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Antifeedant Substances in Plants
Antifeedant Substances in Plants CB Purrington, Swarthmore College, PA, USA Ó 2017 Elsevier Ltd. All rights reserved. This article is a reproduction of the previous edition article by C.B. Purrington, volume 3, pp. 1140–1145, Ó 2003, Elsevier Ltd.
Introduction Although the literature on antifeedancy is dispersed rather thinly under a variety of cognates and related terms (e.g., antifeeding compound, feeding deterrent, feeding rejectant, feeding suppressant, feeding inhibitor, gustatory repellent, phagodepressant), it is unified by the hope that antifeedant sprays might someday become an important pest management tool. In particular, it is hoped that antifeedants might lack the dangers associated with the use of natural or engineered pesticides, both of which frequently have detectable if not substantial toxicity toward other animals, such as ourselves. Unfortunately, most of the literature on this topic has suggested that antifeedants are simply not effective, or not reliably so, against many of the important pests of common crops. Furthermore, insects are capable of habituating to the presence of antifeedants after a given amount of time. For this and several other reasons, the marketing of specific antifeedants has had little success. Although it is easy to be pessimistic about the commercial viability of antifeedants, research on these compounds is producing valuable insight into the ecology and evolution of plant resistance strategies, as well as the corresponding suite of adaptations in herbivores. Knowledge of the presence and diversity of antifeedant compounds in plants is especially important for theories on herbivore host breadth. Early theories on host plant selection highlighted the importance of secondary compounds as feeding cues (i.e., feeding stimulants), but it is now generally believed that antifeedants (and the related repellents, which act on olfactory cells) are even more important in determining host breadth of herbivorous pests. Oddly, however, the word antifeedant has not caught on outside of the field of crop protection and insect neurophysiology. In most recent books and review articles on the ecology and evolution of plant resistance, the word ‘antifeedant’ is never mentioned, and is seemingly displaced by an overemphasis on plant toxins, or rather on the toxic characteristics of compounds that may have other effects such as deterrency. Therefore, a secondary goal of reviewing antifeedants here is to argue for the importance of the term in studies of plant/herbivore ecology and evolution. Although in this article the emphasis is on antifeedant compounds, it should be noted that many morphological structures (e.g., wooliness, spinescence, coloration, etc.) have similar effects on herbivore feeding, and these features were undoubtedly viewed by breeders as antifeedants long before antifeedant chemicals were identified in plant tissues.
Definitions of Antifeedants Most researchers define antifeedants as those chemicals that have antifeedant properties at low concentration, and that act on very specific sensory cells (antifeedant receptors) in the pest. The neurons associated with these antifeedant receptors either prevent insect feeding (feeding deterrent effect) or cause
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cessation or slowing of further feeding (feeding suppressant effect). Another mode of action of some antifeedants is through an apparent ability to block the function of a herbivore’s feedingstimulant receptors, or an ability to bind directly to its normal feeding cues, such as sugars and amino acids. An example of this mechanism is the action of DEET (N,N-diethyl-m-toluamide) in repellent lotions, which deter blood-sucking arthropods by blocking their ability to perceive feeding stimulants. The very potent antifeedant azadirachtin acts in a similar way by reducing the sensitivity of sugar-sensing cells in herbivorous insects and thus causing the insects to incorrectly assess nutritional adequacy of treated host leaves. Only careful electrophysiological studies on the two categories of receptors (feeding deterrent versus feeding stimulant) can determine whether the putative antifeedant has a direct effect on antifeedant receptors, a distinction that is important when asking certain research questions. Another type of ‘false’ antifeedant category is those compounds that act nonspecifically on all gustatory sensilla (e.g., through immediate and general cell toxicity) are also not considered true antifeedants. The finer points of categorizing antifeedants, however, are not usually an issue because data are rarely available on the precise electrophysiological mode of action of antifeedant compounds or extracts. Although antifeedants can belong to a variety of chemical classes, the majority are alkaloid, flavonoid, and terpene secondary compounds. Some of the more commonly known antifeedants are presented in Table 1. In general, antifeedants are often bitter (when tasted by humans), although this is certainly not diagnostic of an antifeedant, and they are usually not involved in primary metabolism (there are exceptions, such as some sugars).
