








key: cord-021013-xvc791wx
authors: Wink, Michael
title: Chapter 1 Allelochemical Properties or the Raison D'être of Alkaloids
date: 2008-05-30
journal: nan
DOI: 10.1016/s0099-9598(08)60134-0
sha: 
doc_id: 21013
cord_uid: xvc791wx

This chapter provides evidence that alkaloids are not waste products or functionless molecules as formerly assumed, but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. These molecules were developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells, which are seen in molecules that mimic endogenous neurotransmitters. The chapter discusses that microorganisms and herbivores rely on plants as a food source. Since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. Many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. Many herbivores have potent mechanisms to detoxify xenobiotics, which allow the exploitation of at least the less toxic plants. In insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. Alkaloids function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. It needs more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants.

organisms. We must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients.

How do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? Because plants do rather well in Nature, this question has often been overlooked. We are well aware of the defensive strategies of higher animals against microbes and predators (1,2,4,15,17,28,494) . The complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. Against predating animals, Nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called "poisons") (1).

It is evident that most of these possibilities are not available for plants with their sessile and "passive" life-style. What then is their evolutionary solution? We can distinguish the following defense mechanisms in plants (3, 4, 7, 15, 17) ; the mechanisms are not independent and may act cooperatively and synergistically. We should be aware that many species have additionally evolved specialized traits in this context. 1 . Mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). 2. Formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. b. Cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. Callose and lignin are often accumulated at the site of infection or wounding (6,7) and form a penetration barrier. c. Synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. These proteins are either stored in the vacuole 1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS 3 or are secreted as exoenzymes into the cell wall or the extracellular space (8, 9) . These compounds are thus positioned at an "advanced and strategically important defense position." In addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. As a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced (2-4, 10-21) . These allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides, glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~O U O synthesis may be induced by elicitors (so-called phytoalexins), infection, or herbivory (4,7, [22] [23] [24] . These products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection (25,26)] or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots (2, 3, 15) ].

In animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals (1,4,12,16,17,28-30, [494] [495] [496] 503) . In many instances, the animals have obtained the toxins from their host plants (4, 12,15,17,27-33). Hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ''toxins" in this context) in animals is that of defense against predators or microorganisms (1, 17, 28, [494] [495] [496] .

These defense compounds are better known as natural products or secondary metabolites. The latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites (34, 35, 38) . Unfortunately the term "secondary" has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. As discussed in this chapter, just the opposite is true.

More than 30,000 natural products have been reported from plants so far (2, 4, 17) . Owing to the sophistication in phytochemical methods, such as chromatography (HPLC, GLC) and spectroscopy (NMR, MS) , new products are reported at rapid intervals. Because only 5-10% of all higher plants, which consist of over 300,000 species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large.

It is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. Within a taxon secondary metabolites often share a common distribution pattern and are therefore of some importance for phytochemical systematics. Classic taxonomy, however, has taken little account of alkaloid distribution: If the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. Because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated (233).

For many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of Nature (34, 35) . In 1887 and 1888, Errera and Stahl (92, 308, 504) published the idea that natural products are used by plants for chemical defense against herbivores. Since the leading plant physiologists of that time were mostly anti-Darwinian, they were not willing to accept the defense argument, which was too much in line with the Darwinian concept. Therefore, this early defense concept was negated and remained forgotten for nearly 60 years. In 1959, Fraenkel(10) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. During the next three decades this concept was improved experimentally, and we can summarize the present situation as follows Although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century (34, 35) . In many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy).

In some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the Centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes (15, 17) . Physiological roles, such as UV protection [by flavonoids or coumarins (4,17)], nitrogen transport or storage (14, 36, 37) , or photosynthesis (carotenoids), may be an additional function.

Allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and 

antimicrobial). Thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). To understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach.

It might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. However, we have to understand and accept that chemical defense is not an absolute process. Rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. Plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. During evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them (14, 15, 32) . This is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. The number of these "specialists" is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. We can compare this situation with our immune system: It works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. Nobody would call the immune system and antibodies useless because of these few adapted specialists! We should adopt the same argument when we consider plants' defenses by secondary metabolites (2) .

Since secondary metabolites have evolved in Nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. In addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds.

About 20-30% of higher plants accumulate alkaloids (505,506). The incidence of alkaloid production varies between taxa to some degree; for example, about 60-70% of species of the Solanaceae and Apocynaceae are 6 MICHAEL WINK alkaloidal, whereas other families contain few alkaloid-producing species. Some alkaloids have a wide distribution in Nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. Alkaloids are not restricted to higher plants (although they are here most numerous); they are also present in club mosses (Lycopodium), horsetails (Equisetum), fungi, and animals such as marine worms (e.g., Nereidae), bryozoans, insects (e.g., Coccinellidae, Solenopsidae), amphibians (toads, frogs, salamanders), and fishes.

Alkaloids thus represent one of the largest groups of natural products, with over 10,000 known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan (38) (39) (40) . In addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates ( [38] [39] [40] . Whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. This is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals.

Alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as W. 0. James and Kurt Mothes ( 3 4 3 , 505,526)l. Because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. The waste product argument probably came from animal physiology: Carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. A similar situation or need, however, is not applicable for plants. In fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves (2,37,506). If alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. On the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals (16,28,494496) . Thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. This idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and Homo sapiens.

