterols (e.g. cholesterol, see Chapter 7) to mammalian hormones like oestrone and testosterone, but most interesting is the cortisone-type cortexone (Figure 7.10). A Dytiscus beetle can contain as much as 1 to 3 mg of cortexone, which it can discharge from glands if attacked by a predator. A cantharid beetle, Cauliognathus lecontei, feeding on Compositae that contain acetylenic esters, has been found to contain dihydromatricaria acid (Figure 10.18), the first acetylenic compound found in insects, and which the beetles use as a defensive secretion.

Chapter 10

172

Figure 10.14 Males of the Orientalfruitfly Bactrocera dorsalisfeeding on aflower of Fagraea berteriana to obtain dimethoxycinnamic alcohol and acetate (Photo R. Nishida. Reproduced from Fig. 1 in J Chem Ecol., 1997, 23, p. 2277, ‘Acquisition of female-attracting fragrance by males of oriental fruit fly from a Hawaiian Lei flower Fagrea berteriana’ by Nishida, Shelley and Kaneshiro. By kind permission of Kluwer Academic Publishers)

CH30

H

O

P

*

H

(a-coniferyl alcohol

Figure 10.15

The production of the sex attractant coniferyl alcohol by fruitfliesfrom dimethoxycinnamic alcohol and acetate fromflowers

Plant Substances Stored, Changed or Unchanged, by Insects

173

The lampyrid beetles (fireflies, Chapter 9) produce lucibufagins (Figure 10.18) to make themselves unpalatable to predators. These substances come from dietary sterols, and therefore represent a group of more highly metabolized compounds. They are related in structure to the cardiac glycosides and toad poisons.

Figure 10.16 A thin layer chromatogram, origin arrowed right, of a solvent extract of the Fagraea berterianaflowers was developed with benzene-ethyl acetate (2:l). The solvent front, arrowed, is on the left. Male B. dorsalis flies were then allowed to gather on the plate to show where the active substances were located (Photo R. Nishida. Reproduced from the same source as Figure 10.14 with the kind permission of Kluwer Academic Publishers)

HO H3C

\

0

',,

'

cucurbitacin 6

dihydrocucurbitacin D

Figure 10.17 An example of a triterpenoid slightly mod$ed and sequestered, dihydrocucurbitacin D in chrysomelid beetles

dihydromatricaria acid R = H3CCH-C0 H3C. CH,CH,CO

H RO

RO romallenone

OH

R ' = CH3C0

CH3CO

CHSCO

CH3CO

CH&O

H

lucibufagins

Figure 10.18 Dihydromatricaria acid is found in cantharid beetles feeding on Compositae which contain polyacetylenic acids. Lucibufagins are similar to cardenolides, sequestered by fireflies. Romallenone is present in the defensivefoam of a grasshopper

174

Chapter 10

The grasshopper Romalea microptera discharges a foam from glands when disturbed. The principal constituent of the foam is romallenone (Figure 10.18) an allenic sesquiterpene. This compound is thought to be OH P-glucosidase

salicin

L.d

salicyl alcohol

salicylaldehyde

Figure 10.19 Two chrysomelid beetles (Phratora vitellinae and Chrysomela tremulae) produce sulicyluldehyde us a defensive secretion.

a degradation product of carotenoids (see Chapter 7) from its food plants. Two chrysomelid beetles (Phratora vitellinae and Chrysomela tremulae) produce salicylaldehyde as a defensive secretion (Figure 10.19). They both feed on willow and poplars, which produce salicin, which they have been shown to convert to salicylaldehyde.

10.1.8 A Parting Thought

A number of highly toxic plant substances manipulated by insects have been considered here. How do they do it? The tobacco alkaloids, represented by nicotine, were considered in Chapter 9. Here are a group of simple substances, highly toxic to higher animals and insects, long used as a commercial insecticide. Nicotine affects acetylcholine receptors in the central nervous system. Yet Lepidoptera of the subfamilies Macroglossinae and Sphinginae are able to tolerate large quantities of nicotine. The tobacco hornworm, Manduca sexta and the cigarette beetle Lassioderma serricorne have adapted to live only on fresh tobacco leaves or cured tobaco respectively. Manduca larvae thrive better on plants with lower nicotine levels than on those on artificially high nicotine, so the alkaloid is no help to them. Yet they prefer to feed on young leaves, and nutritionally thrive much better on them although they have twice a much nicotine in them as older leaves. How do they cope with the toxins? It has been demonstrated for M. sexta that their acetylcholine receptors are not different from those of other insects. They do not tolerate the nerve poisons, rather they appear to have a rich variety of detoxifying enzymes to break down the nicotine, particularly in their central nervous system. Certainly only 10 to 20% of nicotine injected into tolerant larvae can be recovered as nicotine, nicotine N-oxide or cotinine (Figure 9.2). What happens to the rest? We do not know.

Plant Substances Stored, Changed or Unchanged, by Insects

175

We have, as yet, few clues to how insects overcome plant defences. With insects, nothing can be taken for granted and their chemistry still holds many surprises.

BACKGROUND AND FURTHER READING D. Daloze, J. C. Braekman and J. M. Pasteels, Ladybird defensive alkaloids: structural, chemotaxonomic, and biosynthetic aspects (Col.: Coccinellidae), Chemoecology, 1995, 5/6, 173-1 83. J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993, pp. 318 (Chapters 5 & 7, plant defenses). T. Hartmann and D. Ober, Biosynthesis and metabolism of pyrrolizidine alkaloids in plants and specialized insect herbivores, Topics in Current Chemistry, 2000,209,207-243. R. Nishida, Sequestration of defensive substances from plants, Annual Review of Entomology, 2002,47,57-92.

QUESTIONS 1. Leaves of Nerium oleander were injected with sodium [2-2H,]acetate. Aphis nerii were allowed to feed on the leaves and then the aphids were fed to the ladybirds Coccinella undecimpunctata. The cardiac glycoside oleandrin was isolated from the ladybirds and subjected to mass spectrometry. There was a small, but distinct mass spectral peak at M+3. What do you conclude? 2. What experiment do you suggest to discover whether pyrrolizidine alkaloids are reduced from the N-oxide form of the plants to the free base in the gut of an insect and later re-oxidized? 3. By NMR spectrometry, how would you check that the labelling of valine in Figure 10.9 had remained intact in linamarin? 4. In a study of the conversion of phenylalanine to mandelonitrile, benzaldehyde and HCN, DL-phenylalanine (0.17 pmoles) labelled with 14Cin the phenyl ring (specific activity 82 pCi mmol-') was injected into each of 10 individual millipedes. The specific activity of the benzaldehyde isolated from all ten at the end of the experiment was 7.39 x pCi mmol-'. Allowance must be made for the dilution with endogenous precursors, because 2.3 pmoles of benzaldehyde was isolated while only 0.17 pmoles of phenylalanine was injected. Presuming that only the radioactivity in L-phenylalanine is used for incorporation, what was the %age conversion of radio-labelled phenylalanine to benzaldehyde? No radioactivity was found in the HCN.