Identifying Antifeedants through Electrophysiology When studying a particular antifeedant, it is important to determine early on whether the compound’s effect on feeding is simply due to its repellent qualities and not to the activation of receptors involved in taste (gustatory receptors). A simple way of eliminating the olfactory component of a feeding response is to remove or disable the olfactory organs and then compare the responses of disabled and control subjects to a putative compound. When the locations of olfactory and gustatory sensilla are known, however, it is possible to obtain very useful electrophysiological data. These data can establish both that olfactory neurons are not being activated (ruling out a repellency activity) when exposed to the putative antifeedant and that gustatory receptors are transmitting signals for cessation of feeding. In some cases, it is even possible to use electrophysiological data to identify individual gustatory sensilla that are involved with a specific antifeedant or class of antifeedant. With insects, these cells can be located on one or more of a variety of structures including buccal surfaces, mouthparts, tarsi, and antennae. For caterpillars, sensilla
Encyclopedia of Applied Plant Sciences, 2nd edition, Volume 2
http://dx.doi.org/10.1016/B978-0-12-394807-6.00068-X
Secondary Products j Antifeedant Substances in Plants Table 1
Examples of naturally occurring antifeedants and the herbivores on which they are active
Antifeedant
Chemical class
Source plant (family)
Herbivore
Azadirachtin Caffeine Capsaicin Cocaine Gymnemic acid Nicotine Phlorizin Strychnine Tannic acid Warburganal
Limonoid Alkaloid Alkaloid Alkaloid Saponin Alkaloid Phenolic Alkaloid Tannin Sesquiterpenoid
Azadiractha indica (Meliaceae) Coffea arabica (Rubiaceae) Solanum capsicum (Solanaceae) Erythroxylum coca (Erythroxylaceae) Gymnema sylvestre (Asclepiadaceae) Nicotiana tobacum (Solanaceae) Malus domestica (Pomoideae) Strychnos nuxvomica (Loganiaceae) Medicago sativa (Fabaceae) Warburgia salutaris (Canellaceae)
Schistocerca gregaria (Orthoptera) Danaus plexippus (Leipidoptera) Leptinotarsa decemlineata (Coleoptera) Leptinotarsa decemlineata (Coleoptera) Homo sapiens Schistocerca gregaria (Orthoptera) Myzus persicae (Homoptera) Pieris brassicae (Lepidoptera) Hypera postica (Coleoptera) Spodoptera exempta (Lepidoptera)
located on the maxilla are especially important. Unfortunately, because electrophysiological measurements are extremely hard to obtain, even when receptor locations are known (knowledge that applies to only a few of the important insect crop pest species), they are infrequently gathered. More importantly, the nature of collecting such data makes electrophysiology an inefficient tool for mass screening of putative antifeedants, which is a major emphasis in antifeedant research. Another technique for establishing antifeedant action is through the use of in vitro binding assays, in which the strength of antifeedant/receptor interactions are used as indicators of the antifeedant potency. These experiments necessarily require isolation of receptor molecules from the antifeedant-sensing cells, and thus only well-studied organisms, usually insects, are involved in this methodology.