As a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. (1) The alkaloid should have significant effects against microbes and/or animals in bioassays. 7 (2) The compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. (3) The compound should be present in the plant at the right time and the right place. (4) Evidence should be provided that a particular compound is indeed important for the fitness of a plant.

Although more than 10,000 alkaloids are known, only few (-2-5%) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. In most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities.

Furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. These objections have to be kept in mind, because an alkaloid is sometimes termed "inactive" in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). In many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. If the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. Another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. However, if a compound is active against Escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. Nevertheless, most of the data obtained in these studies (Tables I-VIII ) provide important information which at present permits extrapolation to the function of alkaloids in plants.

In this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. In the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail.

It is nearly impossible to cover the literature exhaustively. Therefore, an overview of the allelochemical properties of alkaloids is presented. Because of the large amount of data (literature up to 1990 is included), the selection of examples must remain subjective to some degree. Nevertheless, the author would be grateful to receive information or publications about relevant omissions.

Because Homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (Tables I through V) and alkaloid-containing plants. Previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products (38, 39) . In the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates.

Among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. Entomologists estimate that the number of insects is at least 1 million, but tropical rain forests may harbor up to 20-30 million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists.

Most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( I ,4,10,14,15, 28-33, 494496,503) . Whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. In the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). At the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds.

The close association between plants, especially the angiosperms, and insects evolved during the last 200 million years. Some scientists have called this phenomenon a "coevolutionary" process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved (18,505,506). Applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. Times can also be much longer. If plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. For example, Lupinus polyphyllus from North America has a number of specialized herbivores, but is rarely attacked by herbivores in Europe. This lupine left its enemies behind when it was transferred to Europe three centuries ago. About 10 years ago, however, the North American lupine aphid (Macrosiphum albifrons) was introduced to Europe accidentally. This aphid is specialized to alkaloid-rich lupines with lupanine as a major alkaloid. At present, this aphid has spread over most of Europe and is now colonizing its former host, L. polyphyllus (2, 503) .

Insect herbivores can be divided into two large groups whose strategies with respect to the plant's defense chemistry differ substantially (15). The polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagous insects are often specialized on one or a small number of (often systematically related) hosts.

Polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors (501) that allow the distinction between plants with high or low amounts of "toxins." The receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates (507). These "generalists," as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see Table XI ). Alternatively, they change host plants rapidly and thus avoid intoxication. In addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products (4,15,17,508).

In contrast, mono-and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. For these "specialists" the originally noxious defense compounds are often attractive feeding and oviposition stimulants. These insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes (1,4,10-12,144 7,28,31,33,494-496). These observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as Mothes (35) and Robinson (505). However, these specialized insects are exceptions to the general rule. For these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants (32) . As compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species.

Quite a number of alkaloids have been tested toward herbivorous insects (Table I ). In general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>I%, w/w). Given the choice, insects tend to select a diet with no or only a small dose of alkaloids. Also, 

specialists avoid most "toxins" except those of their host plants. These data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones.

If insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid (15,32). In this case we have the chance to test the toxicity of an ingested alkaloid. If insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection ( Table I) .

As can be seen from Table I a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. Only the specialists can tolerate the respective alkaloids. The tobacco hornworm (Manduca sexta), for example, can grow on a diet with more than 1% nicotine without any adverse effects. Most of the nicotine is either degraded or directly eliminated via the Malpighian tubules and in feces (182). Because nicotine binds to the acetylcholine (ACH) receptor, it is likely that in Manduca this receptor has been modified in such a way that ACH can still bind, but not nicotine (so-called target site modification).

The toxic effects of alkaloids in insects (Table I) can be caused by their interference with diverse cellular and intracellular targets. Since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see Table IV ). With some caution we can extrapolate to insect toxicity.

Because Homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. Many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. Examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. It should be recalled that these taste properties are not identical for all animals. For example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch (185). Conversely, fragrances that are attractive to us are highly repellent to geese (185). Even within a given population taste can differ significantly. It has been observed that a substantial proportion of Homo sapiens cannot detect the smell of HCN, whereas others are highly sensitive. Furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles.

Bitterness varies with the chemical structure of an alkaloid. With the quinolizidine alkaloids (QAs) the following scale was assessed for man: Mean detection levels are 0.00085% for sparteine, 0.0021% for lupanine, and 0.017% for hydroxylupanine (503). Whereas we know a few parameters of olfactory qualities in Homo sapiens, often much less or hardly anything is known for most other vertebrates.

Alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. For a number of alkaloids the respective LD,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. Table I1 presents an overview for 132 alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. Toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per 0s (P.o.)]. Also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin A, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin (1, 28, 29, 259) . Although the general toxicity of alkaloids differs from species to species, the data in Table I1 generally show that many alkaloids are more or less toxic to vertebrates.

The toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly 180 alkaloids (Table 111 ). These data have been obtained by screening many natural products for anticancer activity. However, an alkaloid that can kill a cancer cell is usually also toxic for "normal" cells. Therefore, the data shown in Table I11 are another indication of the general toxicity of alkaloids toward animals. Because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism.