176

Chapter 10

5. The secretion of the grasshopper Romalea microptera contains, in addition to romallenone (Figure 10.18) verbenone, isophorone and 2,6,6-trimethylcyclohex-2-en1,4-dione, below. What is the likely source of these compounds?

verbenone

isophorone

trimethylcyclohexendione

THE BONUS QUESTION Silkworm silk is composed essentially of four amino-acids, glycine, alanine, serine, and tyrosine. The silk, fibroin, is produced from two lateral glands, and the two fibres are glued together with another protein called sericin. The fibres are approximately triangular of dimensions 0.01 mm on each side. A silkworm larva spins its cocoon of silk at the rate of 1 cm sec-’. The unit cell of fibroin has a length of 70 nm and contains two amino-acids, that means the average length of an amino-acid is 35 nm. The amino-acids can be considered as cylinders with a radius of 47 nm. How many amino-acids does the silkworm incorporate into each fibre per second?

Answers to Questions Chapter 1

Ql. From COz to glucose, acetyl CoA, malonyl CoA, fatty acids to hydrocarbons. 4 2 . E=hv of sunlight. Q3. Primary: alanine, deoxyribose, glucose. Secondary: vitamin A, camphor, penicillin. Q4. A synomone, assuming the bee gets nectar from the flower.

Chapter 2

Q1. Haem. More water soluble (and hence more easily excreted). Q2. CH,COCH, + NADH + H’Cl- + CH,CHOHCH, + NAD’ C143. CH,CH,CH,COOH + FAD -+ CH,CH=CHCOOH + FADH2 Q4,

-N

H-enzyme a-keto-isovaleric acid

part of lipoic acid

isobutyryl CoA

For more intermediate steps see Figures 2.11 and 2.13. Q5. Methyl hexanoate. Q6.

177

1

178

Answers to Questions

Note that 2-deuterioacetic acid is not pro-chiral, according to the priority rule, because replacing another hydrogen by deuterium means there is one 'H and two 2Hson the methyl group. Chapter 3

Acetyl-S-ACP to malonyl-S-ACP to stearic acid, desaturated to oleic acid, chain extended by three acetate units to a C,, unsaturated acid, and decarboxylation gives (Z)-9-tricosene. As for question 1, as far as stearic acid, then addition of one propionate (= methylmalonate) unit and two more acetate units gives 6-methyltetradecanoic acid. Decarboxylation gives 5-methyltricosane. Palmitic acid is converted to 11-hexadecenoic acid, then chain-shortened to 9-hexadecenoic acid, and again desaturated by a All-desaturase to (9E, 11E)-tetradecadienoic acid. The remaining steps are obvious. Three deuterium atoms in 9-hydroxydecenoic acid and two in 10hydroxydecenoic acid. The 2-fluoro-atom blocks the first stage of chain-shortening of stearic acid so the whole process is halted. Nine of the ten deuterium atoms would remain.

(a-

p

D3C,C,CD.

3

C D ,

0

.kOH

NH2 Dlo-isoleucine D2

-

p 3 0 D3C.C,CDroH D2

-

0

--c

-..

D

3

7D3 C

.

C

,

C

D

~

o

D2 OH Dg-14-methylhexadecanoic acid

Q7. Bruchin A appears to be made from oleic acid by chain extension with two more acetate units and reduction to the alcohol to give docosanol, which is a-oxidized to an alcohol at the other end of the chain (not necessarily in that order). The 3-hydroxypropionic acid (hydracrylic acid) is an unusual metabolite, with no obvious precursors. It is possibly made here by cooxidation of propionic acid. Both C2* and C24diols are found in the mixture from the insect. Chapter 4 CH3 0 I

enolize

Q2. Seven deuterium atoms maximum.

II

2-hydroxy-6-methylacetoDhenone

'soom

Answers to Questions

e

179

0

H

0

0

0

0

0

0

requires oxidation here polyketide for endocrocin

0 0

0

0 OH

0

O

0 polyketide for griseofulvin

polyketide for alternariol

Q4. 2-Nonanone: Ac-Ac-Ac-Ac-Ac 6-Methyl-3-octanone: Ac-Pro-Ac-Pro 4-Methyl-4-hepten-3-one: Pro-Pro-Pro Q5. Ac-Pro-Pro-Pro

-

*-*-J O

O

+ I

reduction to CH2

O

O

OH 0

H

t

0

Jehy ciration

decarboxylation

pheromone

reduction to CHOH

Q6. A

O

H

Q7. Because of the (@-configuration of the methyl branch, it looks like the chain is begun with 2-methylbutyric acid (see Figure 3.12) but that is without proof. It might be made from acetate and propionate.

CH3COOH

k

O

Y 0

t OH

Chapter 5

Specific incorporation into the lipid fraction was 7.19% and into bombykol 0.1 15%. The amount in bombykol is just about enough to study further by addition of more 'cold' bombykol. If the experiment had been conducted when the females were a few days older, better incorporation might have been achieved.

The propionic acid had not been incorporated intact, or there would have been 13C-13Ccoupling. We must conclude that the propionic acid had been broken down and the label scrambled, so the experiment was unsuccessful. Propionic acid is easily degraded to acetic acid. When using propionic acid

180

Answers to Questions

it is much safer to use it labelled in the carboxyl group. See Question 7 below for an example. Q5. H3C-s

gCH3

> --

m/z 173

m/z 159

The molecule can only be constructed from acetate and propionate by starting on the right-hand side. It contains two acetates and three propionates. Any of a number of ways of labelling could be used. Radio-labelling would be less useful because the molecule would have to be degraded to find where the label was located. CD, labelling of propionic acid would show up in the mass spectrum, but best would be labelling with 2,3['3C]propionic acid and -the 13C-13Ccoupling at the places indicated by asterisks should be seen. 0

HOOC I*

I*

Incorporation is 20.79%. It is much easier to get high incorporations like this when using excised tissues or glands. Chapter 6 Q1. An oxidase will convert a-pinene to cis-verbenol.

42. Made from a-farnesene. An a-oxidation is required (-CH, -+-CH,OH -+ CHO) to produce the aldehyde at the 'wrong' end of the chain. 4 3 . To check, see Figure 6.12, although there can be some mixing of the label in the terminal dimethyl group of geranyl pyrophosphate. *

+

Answers to Questions Q4-

181

rzO

epox'd

p & + , H -

-

h '

-

oxidize

- 2H

periplanone A

Q5.

0

1

caryophylline oxide

H-wor reduce

-

-oxidize

*

enoiize

\ oxid. ancistrodial

\

ancistrofuran

h-&

Q7. The ring closure from gyrinidal to gyrinidone is as for iridoids.

a 2 0 b

farnesol

oxid.

oxid.

OH OH

gyrinidal

Chapter 7

Q1. All the double bonds in the cembrene isomer in Figure 7.2 are trans. There are eight possible isomers, one with all trans double bonds, one all cis, three with one trans and two cis, three with two trans and one cis. 42.

A $J OH

geranylgeraniol

182

Answers to Questions

Q3.

