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, amino-acids, 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, offence 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 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 suggested that pheromones were among the first communication chemicals affecting animal behaviour, and the pheromones of primitive single-cell 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.
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 α-D-glucosides (sucrose is an example) while emulsin hydrolysed β-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. Schoenhemier and Rittenberg showed in 1936 that when an animal was allowed to drink heavy water (D2O) 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.
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 micro-organisms) 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 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. 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 1018 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 1.5); 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 1.5). 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 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.
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 an enzyme that caused bacterial lysis. 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.