For a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. We can distinguish molecular targets and processes that are important for all cells, such as synthesis of DNA, RNA, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (Table IV ). In the following we discuss some of these toxic effects. a. Cellular Targets Nucleic Acids. DNA, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. It is not surprising that a number of secondary metabolites have been selected during evolution which interact with DNA or DNAprocessing enzymes. Some alkaloids bind to or intercalate with DNA/RNA (Table IV) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (Table V) : 9-methoxyellipticine, dictamnine, ellipticine, harmane alkaloids, melinone F, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (PAS), cycasin, olivacine, etc. Many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of AT and GC base pairs. Other alkaloids act at the level of DNA and RNA polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. Whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (Table V) . Besides basic data obtained in Salmonella or Drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. Anagyrine, anabasine, and coniine cause "crooked calf disease" if pregnant cows or sheep feed on these alkaloids during the first period of gestation (329,341,348,349,351,352) . The offspring born show strong malformation of the legs. Some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by Veratrum species, cause the formation of a central large cyclopean eye (329-330, an observation that was probably made by the ancient Greeks and thus led to the mythical figure of the cyclops. It is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. In effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores.

Another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (Tables IV and V) . The alkaloid aristolochic acid, which is produced by plants of the genus Aristolochia, is carcinogenic. The mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine (344,345) , because of its NO, group. Pyrrolizidine alkaloids and their N-oxides, which are abundantly produced by members of the Asteraceae and Boraginaceae but also occur in the families Apocynaceae, Celestraceae, Elaeocarpaceae, Euphorbiaceae, Fabaceae, Orchidaceae, Poaceae, Ranunculaceae, Rhizo- esterified (425,426) . After oral intake, the N-oxides are reduced by bacteria in the gut. The lipophilic alkaloid base is resorbed and transported to the liver, where it is "detoxified" by microsomal enzymes. As a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. The alkaloid can then cross-link DNA and RNA and thus cause mutagenic or carcinogenic effects (especially in the liver) (502). Thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. The PA story is very intriguing, since it shows how ingenious Nature was in the "arms race." The herbivores invented detoxifying enzymes, and Nature the compound which is activated by this process. A herbivore feeding on PA-containing plants will eventually die, usually without reproducing properly. Only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. The protection due to PA can easily be seen on meadows, where Senecio and other PA-containing plants are usually not taken by cows and sheep, at least as long other food is available.

Protein biosynthesis is essential for all cells and thus another important target. Indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (Table IV) , such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. For lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyl-tRNA with amino acids, as well as the elongation step. The inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected (503).

Electron chains. The respiratory chain and ATP synthesis in mitochondria demand the controlled flux of electrons. This target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, CH,-(CH2),,-2,6-methyl-piperidines, capsaicin, the hydroxamic acid DIMBOA, and solenopsine. As mentioned before, however, only a few alkaloids have been evaluated in this context (Table V) .

Biomembranes and transport processes. A cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta-

tion that provides separated reaction chambers. Because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. The controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems.

These complex systems are also targets of many natural products (Table  IV) . Disturbance of membrane stability is achieved by 9-methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. Steroidal alkaloids, such as solanine and tomatine, which are present in many members of the Solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky.

Cells carefully control the homeostasis of their ion concentrations by the action of ion channels (Na+,K+, Ca2+ channels) and through Na+,K+-ATPase and Ca2+-ATPase. These channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. Inhibition of transport processes (ion channels, carriers) is achieved by (Table IV) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin.

A special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (NA), serotonin, dopamine, glycine, and acetylcholine (ACH). We can distinguish two types. Type 1 is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ACH-receptor. In Type 2 the receptor is an integral protein. When a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent Gprotein molecule, which consists of three subunits. The a subunit then activates the enzyme adenylate cyclase, which in turn produces cAMP from ATP. The cAMP molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ACH receptor).

A number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. Targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. Alkaloids relevant here include (Table IV) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga-

lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine, anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. A number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly.

Cytoskeleton. Many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. A number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (Table IV) , namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol.

Colchicine, the major alkaloid of Colchicum autumnale (Liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. As a consequence, chromosomes are no longer separated, leading to polyploidy . Whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. Because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. The derivative colcemide is less toxic and can be employed in the treatment of certain cancers (312). Also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. For normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity .

Another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from Catharanthus roseus (Apocynaceae). These alkaloids also bind to tubulin (312). Both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias.

From Taxus baccata (Taxaceae) the alkaloid taxol has been isolated. Taxol also affects the architecture of microtubules in inhibiting their disassembly (322). Nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin (312). In conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule.

Enzyme inhibition. The inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see Table IV ). Briefly, inhibition of CAMP metabolism (which is important for signal transduction and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by 1-ethyl-P-carboline, P-carboline-1-propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. Inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (Table IV) b. Action at Organ Level. Whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too.

Central nervous system and neuromuscular junction. A remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (Table IV) . The cellular interactions have been discussed above. Disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects.

A herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in Nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. Thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. Some individuals of Homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically.

The activity of muscles is controlled by ACH and NA. It is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. In the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis.

Alkaloids which activate neuromuscular action (so-called parasympathomimetics) include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. Inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) (312) . Skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. The compounds are usually considered to be strong poisons, and it is obvious that they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare LD,, values in Table 11 ).

Inhibition of digestive processes. Food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. The next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. The ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms (312).

Another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. Relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (Table IV) .

Nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. In the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. The liver is also the main site for detoxification of xenobiotics. Lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids (312). These conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination.

Both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (Tables IV and V) . The alkaloids are activated during the detoxification process, and this can lead to liver cancer. Also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins.

Many alkaloids and other allelochemicals are known for their diuretic activity (312). For an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. Since Na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially.