Iycopene

p-carotene

Q5. Specific incorporation was 0.0 19% Q6. Specific incorporation was 0.50%, which is good indication that this compound is further along the route to 20-hydroxyecdysone. Chapter 8 Q1. COOH

O

or

m W C 0 0

mpropylphenol

O 0

O

H

w 0

0

0

rnellein

Q3. Since insects do not have chorismic acid available, both compounds must be derived from tyrosine. Q4. It looks very like a polyketide, so one should try incorporating sodium 2[13C]acetatein the predicted positions without scrambling the label. Both homogentisic acid (Figure 8.1 1) and p-hydroxybenzaldehyde can be Q5derived from tyrosine. Marginalin is a condensation product of homogentisic acid and p-hydroxybenzaldehyde.

Answers to Questions

183

Q7. The sphere represents the remainder of the pyridoxal phosphate co-enzyme.

Chapter 9

The left-hand part is derived from tryptophan and the right-hand part is a phenyl-C, compound.

Rate of incorporation is 0.010% (a) Expected O%, actually found was 0.25%. (b) Expected 11% or 1/9th. Actually found was 10%. See Schroeder, Smedley, Gibbons, Farmer, Attygalle, Eisner and Meinwald, Proc. Natural Academy of Sciences, USA. 1998,95, 13387.

mol. mass 532

mol. mass 530

mol. mass 534

Remember there are two cysteine molecules incorporated into one luciferin molecule. The yield is 4.32%. This maximum is reached after one day of incubation.

Answers to Questions

184 Chapter 10

The oleandrin, which contains an acetate group, was labelled with the trideuterated acetate and this has passed unchanged from plant to aphid to ladybird. The alkaloids are extracted from the plant, reduced to free bases, reoxidized using l 8 0 and fed to the insect. Later the alkaloids are extracted from the insect and examined by mass spectrometry. If the l 8 0 is still intact, there will be a distinct M+2 ion in the spectrum. There should be enhancement of the peaks for the two labelled carbon atoms in the 13CNMR spectrum and there should be 13C-13C coupling. There was 0.002% conversion. Since romallenone is probably a carotene degradation product from the carotenes in the leaves on which the grasshopper feeds, the isophorone and the trimethylcyclohexendione are from the same source. It is difficult to see how verbenone can be formed in the same way, so it has either been made by the grasshopper or from a monoterpene in its food. The Bonus Question

The average volume of an amino-acid in silk is 2.4 x m, and the volume of 1 cm of a silk fibre is 8.66 x m. Therefore there are approximately 360,000,000 molecules per cm length and this number added per second per fibre. The fibres are actually hollow, so a minimum dimension for the fibre has been used. Fibre dimensions vary from 0.023 to 0.009 mm during the spinning process

Appendix Common Abbreviations Ac AcCoA ADP ATP Ci COA-SH D DAHP DMAPP DOPA DXP FAD FADHz GC GC-MS GTP HMG-COA HPLC IPP JH LC-MS MEP MH MVA NAD+,N A D F NADH, NADPH NMR PLP Pr PTTH T

acetyl acetyl coenzyme A adenosine diphosphate adenosine triphophate Curie unit of radioactivity coenzyme A (free form) deuterium (2H) 3-deoxy-D-arabinoheptulosonic acid 7-phosphate dimethallyl pyrophosphate 3,4-dihydroxyphenylalanine 1-deoxy-D-xylulose 5-phosphate flavin adenine dinucleotide reduced flavin adenine dinucleotide gas chromatography linked gas chromatography and mass spectrometry guanosine triphosphate P-hydroxy-P-methylglutarylcoenzyme A high performance liquid chromatography isopentenyl pyrophosphate juvenile hormone linked liquid chromatography (HPLC) and mass spectrometry methylerythritol phosphate moulting hormone mevalonic acid nicotinamide adenine dinucleotide (phosphate) nicotinamide adenine dinucleotide (phosphate), reduced form nuclear magnetic resonance pyridoxal phosphate propyl or propionyl prothoracotropic hormone tritium (3H)

185

Subject Index Normal type entries are page numbers, bold numbers, e g , 1.1 are figure numbers A Abbreviations, 185 Acanthoscelides obtectus, 45,3.25 ACP, see acyl carrier protein Acetic acid, 18,42, 57-58, 64, 85, 106, 130, 2.13,4.1 Acetogenins, 57-68, 129, 130, 131, 1.1,4.5,4.6,4.7 from butyric acid, 62-63,4.11,4.12 from propionic acid, 60-63,4.7, 4.8,4.9,4.10,4.11 Acetylcoenzyme A, see Coenzyme A thioacetat e Acetylenase, 34 Acetylenic acids, 171, 10.18 Actinidine, 92,6.12 Active site, 10, 11, 12, 14, 108 Acyl carrier protein, 29, 31, 60,3.5 Adalia beetles, 47 Adalia punctata, 47 Adaline, 47,3.28 Adalinine, 47, 3.28 S-Adenosyl homocysteine, 19, 76, 2.16 S-Adenosyl methionine, 19, 76, 102, 110, 145, 156,2.16 Adrenalin, 127,8.8 Adynerin, 162, 10.2 Aedes aegyptii, 102 Aenictus rotundatus, 94, 126 Agelenopsis aperta, 152 Aglaostigma sandflies, 163 Agmatine, 156,9.20

Agroporus alutacea, 131,8.13 Albolic acid, 107,7.4 Albolineol, 107,7.4 Alcohol dehydrogenase, 25,61 Alcohol oxidation, 15-1 6, 2.9 Aldolase, 22 Aldol condensation, 63, 85,4.13,6.2 Aleuritic acid, 98,6.22 Alkaloids, 47, 128-129, 143-154,l.l biosynthesis, 145,164,9.3, 9.4, 10.4 bitterness, 166 of insects, 145-1 54 of plants, 143-145, 9.1 metabolized and sequestered, 163-166 precursors, 144,9.2 pyrrolizidines, 164-1 66, 10.4-10.6 toxicity, 143, 149, 166, 174-175 veratrum, 163,10.3 Alkane oxidation, 12 Alkenes double bond position, 40, 82,3.19, 5.12 oxidation, 13,41, 50 Alkylpiperidines, 146, 9.5,9.6 Alkylpyrazines, 152-1 53, 162,9.15, 10.1 Alkylpyrrolidines, 147, 9.7, 9.8 Allelochemicals def., 4 Allenes, 45, 172,3.25, 10.18 Allomone def., 2 , 4 Alydus eurinus, 37,3.14 187

188 Amblyomma americanum, 129, Plate 14 Amblyomma rnaculatum, 129 Amblyomma variegatum, 130 p-Aminobenzoic acid, 126,8.5 5-Aminolaevulinic acid, 138,8.20 Amitermes, 53, 97, 3.37 Ammonia, 20,3.26 Amyrin, 113,7.13 Anabasine, 145,9.4 Ancistrodial, 97,6.20 Ancistrofuran, 97,6.20 A n cistrotermes ca vitho rax, 97 Ancistrotermes pakistanicus, 3.31 Anisomorpha buprestoides, 93 Anisomorphal, 6.12 Anomala cuprea, 45,3.25 Anteiso-acids, 36, 37, 39,3.12,3.17 Anthocyanins, 154,9.17 Anthonomus grandis, 89, Plate 6,6.7 Anthranilic acid, 125, 126, 150,8.5 Ants, 21, 51-52, 57,63-64, 91,98, 105, 126, 144, 146-148,2.19,9.8 Aphaenogaster cockerelli, 125 Aphaenogaster rudis, 145,9.4 Aphidoidea (aphids), 46,96, 134 Aphinin, 135,8.17 Aphins, 134-1 35,8.17 Aphis fabae, 135 Aphis jacobaeae, 165 Aphis neri, 135, 162, Plate 7,8.18 Apis mellifera, 50-5 1, 59, 114 Nasanov attractant, 9 1 queen substance, 50,3.32 sex attractant, 50 venom, 127, 128 Apterygota, 8 Arachidonic acid, 35-36,3.10,3.11 Arachnida, 62 Arctia caja, 128, Plate 11 Argyrotaenia velutinana, 42,3.21 Aromatic compounds, 6, 121-132 amines, 127-1 29,8.9 pheromones, 123,8.2,8.4,8.5 Arthropods, 7, 8, 102, 114