Identifying Antifeedants with Feeding Arenas A far simpler and thus much more common technique for testing putative chemicals and plant extracts for antifeedant properties is the choice arena (Figure 1(a)). This technique uses dishes or cages in which a single herbivore (insect, rabbit, etc.) is presented with unadulterated host plant tissue, as well as host tissue that has been sprayed with the test compound. The antifeedant index is calculated by measuring for each arena the relative amount of each type of disk consumed. In general, an antifeedant is considered effective if, on average, 95% of the treated leaf is left uneaten. As mentioned above, the calculated effect might include strict antifeedant effects (those due to the activation of antifeedant receptors), as well as reduced feeding due to the repellent qualities of the tested compound or extract. If the experimenter wishes to
(a)
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(b)
Figure 1 Choice (a) and nonchoice (b) feeding arenas can be used to estimate the antifeedant effect of test compounds (hatched circles) relative to untreated host material (shaded circles).
separate these effects, the herbivore’s olfactory function must be impaired in some way so that only gustatory contributions to the experimental outcome are measured. A popular variant of this design is to replace the host plant tissue with disks fashioned out of stem pith, artificial diet, filter paper, agar, or glass fiber mat that have been soaked in a feeding stimulant, although this methodology is considerably less meaningful. In particular, using artificial disks probably overvalues the deterrent effect of tested compound because the strength of the applied feeding stimulant can rarely be as strong as that provided by unadulterated, preferred host tissue. Despite these drawbacks associated with artificial disks, use of living material in bioassays has a very important and often overlooked handicap of its own. When living host material is used in bioassays, the phytotoxic effects of putative antifeedants must also be considered. Because many antifeedants are toxins, it is not surprising that some of them are toxic to plants, and thus treated plants may develop an antifeedant quality that is only indirectly caused by the applied compound. For example, plants often produce gamma-aminobutyrate in response to various types of stress, and this compound has antifeedant properties against a variety of tested insects. An additional indirect mechanism of action is via a simple reduction in nutritional content, such as that resulting from a cessation of photosynthesis caused by a test compound that is lethal to plant cells, and which could therefore easily change repellent, antifeedant, and stimulant qualities of plant tissues. For the reasons just described, many of the more careful studies on antifeedants also include control tissue that has been treated with the solvent used to extract or resuspend the antifeedant compound, because many common solvents can stress plant tissue and so decrease their acceptability to insects. Another common experimental methodology is to provide the herbivore with the control and test options singly, thus creating a nonchoice arena (Figure 1(b)). Nonchoice assays are particularly useful in assessing whether an antifeedant is an absolute deterrent, evident when an herbivore’s feeding is completely and permanently inhibited when presented with antifeedant-treated host tissue or artificial disks. Although the nonchoice scenario is often accused of being biologically irrelevant, it is very appropriate when testing the efficacy of antifeedant compounds for use in monocultures, where the pests might not have alternative hosts for miles. Similarly, if the pest of interest does not display significant mobility and host selection in nature, it is experimentally inappropriate to facilitate such movement within a test arena with choices available.
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Secondary Products j Antifeedant Substances in Plants
In this scenario, data from choice arenas would overestimate the true effectiveness of the putative antifeedant substance. As might be expected, collection of data from both choice and nonchoice arenas yields a much more satisfying conclusion about the effectiveness of an antifeedant. Specifically, if the antifeedant effect tends to diminish when assessed with a nonchoice design, the compound might be viewed as a rather poor candidate for use in agricultural settings. The more careful studies will also typically include arenas with disks that have different concentrations of the ‘stimulant’ and the ‘antifeedant,’ because manipulation of these amounts can have very important effects on the antifeedant index. An important complication in interpreting data from both choice and nonchoice arenas is that toxicity effects (antibiosis) can inflate the antifeedant index, which should strictly be a measure of the chemical’s effects on the feeding behavior of the insect. For example, if ingestion of the test disk causes rapid and irreversible muscle paralysis, failure to continue feeding might appear, incorrectly, to be due to antifeedant effects. A useful methodology for estimating the antibiosis component of antifeedant action is to deliver the compound directly to the herbivore’s gut, and then estimate growth rate or survivorship. Of course, many compounds (most notably azadirachtin, which is discussed below) have both antifeedant and toxic activities, so researchers should, ideally, report the LD50 (dosage causing 50% mortality) for compounds together with an index of antifeedancy. Another important consideration in antifeedant research is whether herbivores respond differently to antifeedant compounds over time. One such effect, called habituation or desensitization, occurs when the deterrent effect of a compound on a test insect diminishes over time. Therefore, choice and nonchoice trials of short duration might dramatically overestimate the true antifeedant properties of a compound. For herbivores that are polyphagous (i.e., not specializing on a given plant taxa), habituation to antifeedants is particularly rapid. Because much antifeedant research is directed at developing crop protection chemicals, the prevalence of habituation in such trials has diminished the early hopes that antifeedants would become popular commercial products.