Disturbance of reproduction. Quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. Antihormonal effects could be achieved by mimicking the structure of sexual hormones. These effects are not known for alkaloids yet, but have been confirmed for other natural products. Estrogenic properties have been reported for coumarins, which di-merize to dicoumarols, and isoflavones (4,17) . Insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern Polypodium uulgare, or azadirachtin from the neem tree (4,17) . Juvenile hormone is mimicked by a number of terpenes, present in some Coniferae. Spermatogenesis is reduced by gossypol from cottonseed oil (17) .

The next target is the gestation process itself. As outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo ( Table V) . The last step would be the premature abortion of the embryo. This dramatic activity has been reported for a number of allelochemicals, such as mono-and sesquiterpenes and alkaloids. Some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids (312) .

The antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. Nevertheless, they are defense strategies with long-term consequences. Blood and circulatory system. All animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. Inhibitors of the driving force for this process, the heart muscle, have already been discussed. However, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine (312) .

Some allelochemicals have hemolytic properties, such as saponins. If resorbed, these compounds complex membrane sterols and make the cells leaky. Steroidal alkaloids from Solanum or Veratrum species display this sort of activity as well as influencing ion channels (Table IV) .

Allergenic effects. A number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. Common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds (4,17,312) . Activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. The function of alkaloids in this context is hardly known.

This selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. However, most of the potential targets for the 10,000 alkaloids known at present remain to be established. If no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property.

Summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly.

Dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay (7) . How is this achieved? The aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products (4,7) . This cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. In perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues.

The only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. At the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas (4,17) . In addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity.

Secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. These studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. It may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. In the pharmaceutical context we search for very active compounds which can be employed at low concentrations. Therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. These precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids.

Secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono-and disesquiterpenes, and last but not least, alkaloids (4,17,42,149,322) .

In Table VI 183 alkaloids are tabulated for which antibacterial activities have been detected. The alkaloids usually affect more gram-positive than gram-negative bacteria. Especially well represented are alkaloids which 

3'-Hydroxytabernamine 16Hydroxytetrahydrosecamine 10 Tetrandrine   Thalicarpine  Thalicerbine  Thalidasine  Thalidezine  Thaliglucinone  Thalistine  Thalistyline  Thalmelatine  Thalmirabine  Thalphenine  Thalrugosaminine  Thalrugosidine  Thalrugosine  Tubocurarine   3 ( I +. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); AD, agar diffusion, AL, agar dilution; BG, biogram; LD, liquid culture; MIC, minimal inhibitory concentration; PD, paper disk; SP, suspension; TLC, TLC disk test according to Wolters and Eilert (95) . If more than one value is given, the data refer to different bacterial species tested. derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups (42,50,59,60,63,68,75-84) . Some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline (69), dictamnine ( 9 3 , fagarine (95), stemmadine   (70), yuehchukene (71), liriodenine ( 8 3 , lysicamine (82), oxonantenine   (82), sanguinarine (87), solacasine (50,92) , rutacridone epoxide (95), tryptanthrine (I@#), and tuberin (107,108) (Table VI) .

In many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. Usually yeasts and Candidu are used as test organisms (Table VII) . Table VII lists ( I 19,121), thaliglucinone (79), demissidine  (126,127), solacasine (92), soladulcidine (126,127), solasodine (26,127)tidine (126,127), tomatine (42,126) , verazine (124), cryptopleurine (133) hydroxyrutacridone epoxide ( 9 3 , tryptanthrine (104), and tuberin (107) .

Whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see Table IV ), relevant information for plant-derived compounds is scant. However, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (Table IV) that may be responsible for the antibiotic effects observed. The following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare Tables VI and VII  with Tables IV and V) . Protein biosynthesis in ribosomes is affected by sparteine (56,423, lupanine, angustifoline, 13-tigloyloxylupanine, and 13hydroxylupanine (56,98,99,417,421,422) . Intercalation or binding to DNA is influenced by fagaronine, dictamnine (367), harman alkaloids (376,378) [binding to DNA is light dependent (66)], berberine (396-3981, chelerythrine (400), and sanguinarine (400,409) ; these compounds may thus inhibit important processes such as DNA replication and RNA transcription that are also vital for microorganisms. The stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( 4 3 3 , solanine (430,432,433), and solasonine (435) , thus leading to an uncontrolled flux of metabolites and ions into microbial cells. Inhibition of metabolically important enzymes is affected by berber- ine (399), chelerythrine (259,401), chelidonine (402), palmatine (399), sanguinarine (143,259), solacongestidine (434) , and papaverine.

In contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (Table IV) , such an interaction has not been described for plantderived compounds. Since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids.

We can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. Inducing agents, which have been termed "elicitors" by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents (4,17,22-24) . The induced compounds are called "phytoalexins," which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. In another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below).

Many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. Using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors (4,17,22-24) . Among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see Table XI ). Quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense (503) (see Table I ). On wounding, QA production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (Table XI) (2,184,503) .

The reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail (4,17,22-24) . These studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: Within 20 min of elicitation, mRNAs coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites (4,17,22-24) . Similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated.