Subject Index

Asparagine, 24, 168,2.21, 10.10 Aspartic acid, 155,9.19 Aspidiotus nerii, 162 Asymmetric induction, 24-26, 2.23 Atrax robustus, 151 Atta cephalotes, 61 Atta texana, 61 Autoradiography, whole-body, 72

B Bactrocera dorsalis, 171, 10.14, 10.16 Becquerel unit, 70 Bees bumblebees, 104, 105 colletine, 54-55 halictine, 54 honeybees, see Apis mellifera Nomada, 96 solitary, 65 stingless, 94, 104, 105, 107 Biacetyl, 135 Bilins, 138, 8.20 Bilirubin, 138,8.20 Biliverdin, 138,8.20 Biopterin, 137,8.19 Biosynthesis, def., 1 analytical aspects, 82 compounds of mixed pathway, 154-158 de novo, 90,93 dynamic equilibrium, 5 in plants, 6,40, 87-88, 110, 115, 121-123, 143-145,154, 158, 161, 164, 166, 169,6.6,9.17, 10.12 reaction types, 5, 6, 1.3 routes, 1.1 sequence of events, 72 site of, 69, 71 Blatta germanica, 48, 84, 111, 145, 7.9 Blattodea (cockroaches), 48, 102, 130, 131 Blister beetles, see Coleoptera Bombykol, 9,44, 83-84,3.23

Subject Index Bombyx mori, 9,44, 140, Plate 2 Brevicomin, 90,6.10 Brood cells, 55 Bruchus pisorum, 56 Bugs, see Hemiptera C 13C-2H coupling, 77,5.9 13C-13Ccoupling, 74, 80-81,5.11 13Clabelling, 70, 74, 93, 168, 10.9 and NMR spectroscopy, 77,78, 5.9,5.11 14Clabelling, 40-41,46,65,7 1,73-74, 93, 106, 112, 113, 125, 127, 129, 145, 146, 149, 155, 157, 167, 168, 171,3.20,5.2, 5.3,5.4, 7.12,8.6,9.3,9.22, 10.9 36Cllabelling, 130 Caddis flies, see Trichoptera Cadaverine, 144, 152,9.2 y-Cadinine, 97,6.20 Cahn-Ingold-Prelogrules, 24-25,2.22 Calliphora stygia, 120 Calotropin, 162,lO.l Campesterol, 110,7.8 Camponotus clarithorax, 23, 123 Camponotus herculeanus, 4.14 Camponotus inequalis, 4.14 Camponotus ligniperdus, 4.14 Camponotus nearticus, 126 Camponotus rujpes, 64,4.14 Camponotus silvicola, 64,4.14 Camponotus ants, 64 Cantharidin, 97-98,6.21 Caparrapioxide, 97, 6.20 Carabid beetles, 57 Carboxypeptidase, 12-1 3,2.3 N-Carboxybiotin, 29,3.4 Cardiac glycosides, 112-1 13, 162, 165, 172, 7.12, 10.1, 10.2 Carminic acid, 134,8.16 Carotenes, 6, 116-118, 132,1.1,7.16, 7.17 Carpophilusfreernani, 62,80,4.11, 4.12

189 Carvone, 24,2.21 Caryophylline oxide, 97,6.20 Cauliognathus lecontei, 171 Cedrene, 98-99,6.22 Cembrene, 105, 106,7.2, 7.3 Centipedes (Chilopoda), 64, 128, 129, 166 Centipedin, 64, 129,4.15,8.10 Cerambycid beetles, 57 Ceroplasteric acid, 107, 7.4 Ceroplastes albolineatus, 107,7.4 Ceroplastols, 107,7.4 Chalcogran, 65,4.17 Chelicerata, 7 Chemical parsimony, 50 Chemochromes, 132 Chilocorine, 148,9.9 Chilocorus cacti, 148 Chirality, 5,23-26, 36,45, 61, 82, 2.22,3.3 chiral shift reagent, 83 diastereomers, 83 optical rotation, 83 optical rotatory dispersion, 83 circular dichroism, 83 Chironomus flies, 138 Cholesterol, 24, 85, 109-113, 7.7,7.8 Chorismic acid, 123, 156,8.1 compounds derived from, 125-127, 156,8.5 Choristoneurafumiferana, 3.23 Chrysolina carnifex, 116 Chrysolina coerulans, 112-1 13,7.12 Chrysomela tremulae, 15&155, 174, 9.19, 10.19 Chrysomelidial, 93,6.12,6.13 Chrysophtarta beetles, 166 Chrysopterin, 138,8.19 Cinnabarinic acid, 140,8.21 Citronellol, 91,6.1,6.4 Claisen condensation, 28, 138,6.2 Claisen rearrangement, 123 Coccinella septempunctata, 47, 162, 165, Plate 3 Coccinella undecempunctata, 162,175

190 Coccinellids (ladybirds), 4 6 4 8 , 148 Coccinellines, 4647, 67,3.26,3.27 Coccoidea (scale insects and mealy bugs), 107, 133 Cochineal, 134,8.16 Co-enzyme A, 14,31,2.6 thioacetate, 14, 29, 85, 1.1, 2.6, 3.3,6.2 Co-enzymes, 10, 13-2 1 Cold-hardy insects, 23 Coleoptera (beetles), 4, 8,45 bark beetles, 65, 89-90 benzaldehyde in, 125 benzoic acid in, 125 blister beetles, 66-67, 97-98 bombardier beetles, 131,8.12 cantharid beetles, 173 carabid beetles, 21 Chrysolinina, 112-1 13,7.12 chrysomelids and cucurbitacins, 171, 10.17 click beetles, 104 defensive secretions, 4 5 4 8 , 91 lampyrid (fireflies), 173, 10.18 leaf beetles, 93-94 luciferin in, 157 mandelonitrile in, 167 meloid beetles, 97, Plate 10 phenols in, 129 pheromones, 45, 54,3.25,3.38 quinones in, 130 sequestering plant toxins, 161 water beetles, 111, 171 Collembola (springtails), 130 Communication chemicals, 4 Coniferyl alcohol, 125, 171, 8.2, 10.15 Coniine, 143, 145, 9.1, 9.3 Convergine, 47, 3.26 Corpus allatum, 101 Cotinine, 145, 174,9.4 Crematofuran, 105,7.2 Crematogaster ants, 51-52, 105 Crematogaster brevispinosus, 105,7.2 Crematogaster deformis, 129,S.lO