Antifeedants Can Be Feedants to Other Pests For those wishing to make antifeedants commercially viable, a constant source of disappointment is the fact that some antifeedants, despite being very active against many herbivore species, are potent feeding stimulants to a few economically important pests. For example, gossypol, a dimeric sesquiterpene found in cotton (Gossypium spp.), has antifeedant action toward generalist insect pests (e.g., Heliothis spp. and Epicauta spp.) but acts as a feeding stimulant for the boll weevil (Anthonomous grandis), a specialist on this species. In the end, it might be necessary to treat crops with both ‘specialist-targeted’ and ‘generalist-targeted’ antifeedants to achieve full protection of a given crop species. Because the vast majority of research on antifeedants is performed with generalist Lepidopteran larvae (Spodoptera spp., Heliothis spp., and Pieris spp.) and Orthopterans (Locusta migratoria and Schistocerca gregaria), our knowledge of antifeedants affecting specialists is limited.
Evolution of Antifeedants Because most identified antifeedants are toxic, it is not particularly surprising that herbivores have evolved gustatory senses that can perceive such toxins. The response of many insect species to specific antifeedants, therefore, may be explained as an adaptation to avoid further toxicity. In other words, genetic variants in a herbivore population that cannot perceive an existing or new plant toxin tend to have low fitness, and will not contribute many offspring to the next generation. However, there are numerous examples showing that chemicals (e.g., strychnine) can have strong antifeedant effects even though the insect species has probably had no prior encounter with the chemical throughout evolutionary time. In these instances, it is necessary to invoke the evolution of a generalized antifeedant receptor. Most hypotheses on the evolution of antifeedant sensitivity suggest that deterrent sensilla were originally sensitive to a wide array of compounds. Indeed, at first they were not even involved in deterrent activity, but rather in general gustatory identification of food. It is assumed that over evolutionary time these cells gradually lost their sensitivity to certain compounds (e.g., water, sugars, amino acids, etc.) but retained their sensitivity to other classes of compounds. Coupled with this restriction of sensitivity is the development of an antifeeding neural activity for these receptors. For some herbivores, these antifeedant receptors still identify large numbers of compounds as antifeedants. Why do some plants apparently lack strong antifeedants? Even for antifeedants that are active at very low concentrations, it is likely that there is a metabolic cost involved in their production and storage. Therefore, protection comes at some fitness cost, and plant species that lack such antifeedants might enjoy high levels of growth and reproduction, at least in areas where herbivore pressures are absent or minimal. Like other types of chemicals involved in plant resistance, antifeedant content (e.g., phenolics, hydrogen cyanide, cucurbitacins) of some plants can be increased by herbivory. Induction thereby increases resistance when insects are present, but minimizes the fitness costs of producing a resistance phenotype when pests are absent or infrequent. When considering the adaptive benefit of toxic secondary compounds, selection might reasonably favor those compounds that are easily identified by existing deterrent cells in herbivorous species. Plants possessing compounds that are not easily perceived by pests would therefore be subject to the occasional massive loss of tissue that occurs before the herbivore ceases feeding. Therefore, one could make the prediction that natural selection imposed by herbivore sampling (ingestion of small amounts of tissue) could add to the selective advantage of those toxic compounds that can be perceived by common pests.