We assume that a substantial number of the 10,000alkaloids have antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not Table VI . If a range is given, the first value gives a 10% inhibition, the second value a 100% inhibition. specialized on a particular host plant. However, alkaloid production does not necessarily have to be involved with antimicrobial defense. For example, Phytophthora or Fusarium will attack alkaloid-rich plants of Nicotiana, Solanum esculentum, and S . tuberosum. Cladosporium and Fusarium can develop in nutrient-containing media enriched with alkaloids, and Aspergillus niger can utilize alkaloids as a nitrogen source (506).

In addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. In these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. We suggest that these specialists have adapted to the chemistry of their host plants. Mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). These mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. Some associations between plants and fungi are symbiotic in nature, such as Rhizobia in root nodules of legumes or microrhizal fungi in many species. In lupines, nitrogen-fixing Rhizobia are present both in alkaloid-rich and alkaloid-free plants. They must therefore be able to tolerate the alkaloids, which are also present in the root. Alkaloid production in lupines is more or less unaffected whether or not the plants harbor Rhizobia (185,506) .

An ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. Graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi (4). A similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity.

From the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (Tables I1 and  111 ). Since many alkaloids are antibacterial and antifungal (Tables VI and  VII) and are present in plants at relatively high concentrations (Section IILA), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. It should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes (2-4,7) . A cooperative, or even synergistic, process could thus be operating.

C. ANTIVIRAL PROPERTIES Plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (Heteroptera). Resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place.

The assessment of antiviral activity is relatively difficult. As a result, only a few investigators have studied the influence of alkaloids on virus multiplication. Nevertheless, at least 45 alkaloids have been reported with antiviral properties (Table VIII) . Only sparteine (527) and cinchonidine (142) have been tested for antiviral activities against a plant virus, the potato X virus. All other evidence for antiviral activities (Table VIII) Table VIII are difficult to interpret at present. Polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum-and Golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins.

Because alkaloids often deter the feeding of insects, such as aphids and bugs (Table I ), viral infection rates may be reduced in alkaloid-rich plants. Such a correlation exists for alkaloid-rich lupines (so-called bitter 141  109  109  147  148  147  147  148  146  147  109  147  147  148  147  148  150 150 150 150 lupines) and low-alkaloid varieties (the so-called sweet lupines) (see Table XII) .

Plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. This phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense (4,17,498-500) . A number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated.

It was observed by Molisch in 1937 (497) that plants can also influence each other by their constituent natural products, and he coined the term "allelopathy" for this process. Secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain (4,17) . Both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. Allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments (4,17,19,497-500) , but how it operates in ecosystems is still often a matter of controversy. It is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. Thus allelochemicals might never reach concentrations high enough to be allelopathic.

Allelopathic natural products have been recorded in all classes of secondary metabolites. Few research groups have studied the effect of alkaloids in this context, but at least 50 alkaloids have been reported with allelopathic properties (Table IX) . As can be seen from Table IX , allelopathic activities can be found within nearly all structural types of alkaloids. At higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. More sensitive, however, is the growth of the radicle and hypocotyl. They respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. In species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in Colchicum autumnale (503, 506) . It is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms. alkaloids (56,166,378), berberine (396-398), sanguinarine (400,409) and Veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine (404) and quinolizidine alkaloids (56,99,416-418,422) Tables IV and  IX) .

The inhibitory action of quinolizidine alkaloids should be explained in this context (184, 503) . They are very abundant in lupine seeds (up to 3-8% dry weight). During germination, 13-hydroxylupanine is converted to ester alkaloids, such as 13-tigloyloxylupanine. The latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic QA. These alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. When lupine and Lepidium seeds were grown together in the same pot, growth of the Lepidium seedlings was much reduced and inhibited, indicating that QAs may also be relevant in the ecological context (184) .

Although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from Table IX that alkaloids can be toxic to plants, probably by interfering with basic metabolic or molecular processes.

Although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (Tables  I-IX) is far from complete, we can safely state that alkaloids have potent deterrent or poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. The next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in Nature.

Because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. It is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. We must remember that alkaloid composition and levels are often tissue or organ specific (4,25,38) . They may vary during the day [a diurnal cycle has been observed for QAs and tropane alkaloids (185,503,506)l or during the vegetation period (39.  505,506) . Furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. Unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. In addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. However, herbivores or pathogens do not feed on the dry plant in general, but on the "wet" fresh material. In the context of chemical ecology we urgently need data on a fresh weight basis. As an approximation, in this chapter we use a conversion factor of 10 to convert dry weight to fresh weight data if only the dry weight data are available.

Summarizing the relevant phytochemical literature, we find that alkaloid levels are between 0.1 and 15% (dry weight), which is equivalent to O.OI-lS%fresh weight, or 0.1-15 mg/gfresh weight. For plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (Table X ) , which fall in the range deduced before. We have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore (2, 184, 503, 527) . This means that plants obviously play safe and have stored more defense chemicals than actually needed. If we look at the ED,, and LD,, values given in Tables 1 through IX, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances.

It seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. Achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. Most allelochemicals would probably 

interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made (25). Whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (QAs, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) (4,25) .

In this context it should be recalled that many alkaloids are charged molecules at cellular pH and do not diffuse across biomembranes easily. During recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. The next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. Proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally (503,510,512) . Thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness.

As a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. Translocation via the phloem, xylem, or apoplastically must have therefore occurred. Phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids (36,39,511) .

If the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. Alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis (25, 38, 39) . In lupines, but also in other species (486,4891, alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between 20 and 200 mM (Table  X) , which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. The accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way.