Subject Index Crematogaster scutellaris, 3.34 Crustacea, 7, 102, 115,7.14 Cryptolaemus montrouzieri, 47 Cryptolestes beetles, 3.38 Cryptolestes ferrugineus, 95,3.38, 6.19 Cucujolides, 95-96,6.19 Cucurbitacins, 171, 10.17 Cupiennius salei, 52 Curie unit, 70 Cuticular hydrocarbons, 3 7 4 0 , 8 4 Cyanoalanine, 168,lO.lO Cyanogenic glucosides, 166-1 69, 10.7-10.10 Cybister limbatus, 112 Cycasin, 169, 10.11 Cycloartenol, 110 Cysteine, 157,9.22 Cytochromes, 33,41, 5 1,2.5 cytochrome P450,13, 38, 51, 102, 109, 114, 151, 164

D Dactylopius coccus, 134 Dacus oleae, 65, 4.16 Danaidone, 165, 10.4 Danaus chrysippus, 162 Danaus plexippus, 162, Plate 12 Decarboxy lation of amino-acids, 20,2.18 of fatty acids, 38,3.15 of pyruvic acid, 16-18, 2.13 of sterols, 109,7.7 Defensive substances, 2, 5, 7, 2 1, 36, 37,40,4648, 51-52, 53, 57,62, 66-67,91,97, 105, 111-1 12, 116, 125, 126, 129, 131, 161-170, 171-174,6.17,7.3, 7.10 Dendroctonus frontalis, 90,6.9 Dendroctonus ponderosae, 90,6.9 Dendrolasin, 69,95,6.18 Deoxyarabinoheptulosonic acid phosphate, 122,S.l Deoxyxylulose 5-phosphate, 88,6.6

Subject Index

Desaturase, 32-35,43,3.6-3.8,3.22, Plate 1 A9 type, 32-34 A1' type, 34,43,3.22 Desmogramma beetles, 113 Deuterium labelling, 17,26,37,40,47, 48,62, 64, 75-76, 80, 81, 87,93, 96, 97, 113, 125,2.24,2.25,3.13, 3.19,3.28,3.29,4.14,6.13,6.15 and gas chromatography, 76 and stereochemistry, 8 1-82 and volatility, 81 Deuterium oxide (heavy water), 6, 81 Diabrotica speciosa, 171 Dichlorophenol, 129,8.10 Dimethallyl pyrophosphate, 87, 99, 6.2 Dimethyl disulphide, 74, 82, 5.5 Dimethylquinazoline, 126,8.5 Dinoponera australis, 63 Diplopoda (millipedes), 53, 125, 126, 129, 130, 131,151,166, 167,8.13 Diptera (flies), 40,65, 101 Disparlure, 3.23 Diterpenes, 85, 104-106,7.1-7.3 Dolichodial, 9 1, 6.12 DOPA, 127, 132,8.7,8.8,8.14 Dopamine, 127,8.8 Double bonds, 82,5.12 reduction, 16,2.10 oxidation, 41,3.20 Drosophila buzzatii, 71,5.1 Drosophila melanogaster, 140, 7.15 Dytiscus marginalis, 141 E Ectatoma ruidum, 104 Ecdysone, 114,7.14 Ecdysteroids, 5, 114-1 16,7.14,7.15 conjugates, 114,7.15 inactivation, 114,7.15 phytoecdysteroids, 115-1 16,7.15 Eicosanoids, 35, 1.1,3.11 Eloides longicollis, 130 Emodin, 134, 8.16

191 Emulsin, 5 Enantiomers, 23, 50, 61, 66, 2.22 Endopterygota, 8 Enzymes, 5, 10-13 Epiblema scudderiana, 23 Epilachna borealis, 156 Epilachna varivetis, 47, Plate 4 Epilachnine, 47,3.28 Epiphytas postvittana, 55 Epoxidase, 34 Ericerus pela, 107 Eriococcus scale insects, 134 Erythropterin, 138, Plate 9,8.19 Erythrose 4-phosphate, 121-1 22, 1.1,8.1 Estigmene acrea, 44,3.24 Ethanol, 18, 22, 25-26, 2.24 Eucondylodesmus elegans, 125, 8.4 Eumaeus atalajlorida, 169 Eumelanin, 133 Eupoecilia ambiguella, 42 Eutetramorium mocquerysi, 153 Evolution chemical, 17, 102 parallel, 7, 113, 168 Exochomine, 148,9.9 Exochomus quadripustulatus, 148 Exopterygota, 8 F Faranal, 99,6.25 Farnesyl pyrophosphate, 95, 99, 101, 104,6.17, 7.1 Farnesoic acid, 102 Farnesol, 98,6.1,6.17,6.21 Farnesenes, 96,99,6.17,6.20,6.24 Fatty acids, 28-35, 1.1 allenic, 3.25 biosynthesis, 29-30,3.3,3.5 branched, 36-38,3.12 chain lengthening, 38,3.15 degradation, 29-30,3.3 odd-numbered, 31, 33, 36, 37 synthetase, 29

192 unsaturated, 31-35,40, 53,3.8, 3.19 Feeding deterrence, 143, 170 Flavin adenine dinucleotide, 16, 32, 33, 137, 2.10,3.3 Flavones, 154,9.17 Fluoromevalonolactone, 87,6.4 Formic acid, 8, 19,40 biosynthesis, 21,2.19 Formica rufa, 4.14 Formyl group, 19, 109 Frontalin, 6.9

G Gascardia cerifera, 107 Gas chromatography, 76,81, 82,93 chiral stationary phase, 83 -mass spectrometry, 76, 82,93 radio-, 71 Geraniol, 87,91,6.1,6.4,6.7,6.9, 6.13 Geranylcitronellol, 104,7.1 Geranylfarnesol, 107 Geranylgeraniol, 104,7.1 Geranylgeranyl pyrophosphate, 104, 116, 7.1,7.6 Geranyllinalool, 104,7.1 Geranyl pyrophosphate, 87, 94, 6.3, 6.17 Germacrenes, 97, 6.20 Glands (secretory) of insects, 61 abdominal, 42, 125 corpus allatum, 101 defensive, 62,93, 113, 116, 165, 167 Dufour, 51, 54, 63, 99, 104, 105, 107 ejaculatory bulb, 7 1 elytra, 154, frontal, 105, 106, 107 hairs, 48, 128, 144, Plate 4 hairpencils, 165 labial, 104 mandibular, 61, 62,95, 123, 126, 152,4.3

Subject Index

metapleural, 129,4.3 metathoracic, 37 Nasanov, 91 osmeterium, 97 prothoracic, 112, 114, pygidial, 37, 58, 131, 151 rectal, 64, 171 sternal, 49 thoracic, 150 venom or poison, 51,63, 148, 152 ventral, 130 Glomerine, 126,8.5 Glomeris marginata, 126 Glucose, 17, 22, 1.1 Glucosidase, 93, 166 Glucosinolates, 169-170,10.12,10.13 detoxified, 170, 10.13 Glutamine, 46, 158 Glyceraldehyde 3-phosphate, 22, 88, 1.1,2.20,6.6 Glycerol, 23,24, 79,2.23,5.10 Glycine, 19,21, 138 Gonypteryx rhamni, 136, 137 Gnamptogenys striatula, 101, 6.26 Grandlure, 89,6.7 Grapholitha molesta, 123 Green leaf volatiles, 53,3.36 Griseofulvin, 73-74, 5.2,5.3 Guanosine triphosphate, 137,8.19 Gyrinidal, 103 Gyrinidone, 103