Commercialization of Plant-Derived Antifeedants The ultimate goal of antifeedant research is to isolate or synthesize a compound that functions as an absolute antifeedant, i.e., one that completely inhibits herbivores from ever attempting to feed again, but is at the same time nontoxic to even the targeted pest species (and therefore unlikely to
Secondary Products j Antifeedant Substances in Plants
O
O
OCH3 C
C
O
O
OH
OH O
O O H
O H3C
C
O
O
OH
H
O
O Figure 2 Chemical structure of azadirachtin, a potent antifeedant isolated from the neem tree.
be toxic to other animals such as ourselves). But if that antifeedant is not acutely toxic, evolution of countermeasures in the pest populations are likely to rapidly occur, and fix, as soon as mutations appear that diminish the ability of sensory cells from perceiving the antifeedant. To date, only azadirachtin (Figure 2) derived from the neem tree (Azadirachta indica) has received any commercial success; this is marketed under a variety of trade names, e.g., Margosan-O, Azatin, Bioneem, Neemesis, Neemgard. Azadirachtin probably owes much of its success to its multiple modes of action. In addition to its much-touted antifeedant qualities, it is repellent and also toxic to many insects, in part due to its deleterious effects on insect growth regulation and maintenance of circadian rhythm. Azadirachtin is also an ‘indirect’ antifeedant due to its toxic effects on treated plants, which thus become less advantageous hosts for herbivores. Resistance to azadirachtin, therefore, is regarded as being unlikely to develop rapidly in natural populations because it acts at many different points in the life cycle of affected insects. But despite statements that insects will never be able to mount countermeasures, laboratory selection experiments (with aphids) have shown that the antifeedant and toxic effects on insects can diminish after 40 generations. Interestingly, aphids exposed to a blend of neem compounds, rather than just to azadirachtin, did not develop resistance, indicating that synergistic effects of antifeedants are important in plant resistance. In general, there are several reasons why antifeedant research has not generated successful products. First, as mentioned above, potent antifeedants that naturally occur are likely to be toxins also, and thus are unlikely to be viewed as
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‘safe’ in the eyes of regulatory agencies or the consuming public. Azadirachtin, for example, has toxic effects on certain mollusks, nematodes, fungi, protozoa, and even viruses. Although many of these groups can be pestiferous, the wide range of activity also warrants concerns about additional, untested toxicity against neutral or beneficial organisms in sprayed fields. Second, because toxic secondary compounds often exist in plant vacuoles and other cell macrostructures, spraying extracted antifeedant chemicals on plant leaf surfaces (even species that normally produce the antifeedant) may cause plant stress or mortality. Third, if an antifeedant is to be successfully commercialized, it is important to use choice trials of long duration to rule out the possibility that insects will habituate to its presence. Most trials do not last for the entire period in which feeding takes place, and thus promising antifeedants are often advanced to field tests only to reveal that herbivores habituate rather quickly to their presence. Fourth, attempts at cloning antifeedant genes and incorporating them into transgenic plants is unlikely to be easy, because many of the secondary products involved in antifeedancy are synthesized by the sequential action of multiple genes; to date, there have been few successes of reliably transferring whole biosynthetic pathways into plants that may lack the necessary precursors. Finally, as stated earlier, antifeedants identified to date do not have entirely predictable effects on all herbivores; in contrast, many insect toxins are reliably toxic against the majority of insect targets.
See also: Crop Diseases and Pests: Phytoalexins, Natural Plant Protection.