A number of plants produce laticifers filled with latex. For example, isoquinoline alkaloids in the family Papaveraceae are abundant in the latex (39), where they are sequestered in many small latex vesicles. In latex vesicles of Chelidonium mujus the concentration of protoberberine and benzophenanthridine alkaloids can be in the range of 0.6-1.2 M, which is achieved by their complexation with equal amounts of chelidonic acid (512). If a herbivore wounds such a plant, the latex spills out immediately. Besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, Chelidonium plants are hardly attacked by herbivores. In addition, as these alkaloids are also highly antimicrobial (Table IV) , the site of wounding is quickly sealed and impregnated with natural antibiotics. Other well-known plants that have biologically active alkaloids in their latex belong to the families Papaveraceae (genera Papauer, Macleya, and Sanguinaria) and Campanulaceae (genus Lobelia) (39) .

It is intuitively plausible that a valuable plant organ must be more protected than others. Alkaloid levels are usually highest during the time of flowering and fruit/seed formation. In annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. All these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. Spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones (15); examples are isoquinoline alkaloids in cacti or QAs in legumes (184) . If a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds (15,184); thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis.

These few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. Thus, specialized locations must be regarded as adaptive.

Alkaloid concentrations can fluctuate during the vegetation period, or even during a day (36,506). but in biochemical terms their biosynthesis and accumulation are constitutive processes. This ensures that a certain level of defensive compounds is present at any time. Furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. The same seems to be true for a defense compound. An alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. Only by replacing these molecules continuously can the presence of the active compounds be guaranteed. For example, it was suggested that nicotine has a half-life of 24 hr in Nicotiana plants, and that more than 10% of the CO, fixed passes through this alkaloid (505).

In other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. These compounds are termed "phytoalexins" in phytopathology (22) (23) (24) . Classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. Using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin-6-one, rutacridone alkaloids) when challenged with elicitors from bacterial or fungal cell walls (Table XI) . But what is the situation after herbivory? When plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. The situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. Here the de nouo production of an allelochemical would be worthwhile. There are indeed some preliminary experimental data that support this view.

In Liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present (506). In tobacco the produc- " CC, Cell culture; PL, plant tion of nicotine, in lupines that of QAs, and in Atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of 5. Whereas the response was seen after 2-4 hr in lupines, it took days in Nicotiunu and in Atropei (Table XI) . We suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system.

The induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. Many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future.

Although the physiology and metabolism of most alkaloids are extremely intricate (3839) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. In other words, the alkaloids are present at the right time, the right place, and the right concentration.

The aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. Besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. We suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart.

For one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed (2,184,484,503,527) . As mentioned before, QAs constitute the main secondary products of many members of the Leguminosae, especially in the genera Lirpinus, Genistu, Cyfisiis, Bccptisiu, Thrrmopsis, Sophoru, Ormosici, and others (503).

Lupines have relatively large seeds which contain up to 40-50% protein, up to 20% lipids, and 2-8% alkaloids. To use lupine seed for animal or human nutrition, Homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. This habit has been reported for the Egyptians and Greeks in the Old World, and for the Indians and Incas of the New World. The resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. In Mediterranean countries people still process lupines in the old way, and sometimes the seeds are salted afterward and served as an appetizer, comparable to peanuts.

At the turn of the twentieth century, German plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. Although sweet lupines are extremely rare in Nature ( 1 in >100.000), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than 0.01% exist for Lupinus albus, L. mutabilis, L . luteus, L. angustifolius, and L . polyphyllus. As far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. This offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores.

The results of these experiments were clearcut (2,184503,506,527 ) (Table XU). In the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. When lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores (2,184,503,527) . Rabbits (Cuniculus europaeus) and hares (Lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. Before dying rabbits will certainly try to eat bitter lupines.

A similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (Table XII) , namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. The alkaloid-poor variety of L . luteus also became a host of Acyrthosiphon pisii (506). In Poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. The disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants' blossoming. Thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. Infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production (506). This observation again stresses the importance of alkaloids for the fitness of lupines.

Plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail. These experiments and observations clearly prove the importance of QAs for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects.

The lupine example also tells us about the standard philosophy and problems of plant breeding. With our present knowledge on the ecological importance of QAs for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. In order to grow them we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. It can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced (2). We can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats.

There are, however, alternatives. Taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). At present a few companies are actively exploring these possibilities. One idea is to produce pure protein, lipids, dietary fibers from bitter seeds. A spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide (185,503) .

It is evident, however, that each plant has developed its own strategy for survival. If all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. This specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. We regularly see one to five main alkaloids in a plant, but also several (up to 80) minor alkaloids. This qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. Normally, we classify the compounds as belonging to one or two chemical groups. This does not mean, however, that their biological activities are identical. On the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. In consequence, its toxicity may be higher (see QAs in Table I ). Thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. As the composition of these chemicals changes, it is even more difficult for them to cope. Therefore, we suggest that structural diversity and continuous variation are means by which Nature counteracts the adaptation of specialists.

In medicine, we do a similar thing if we want to control microbial diseases. To overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. If only one antibiotic were given to all patients, the development of resistance would be much favored.

It has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only 30% of all plants do). These authors, such as Robinson ( 5 0 3 , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. Only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. The arguments of Robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in Nature; however, these plants are not known. From an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens.

Although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. Since Nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. For quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (Tables I-X) .

Plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. It is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts (4, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . As compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a "status quo" or equilibrium can be observed between the specialists (or parasites) and their hosts. A specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival.

Host plant-specific specialists occur within bacteria, fungi, and herbivores. The interaction of the former two groups is a central topic for plant pathologists. They often find that susceptible and nonsusceptible microbe strains exist. In most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-ate them. Many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. It is assumed that these phytotoxins serve to weaken the host plants' defense, but may be this is not the whole story.

Many grasses are infected with fungi that produce ergot alkaloids. It has been assumed that these fungi (e.g., Clauiceps) are proper parasites. In recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature (513). Ergot alkaloids are strong vertebrate toxins (Tables I-IV) ; they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (Table IV) . In fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. This means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. Since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. We expect that similar relationships are likely to be detected in the future.

As mentioned earlier, a large number of mono-and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. In general, we can see the following main schemes (4,15, 17, 32, 507, 508) . In Type 1 adaptations, a species "learns" (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the Malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. Alternatively, in Type 2 strategies a species not only tolerates a plants' defense compound, but exploits it for its own defense or for other purposes, such as pheromones I7,494496,506) .

Examples of Type 1 include Manduca sexra, whose larvae live on Nicoriana and other solanaceous plants. The alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces (182). In addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in "normal" animals (17). The potato beetle (Leptinotarsa decernlineata) lives on Solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, The bruchid beetle Callosohruchus fasciarus predates seeds of QA-rich plants, such as Laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces (492).

Examples of Type 2 are to some degree more interesting. In a number of plants alkaloids are translocated via the phloem (511). When aphids live on these plants they are in direct contact with the alkaloids present. A number of examples are known at present which show that adapted aphids can store the dietary alkaloids. Examples are the quinolizidines in Aphis cytisorum, A. genistae, and Macrosiphum albifrons, the pyrrolizidines in Aphis jacobaea, A . cacaliaster, and aconitine in Aphis aconiti (185, 511) . For alkaloid-storing M . albifrons it was shown experimentally that the QAs stored provide protection against carnivorous beetles, such as Carabus problematicus or Coccinella septempunctata (465, 503) . Acyrthosiphon spartii prefers sparteine-rich Cytisus scoparius plants (506); although it is likely that this species also stores QAs, it has not been demonstrated to do so.

Larvae of the pyralid moth Uresiphita reversalis live on QA-producing plants, such as Teline monspessulana. The larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. The uptake is both specific and selective and is achieved by a carrier mechanism. Whereas alkaloids of the 10-oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the 10oxosparteines being eliminated with the feces (503,514) . The larvae gain some protection from storing QAs, as was shown in experiments with predatory ants and wasps. When the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage (503,514). The emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. In contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants (503,514) .

The larvae of the blue butterfly (Plebejus icaroides) feed only on lupines, rich in alkaloids. As far as we know, the larvae do not sequester or store the dietary alkaloids (506). Helopeltis feeds on Cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense (506). Larvae of the butterflies Pachlioptera aristolochiae, Zerynthia polyxena, Ornithoptera priamus, and Battus philenor live on Arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound (4,28,236) .

The best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families Asteraceae and Boraginaceae (502). Some arctiid larvae of Tyria jacobaea, Cycnia mendica, Amphicallia bellafrix, Arginia cribaria, and Arctia caja were shown to store the dietary PAS and exploit them for their own defense (4,17,28,31,222-224,237) . In Tyria jacobaea, Arctia caja, Diacrisia sannio, Phragmatobia fuligonosa, and Callimorpha dominula PAS are taken up and stored in the integument (523).

Monarch butterflies (e.g., Danaus plexipus) combine two sets of natural compounds. Larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. Adult butterflies visit plants with PAS, where they collect PAS that are converted to pheromones or transferred to their eggs (4,f 7,31,33,36f,515) . A similar PA utilization scheme was observed with larvae of the moth Utetheisa ornatrix (367, 516) , where the compounds were shown to be deterrent for spiders and birds (225, 525) . The chrysomelid beetle Oreina feeds on PA-containing plants, such as Adenostyles, and stores the dietary PAS in the defense fluid (463,524).

In the arctiid Creatonotos transiens was observed an advanced exploitation of PAS (31,33,429,517-521) . The alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. Dietary pyrrolizidine N-oxides are resorbed by carrier-mediated transport. After resorption, free PAS are converted to the respective N-oxides and (7S)-heliotrine to (7R)-heliotrine. The latter form is later converted to a male pheromone, (7R)-hydroxydanaidal. PAS are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. In the adult moth, however, the PAS are mobilized. In the female adult, PAS are translocated into the ovary and subsequently into the eggs. In the male, PAS are necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of PA-derived pheromones, which are dissipated from these coremata. In addition, PAS are transferred into the spermatophore and thus donated to the female. A significant amount of PAS is further transferred to the eggs, which thus obtain chemical protection from the PAS previously acquired by both male and female larvae.

Marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (Table IV). These algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins (4,17,28,29,494,495) . As a consequence, these animals have acquired chemical defense compounds, which they can use against predators.

This discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. These insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. However, many of the respective plant-insect interactions have not yet been studied, and it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in Nature than anticipated.

Whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. For example, grouse (Lagopus lagopus) or capercaillies (Tetra0 urogallus) prefer plants of the families of Ericaceae or Coniferae, and crossbills seeds of Picea and Abies species, which are rich in terpenes. The Australian koala is oligophagous and prefers terpene-rich species of the genus Eucalyptus.