H Habrobracon hebetor, 105 Haem, 12,42,46, 166,2.4, 2.5,8.20 Haemolymph, 38,42,46,66,97, 135, 163, 165, 168 Harpaphe haydeniana, 167, Plate 13, 10.8 Harpogoxenus sublaevis, 147,9.8 Heavy water, see deuterium oxide Heavy-atom labelling, 42,69, 75-8 1 Heliconius butterflies, 168 Helicoverpa zea, 3.23 Hemimetabola, 8,1.5

Subject Index Hemiptera (bugs), 65 aggregation, 53 defensive secretion, 53 Hexapoda, 7 Hippodamia convergens, 47, Plate 7 Hippodamine, 47,3.26,3.27 Histamine, 144,9.2 Hololena curta, 155,9.20 Holometabola, 8, 1.5 Homofarnesenes, 99, 6.24 Homohimachalene, 100,6.25 Homomevalonic acid, 99, 101, 105, 6.23 Homomonoterpenes, 101,6.26 Homoptera (plant-sucking bugs, scale insects), 107, 133-134, Plate 8 Homoterpenes, 99-101, 106,7.3 Honeybees, see Apis mellifera Hormones, 2,4, 35, 1.1 def., 2 Hospitalitermes umbrinus, 106 Humulone, 154,9.17 Hydrocarbons, 1.1 chiral, 38, 39,45,3.24 of cuticle, 37-39 melting temperature, 39 methyl-branched, 38-40,48,3.16, 3.17 pheromones, 40-41,44 Hydrogen cyanide, 166-1 69 detoxified, 168, 10.10 possibly not deterrent, 168-169 Hydrogen peroxide, 131,8.12 Hydroxydanaidal, 165, 10.4 20-Hydroxyecdysone, 114,7.14 Hydroxylase, 34,3.9 2-Hydroxy-6-methylacetophenone, 74, 5.4 p-Hydroxy -p-methylglutaryl coenzyme A, 85,6.2 Hymenoptera, see ants, bees and wasps, 89, 104 pigments, 137

193 Hyperaspine, 148,9.9 Hyperaspis campestris, 148

I Ilybius fenestratus, 112, 7.11 Immune response, 35 Indole, 126,8.5 Indolizidines, 148, 150,9.10, 9.12 Ingested substances, 13 Insecta, 7, 8 Invertin, 5 Invictolide, 64,4.14 Ips paraconfusus, 90,6.8 Ipspini, 90,6.8 Iridoids, 9 1-94 Iridomyrmecin, 9 1,6.12 Iridomyrmex, see Linepithema Iso-acids, 36, 39,3.12,3.17 Isobutyric acid, 36, 37,75,3.12,3.13, 3.23 Isocrematofuran, 105,7.2 Isoflavones, 154, 9.17 Isoleucine, 36, 99, 148, 151, 168, 3.12, 10.9 Isopentenyl pyrophosphate, 87, 88, 94,99, 104,6.2,6.17,7.1 Isoprene, 85,6.1 Isoprenoids, 1.1 Isoptera (termites), 49-50, 9 1, 104, 105-1 06 Isothiocyanates, 170, 10.13 Isotopes, 69-70, Table 5.1 isotope effect, 76,81-82 isotopic enrichment, 77 kinetic isotope effect, 8 1-82 Isovaleric acid, 52, 78,5.10 Isoxazole glycosides, 154-1 55,9.19 J Jalaric acid, 98, 6.22 Juvabione, 95,6.18 Juvenile hormone, 24, 101-102, 1.5, 6.27 inactivation, 102 mimics, 5,7,95

194 K Kairomone, def., 4 Kermesic acid, 8, 134 Kermococcus ilicius, 134 Ketals, cyclic, 65-66, 94,4.16,4.17, 6.16 P-Ketosynthase, 30,3.5 Kuhn-Roth degradation, 74 Kynurenine, 139-140,8.21

L Laccaic acids, 134, 8.16 Laceifera lacca, 98 Laccijalaric acid, 98,6.22 Lac insects, 98-99, 134 Lactic acid, 16-18,22, 2.12 Lactones, 4.14 macrocyclic, 53-55, 96, 3.37,3.38, 3.39,6.19 Ladybirds, see coccinellids Lanosterol, 109,7.6,7.7 Lasioderma sericorne, 144, 174 Lasius fuliginosus, 4.14 Lasius niger, 64,4.14 Lepidoptera, 4, 8,40, 117, 136, 144, 158, 161, 171, 3.18 cardiac glycosides in, 162, 10.1 copulating pheromones, 125 cyanogenesis, 167, 168,10.9 and cycasin, 169 juvenile hormone, 101 pigments, 137, 138,8.20 pyrrolizidine alkaloids in, 164-165, 10.4 sequestering plant toxins, 121 sex pheromones, 4 1 4 5 Leptinotarsa beetles, 113 Leptoglossus bugs, 125 Leptothorax acervorum, 147,9.8 Leptothorax muscorum, 147,9.8 Leucine, 52, 148, 153 Leucopterin, 136,8.19 Limonene, 6.1,6.4 Linalool, 6.1,6.4 Linalyl pyrophosphate, 89,6.4

Subject Index Linepithema humile, 54, 91,3.39 Linoleic acid, 35,49,3.10,3.31 Linolenic acid, 35,44, 66, 158,3.10, 3.26,4.17 Limonene, 24 Linamirin, 167, 10.9, 10.10 Linamirinase, 168 Linyphia triangularis, 52 Lipoic acid, 18,2.14 Lipophorin, 38,49 Loganin, 93,6.12 Lotaustralin, 167, 10.9 Lucibufagins, 157, 173, 10.18 Luciferin, 157,9.22, 9.23 Luciferase, 157 Lutzomyia longipalpis, 100 Lymantria dispar, 3.23 Lysine, 144,9.2 Lysozyme, 10-12,2.1,2.2

M Macrosiphon Eiriodendri, 118,7.17 Macrosiphon rosae, 135 Macrotermitinae, 49 Makisterone A, 114,7.14 Malacosoma moths (tent caterpillars), 111,7.9 Malonyl CoA, 29, 85,3.4,3.5, 6.2 Mamestra configurata, 124 Mandelonitrile, 166-1 67, 10.7, 10.8 Manduca sexta, 143, 174-1 75, Plate 15 Manica rubida, 67,99 Mannich reaction, 47, 3.28 Marginalin, 141 Mass spectrometry for heavy isotope labelling, 64, 75, 76, 80, 82, 93, 125, 150, 5.7,5.8 Mating disruption, 4,43 Megalomyrmex ants, 147 Melanargia galanthea, 154 Melanin, 132-1 33, 138,8.14 Mellein, 64, 129,4.3,4.14 Messor ants, 145 Methacrylic acid, 37, 75,3.13