Further Reading Chapman, R.F., 1974. The chemical inhibition of feeding by phytophagous insects: a review. Bull. Entomol. Res. 64, 339–363. Chyb, S., Eichenseer, H., Hollister, B., Mullin, C.A., Frazier, J.L., 1995. Identification of sensilla involved in taste mediation in adult western corn rootworm (Diabrotica virgifera virgifera LeConte). J. Chem. Ecol. 21, 313–329. Dethier, V.G., 1947. Chemical Insect Attractants and Repellents. The Blakiston Company, Philadelphia. Mordue (Luntz), A.J., Blackwell, A., 1993. Azadirachtin: an update. J. Insect Physiol. 39, 903–924. Norris, D.M., 1986. Anti-feeding compounds. In: Kato, T., Kramer, D.W., Kuck, K.-H., Morris, D.M. (Eds.), Chemistry of Plant Protection: Sterol Biosynthesis, Inhibitors and Anti-feeding Compounds. Springer-Verlag, New York, pp. 97–146. Schoonhoven, L.M., 1982. Biological aspects of antifeedants. Entomol. Exp. Appl. 31, 57–69.
Introduction Although the literature on antifeedancy is dispersed rather thinly under a variety of cognates and related terms (e.g., antifeeding compound, feeding deterrent, feeding rejectant, feeding suppressant, feeding inhibitor, gustatory repellent, phagodepressant), it is unified by the hope that antifeedant sprays might someday become an important pest management tool. In particular, it is hoped that antifeedants might lack the dangers associated with the use of natural or engineered pesticides, both of which frequently have detectable if not substantial toxicity toward other animals, such as ourselves. Unfortunately, most of the literature on this topic has suggested that antifeedants are simply not effective, or not reliably so, against many of the important pests of common crops. Furthermore, insects are capable of habituating to the presence of antifeedants after a given amount of time. For this and several other reasons, the marketing of specific antifeedants has had little success. Although it is easy to be pessimistic about the commercial viability of antifeedants, research on these compounds is producing valuable insight into the ecology and evolution of plant resistance strategies, as well as the corresponding suite of adaptations in herbivores. Knowledge of the presence and diversity of antifeedant compounds in plants is especially important for theories on herbivore host breadth. Early theories on host plant selection highlighted the importance of secondary compounds as feeding cues (i.e., feeding stimulants), but it is now generally believed that antifeedants (and the related repellents, which act on olfactory cells) are even more important in determining host breadth of herbivorous pests. Oddly, however, the word antifeedant has not caught on outside of the field of crop protection and insect neurophysiology. In most recent books and review articles on the ecology and evolution of plant resistance, the word ‘antifeedant’ is never mentioned, and is seemingly displaced by an overemphasis on plant toxins, or rather on the toxic characteristics of compounds that may have other effects such as deterrency. Therefore, a secondary goal of reviewing antifeedants here is to argue for the importance of the term in studies of plant/herbivore ecology and evolution. Although in this article the emphasis is on antifeedant compounds, it should be noted that many morphological structures (e.g., wooliness, spinescence, coloration, etc.) have similar effects on herbivore feeding, and these features were undoubtedly viewed by breeders as antifeedants long before antifeedant chemicals were identified in plant tissues.
Definitions of Antifeedants Most researchers define antifeedants as those chemicals that have antifeedant properties at low concentration, and that act on very specific sensory cells (antifeedant receptors) in the pest. The neurons associated with these antifeedant receptors either prevent insect feeding (feeding deterrent effect) or cause
364
cessation or slowing of further feeding (feeding suppressant effect). Another mode of action of some antifeedants is through an apparent ability to block the function of a herbivore’s feedingstimulant receptors, or an ability to bind directly to its normal feeding cues, such as sugars and amino acids. An example of this mechanism is the action of DEET (N,N-diethyl-m-toluamide) in repellent lotions, which deter blood-sucking arthropods by blocking their ability to perceive feeding stimulants. The very potent antifeedant azadirachtin acts in a similar way by reducing the sensitivity of sugar-sensing cells in herbivorous insects and thus causing the insects to incorrectly assess nutritional adequacy of treated host leaves. Only careful electrophysiological studies on the two categories of receptors (feeding deterrent versus feeding stimulant) can determine whether the putative antifeedant has a direct effect on antifeedant receptors, a distinction that is important when asking certain research questions. Another type of ‘false’ antifeedant category is those compounds that act nonspecifically on all gustatory sensilla (e.g., through immediate and general cell toxicity) are also not considered true antifeedants. The finer points of categorizing antifeedants, however, are not usually an issue because data are rarely available on the precise electrophysiological mode of action of antifeedant compounds or extracts. Although antifeedants can belong to a variety of chemical classes, the majority are alkaloid, flavonoid, and terpene secondary compounds. Some of the more commonly known antifeedants are presented in Table 1. In general, antifeedants are often bitter (when tasted by humans), although this is certainly not diagnostic of an antifeedant, and they are usually not involved in primary metabolism (there are exceptions, such as some sugars).