For approximately 65 million years, the only true herbivorous vertebrates have been the mammals. The Mesozoic reptiles disappeared following the mesophytic flora. Birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source (18).

Although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. The strategies of the polyphagous species include the following.

Avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic or poisonous by man) by olfaction or taste discrimination. Often such compounds may be described as bitter, pungent, bad smelling, or in some other way repellent.

2. Sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin.

3. Detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. This evolutionary trait is very helpful for Homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. Carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons (505). It was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals.

Some animals, such as monkeys, parrots, or geese, ingest soil. For geese (185) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids (185). This procedure would reduce the allelochemical content available for resorption. 5. Animals are intelligent and can learn. The role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species.

For most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored.

Sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals.

More evidence on this subject is available for Homo sapiens, who has evolved a number of "tricks," some of them obviously not anticipated by evolution. First, man tends to avoid food with bitter, pungent, or strongly scented ingredients. As a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. Second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. Furthermore, some of these enzymes, such as cytochrome P.450 oxidase, is inducible by dietary xenobiotics. Third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) Many fruits or vegetables are peeled. As many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) Most food is boiled in water. This leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. Many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) South American Indians ingest clay when alkaloid-rich potato tubers are on the menu. Since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet (522) . (d) Man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. From the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. These plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides.

In general, only a few plants are exploited by man as food, as compared to the 300,000 species present on our planet. This means that even Homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses.

In this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids (4,17,28,494-496) . These animals have the common feature that they are usually slow-moving, soft-bodied organisms. Marine animals, such as mol-luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals (4.17,28,29,221,226,229,232,233,234,495) .

A number of nemertine worms, such as Amphiporus or Nereis, produce alkaloids such as 2,3-bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( 4 1 7,28,230,226,) . Arthropod-made alkaloids include glomerine and homoglomerine in Glomerus (215) , adaline in Adalia (227), coccinelline, euphococcinine, and derivatives in Coccinella, Epilachna, and other coccinellid beetles (28,226,227,235) , and stenusine in Stenus (215) , which are considered to be antipredatory compounds (4,17,28,494-496) .

Solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. These alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (Table IV) (28, 494) .

Many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines (4,17,231,494) .

Not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class Amphibia. Tree frogs of the genus Dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins A-C, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (Table IV) (4,17,28) . Natives have used the alkaloids as arrow poisons. Similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus Pitohui (528) . Salamanders, Salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group (4,17,28). Salamandrine is both an animal toxic (paralytic) and an antibiotic. Toads (Bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin (4,17,28). Except for bufotoxin, the other chemicals are, or mimic, neurotransmitters.

These examples show that alkaloids found in animals can either be derived from dietary sources (see Section 111,D,2) or be made endogenously. Common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. In animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals (28,29,494,495) , but not so those with weapons, armor, or the possibility for an immediate flight. Plants merely developed a similar strategy as these "unprotected"

animal species. In this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned.

Evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed (34,35), but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. These molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (Table IV; Section II,A,3,a).

On the other hand, microorganisms and herbivores rely on plants as a food source. Since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. Many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. In addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. In insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. Alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant.

The overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants.

Defense in Animals

Introduction to Ecological Biochemistry

Defense Mechanisms in Plants

Herbivores: Their Interaction with Secondary 16

Okologische Biochemie

Allelochemicals: Role in Agriculture and Forestry

Cell Culture and Somatic Cell Genetics of Plants" (F. Constabel and 25. M. Wink

Gifttiere und ihre WafTen

Perspectives in Chemoreception Behavior

Biochemie und Physiologie der sekundaren Pflanzenstoffe

Plant Metabolites

Biosynthese der Alkaloide

Biochemistry of Alkaloids

The Alkaloids: The Fundamental Chemistry

Antiseptika

Baerheim Svendsen

Lloydia 51. N. M. Rojas Hernandez

Vallejos and 0. A. Roveri

The Merck Index

Proc. 3rd Inf. Lupin Conf

Insect Biology in the Future

Micromolecular Evolution, Systematics and Ecology

Phytochernical Ecology: Allelochernicals, Mycotoxins and Insect Pheromones

Phytochem

Biogene Gifte

Rozniki Nauk Rolnikzych

Die Gift-und Arzneipflanzen in Mitteleuropa

Antibiotics: Mechanism of Action of Antimicrobial and Antitumour Agents

Antimicrob. Agents Chemo

Pharmazeutische Biologie 11, Biogene Arzneistoffe

Pharmazeutische Biologie

Drug Use in Pregnancy

The Alkaloids

Handbook of Enzyme Inhibitions

Cold Spring Harbor Conf

Primary and Secondary Metabolism of Plant Cell Cultures

Chemical Defenses of Arthropods

Der Einfluss einer Pflanze auf die Andere-Allelopathie

The Science of Allelopathy

Allelopathy

Lectures on Insect Olfaction

Focus on Insect-Plant Interactions

Alkaloid Biology and Metabolism in Plants

Molecular Aspects of Insect Plant Associations

Methods of Plant Biochemistry

Secondary Products in Plant Tissue Culture

Proc. Natl

The Alkaloids

The work of the author was supported by the Deutsche Forschungsgemeinschaft. I thank Dr. Th. Twardowski for reading an earlier draft of the manuscript.