Subject Index Methionine, 148 deuterated, 64 doubly labelled, 74,5.5 2-Methylbutyricacid, 36,54,164,3.12 3-Methylenepentyl pyrophosphate, 99,6.23 Methylerythritol phosphate pathway, 6, 87-88, 116, 154,6.6 Methyl group transfer, 19, 2.16 6-Methyl-6-hepten-2-one, 94,6.16 Methylmalonyl CoA, 38, 60 Methyl 4-methylpyrrole-2carboxylate, 153,9.16 Methy1 6-methylsalicylate, 57, 76-77, 1.2, 5.8,5.9 Methyl oxidase, 34 6-Methylpelleterine, 47,3.28 Mevalonic acid, 69, 85, 87, 1.1,6.2 Mevalonolactone, 87,93, 106, 112, 6.5,6.13 Migdolus fryans, 151 Millipedes, see Diplopoda Monomorium ants, 147, 148 Monomorium pharaonis, 99, 105, 148,9.10 Monoterpenes, 85-94,6.1,6.2,6.3, 6.4,6.6 biosynthesis, 85-89,6.2-6.4 defensive compounds, 9 1-94 pheromones, 89-91 Morpho didius, 132 Morpho rhetenor, 132 Moulting hormone, see also ecdysteroids, 114-1 16, 1.5 Musca dornestica, 40, 145 Myrcene, 6.1,6.8 Myriapoda, 7, 126 Myristic acid, 28,3.1 Myrmica ants, 99, 6.24 Myrmica rubra, Plate 8 Myrmica scabrinodis, 61 Myrmicaria ants, 150,9.10, 9.12 N 15Nlabelling, 168, 10.9

195

NIH shift, 123, 8.3 NMR spectroscopy, 75-77, 78, 80-81, 83, 150,5.9,5.11 Nannotrigona testaceicornis, 104 Naphthoquinones, 131,8.13 Narceus gardanus, 131 Nasutitermes octopilis, 106 Nasutitermitinae, 105 Natural products, 2 Neanura muscorum, 130 Nepetalactone, 9 1,6.12 Neriaphin, 135,8.18 Nerol, 6.1,6.7 Nerolidol, 6.17 Neryl formate, 91 Neurotransmitters, 127, 149 Nicotinamide adenine dinucleotide (and its forms), 15-16, 17, 22, 30, 33, 82,85, 101, 108, 145,2.8, 2.9,3.3,7.5 Nicotine, 143, 145, 174-175,9.4 Nicotinic acid, 144,9.2 Nomadone, 96,6.20 Norgeraniol, 94,6.15 0 '*Olabelling, 75, 90, 96, 5.6, 6.10, 6.19, 9.23 Ocimene, 6.1 2-Octynoic acid, 46 Odour receptors, 23,2.21 Oleandrin, 162, 10.2 Oleic acid, 28, 38,45,48,49, 65, 81-82, 148,3.1,3.10,3.29,3.31 Ommins, 139, 8.21 Ommochromes, 139,8.21 Oncopeltusfasciatus, 138, Plate 17 One-carbon fragment, 19,21,2.15 Opilionids, 62, 129, 130,4.10 Oreina beetles, 165, 10.5 Oreophoetesperuana, 151 Orgyia pseudotsuga, 3.23 Ornithine, 144, 9.2 Orthoptera (grasshoppers and locusts), 129, 130,131, 138

196 Oxidative dehydrogenation, 133, 134, 8.15 Oxidus gracilis, 167 Oxygenases, 12 Oxynitrilase, 166, 10.7 P Paederus beetles, 66-67 Palmitic acid, 28, 32,42,44, 98, 3.1 Palmitoleic acid, 32,45,3.25 Paltothyreus tarsatus, 74 Papilio memnon, 97 Papilio protenor, 97 Paropsis beetles, 166 Pect inophora gossyp iella (pink bollworm), 117,3.23 Pederine, 66,4.18 Periplanata americana, 96, 129, 145 Periplanones, 96-97,6.20 Pest control, 2,4, 63, 66, 143 Phaedon amoraciae, 93,6.13 Phaeomelanin, 133 Pharmacophagy def., 161 Phenols, 129-1 30,S.lO Phenylacetic acid, 125,8.2 Phenylalanine, 121, 123, 125, 127, 137, 144, 148, 167,8.2, 10.8 Phenyl-C, compounds, 122, 123-125, 154, 1.1, 8.2 Phenylpyruvic acid, 123, 129,8.1,8.2 Pheromones, 2,4,94, 1.1 aggregation, 54, 62,65-66, 7 1, 89, 90, 95,4.12,4.17, 6.7,6.8 alarm, 9697, 130,6.20 attractant, 37, 91, 111 blends, 43 and chirality, 82-83,90 contact, 48 copulating, 125, 165 def., 2 dispersing, 9 1 hydrocarbon, 40 oviposition deterrent, 154,9.18 queen, 64, 105 response inhibition, 42-43

Subject Index

sex attractant, 8, 37,4045, 50, 52, 65, 89,91, 100, 104, 105, 123, 124, 130, 151,3.12,3.21-3.25, 3.31 swarm, 3.33 trail, 49-50, 57,61,64,76,99, 101, 105, 111, 125, 126, 145, 150, 152, 153, 3.31, 4.14, 6.25, 6.26 volatile, 61-65 Philanthus triangulatus, 151 Philanthotoxins, 151,9.14 Phorcabilin, 8.20 Phosphoenol pyruvate, 22,121, 123, 1.1,2.20,8.1 Photosynthesis, 16, 75, 1.1, 1.4,5.6 Phragmatobia fuliginosa, 44,3.24 Phratora vitellinae, 174, 10.19 Phyllopertha diversa, 15 1 Phytoecdysteroids, see ecdysteroids Pieris brassicae, 136, 138, 170 Pieris rapae, 136 Pigments of insects, 117-1 18, 132-140,7.16,7.17,8.14, 8.16-8.21 a-Pinene, 90,6.8 Pityogenes chalcographus, 65,4.17 Plagiodial, 94,6.14 Plant substances metabolized by insects, 35, 44, 53, 89-90, 110-115, 116, 121, 125, 127, 129, 130-131, 132, 154, 158, 161-167,169, 170, 171-174 Platyphora beetles, 113, 165 Platyphora boucardi, 10.6 Platyphora kollari, 113,7.13 Plectreurys tristis, 9.20 Plutella xylostella, 170 Polistes dominulus, 137, Plate 16 Polyamines, 151-152,9.12,9.14 Polyketides, 57, 78, 121, 131, 145, 154,1.1,4.1-4.4,4.6,4.8,4.9,9.3 Polyketide synthase, 61 type I, 59, 145 type 11, 59 type 111, 154,9.17

Subject Index Polyomrnatus icarus, 154 Polyphenol oxidase, 132 Polyunsaturated acids, 6, 35,44 Polyzonamine, 151,9.13 Polyzonium rosalbum, 151 Ponasterone A, 115,7.14 Porphobilinogen, 138,8.20 Precoccinelline, 47,3.26,3.27 Prephenic acid, 123,g.l Prenyl transferase, 87,94 Primary metabolites, 1, 28 Pro-chiral atoms, 15,25,82, 87, 2.9,2.23-2.25,3.9,6.3, 8.1 def., 25 Propionic acid, 37,38,48, 60,62, 64, 99, 130,3.16,3.17,3.30, 4.7,4.9,4.10-4.12 Prostaglandins, 35,3.11 Prothoracotropic hormone, 114 Protoaphins, 134-1 35,8.17 Protoporphyrin IX, 138,2.4, 8.20 Prunasin, 166, 10.7 Pterins, 136-138, Plate 17,8.19 Pterobilin, 138,8.20 Pterygota, 8 Putresine, 144, 151, 164,9.2 Pyrearinus termitilluminans, 160 Pyridoxal phosphate, 19-21,88, 138, 2.17,2.18, 10.10 Pyridoxamine, 20, 123, 2.17 Pyrophosphatase, 87, 101 Pyrrhocoris apterus, 138, Plate 9 Pyrroles, 153 Pyrrolizidine alkaloids, see alkaloids Pyrrolizidines, 148,9.10 Pyruvic acid, 17-18,22,88, 1.1,2.12, 2.13,2.20,6.6

Q

Queen substance, 50,3.32 Quinoline, 150 Quinones, 130-132, 157 pigments, 133-1 36

197

R Radio-labelling, see also I4Cand tritium tritium, 21,42, 57, 69, 72 dilution, 72,73, 147 double labelling, 74 specific, 72 uniform, 72, 149 Reflex bleeding, 46, 66,98 Reticulitermes santonensis, 107 Reticulitermes termites, 105 Retronecine, 166, 10.4, 10.6 Rhadinoceraea nodicornis, 163, 10.3 Rhugoletis cerasi, 154, 9.18 Rhinotermitidae, 50 Rhodommatin, 140,8.21 Rhodanese, 168 Rhytidoponera aciculata, 58, 74, 78, 5.5 Rhytidoponera chalybaea, 4.3 Romalea micropteru, 129, 174, 176 Romallenone, 174, 10.18 Royal jelly acid, 5 1,3.33

S 35Slabelling, 69,70,73, 140,5.5 Salicylaldehyde, 174, 10.19 Sandflies (Diptera), 105 Saponins, 113, 165,7.13 Sarpedobilin, 138 Scale insects, see Homoptera Scarites subterraneus, 37 Schemochromes, 132 Schizura concinna, 21 Scolopendra subspinipes multilans, 64-65, 129, Plate 5,4.15,8.10 Secondary metabolites def, 1 Seirurctia echo, 169 Semiochemistry,4,5 1 Sequestered substances from plants, 161-1 74 Serine, 19,21,48 Serotonin, 128-129,8.9 Sesquiterpenes,85,94-99,6.17,10.19 pheromones, 96-97,6.20

198 Sesterterpenes, 85, 107,7.4 Shellac, 98, 134 Shikimic acid, 6, 69, 1.1 pathway, 121-123,S.l Silk, 176 Sinigrin, 170, 10.13 Sitosterol, 110,7.S Skatole, 126,S.5 Social insects, 37, 61 Solenopsins, 146-1 47,9.5,9.6 Solenopsis ants, 148 fire ants, 146, 9.5 thief ants, 147,9.7 Solenopsis geminata, 146,9.5,9.6 Solenopsis invicta, 64 Species recognition, 125 Specific activity def., 70 Specific incorporation, 70, 112 Spermidine, 151, 164, 9.14, 10.4 Spermine, 151,9.14 Spider mites, 90 Spiders, 52 sex attractant, 52 venom, 128, 151, 152, 155,9.14 Spodoptera eridania, 39,3.18 Spodoptera exigua, 158 Springene, 104, 105, 7.1 Squalene, 108, 114,7.5,7.6 Stearic acid, 28,32,38,48, 51,81, 82, 3.1,3.15,3.33 Stegobinone, 84 Stegobium paniceum, 84 Stenus comma, 151 Stenusin, 151,9.13 Steroids, 6, 12, 109-1 16,1.1,7.7-7.11 as insect nutrient, 110,7.S as defensive secretions, 111-1 13, 116 as pheromones, 111 Stigmasterol, 110,7.S Subcoccinella vigintiquatuorpunctata, 148, 159 Substrate, 10, 33 Succinyl CoA, 138 Sulcatol, 94, 6.16

Subject Index Sulphatase, 170 Symbionts, 67,98, 135 Synomone def., 4

T Taste receptors, 23, 2.21 Tenebrio molitor, 67 Termites, see Isoptera Terpenes, 85, 158, 1.1 degraded, 94,6.16 Tetradecenyl acetate, 44,3.23 Tetrahydrofolic acid, 19, 21, 109, 137, 2.15,2.16 Tetramor ium impurum, 76 Tetraponerine ants, 148 Tetraponerines, 148,9.10,9.11 Tetrapyrroles, 138,8.20 Tetraterpenes, 85, 116-1 18,7.16 Thiamine diphosphate, 17, 88, 2.11, 2.13 Thin layer chromatography, 171, 10.16 Thioesters, 14, 29, 79, 2.6,2.7,3.2, 5.10 Thioglucosides, 93 Threonine, 152 Ticks (Ixodoidea), 102, 111, 127, 129-130, 8.10 Tocopherol acetate, 156,9.21 Transaminase, 19-20,2.17 Triatoma bugs, 126,8.5 Tribolium confusum, 40 Trichoplusia ni, 43 Trichoptera (caddis flies), 62, 126, 130 9-Tricosene, 40,41,3.20 Triglycerides, 28,42,3.1 Triterpenes, 85, 108-1 10, 171, 10.17 Tritium labelling, 26,41, 76, 87,2.25, 3.20 Trogoderma beetles, 37,3.12 Trogoderma granarium, 37 Tryptophan, 121, 125-128, 139, 144, 151,S.5 Tuberolachnus salignus, 135 Tyrophagus putrescentiae, 9 1,6.11

Subject Index Tyrosine, 123, 127, 130-132, 134, 137, 140, 151, 8.2,8.3 U Ubiquinones, 157, 9.21 Unsaturated acids, 32-35,3.8, 3.10 Uric acid, 20 Utethesia ornatrix, 165 V Valine, 36, 37, 75-76, 99, 168,3.12, 10.9 Venetian red, 8, 134,8.16 Venom components, 5,21, 127, 128, 144-148, 151,152, 155, 156 Veratrum alkaloids, see alkaloids cis-Verbenol, 90,6.8 Vespa crabro, I37 Vespa vulgaris, 137 Violaxanthin, 117 Virginae butanolide A, 77-79, 8 1, 5.10

199 Visual pigment, 117, 139-140, 7.16,8.21 Visual spectrum, 132 Vitamins, 21,29, 117, 156 Volicitin, 158,9.24

W Wasps, 62,65, 127 social, 128 parasitic, 53, 105, 158 pigments, 137

X Xanthommatin, 139,8.21 Xanthophyll, 118,7.16 Xanthopterin, 136, Plate 16, 8.19 Z Zophobas rugipes, 130 Zygaena trifolii, 167, 168, 10.9