Identifying Antifeedants through Electrophysiology When studying a particular antifeedant, it is important to determine early on whether the compound’s effect on feeding is simply due to its repellent qualities and not to the activation of receptors involved in taste (gustatory receptors). A simple way of eliminating the olfactory component of a feeding response is to remove or disable the olfactory organs and then compare the responses of disabled and control subjects to a putative compound. When the locations of olfactory and gustatory sensilla are known, however, it is possible to obtain very useful electrophysiological data. These data can establish both that olfactory neurons are not being activated (ruling out a repellency activity) when exposed to the putative antifeedant and that gustatory receptors are transmitting signals for cessation of feeding. In some cases, it is even possible to use electrophysiological data to identify individual gustatory sensilla that are involved with a specific antifeedant or class of antifeedant. With insects, these cells can be located on one or more of a variety of structures including buccal surfaces, mouthparts, tarsi, and antennae. For caterpillars, sensilla
Encyclopedia of Applied Plant Sciences, 2nd edition, Volume 2
http://dx.doi.org/10.1016/B978-0-12-394807-6.00068-X
Secondary Products j Antifeedant Substances in Plants Table 1
Examples of naturally occurring antifeedants and the herbivores on which they are active
Antifeedant
Chemical class
Source plant (family)
Herbivore
Azadirachtin Caffeine Capsaicin Cocaine Gymnemic acid Nicotine Phlorizin Strychnine Tannic acid Warburganal
Limonoid Alkaloid Alkaloid Alkaloid Saponin Alkaloid Phenolic Alkaloid Tannin Sesquiterpenoid
Azadiractha indica (Meliaceae) Coffea arabica (Rubiaceae) Solanum capsicum (Solanaceae) Erythroxylum coca (Erythroxylaceae) Gymnema sylvestre (Asclepiadaceae) Nicotiana tobacum (Solanaceae) Malus domestica (Pomoideae) Strychnos nuxvomica (Loganiaceae) Medicago sativa (Fabaceae) Warburgia salutaris (Canellaceae)
Schistocerca gregaria (Orthoptera) Danaus plexippus (Leipidoptera) Leptinotarsa decemlineata (Coleoptera) Leptinotarsa decemlineata (Coleoptera) Homo sapiens Schistocerca gregaria (Orthoptera) Myzus persicae (Homoptera) Pieris brassicae (Lepidoptera) Hypera postica (Coleoptera) Spodoptera exempta (Lepidoptera)
located on the maxilla are especially important. Unfortunately, because electrophysiological measurements are extremely hard to obtain, even when receptor locations are known (knowledge that applies to only a few of the important insect crop pest species), they are infrequently gathered. More importantly, the nature of collecting such data makes electrophysiology an inefficient tool for mass screening of putative antifeedants, which is a major emphasis in antifeedant research. Another technique for establishing antifeedant action is through the use of in vitro binding assays, in which the strength of antifeedant/receptor interactions are used as indicators of the antifeedant potency. These experiments necessarily require isolation of receptor molecules from the antifeedant-sensing cells, and thus only well-studied organisms, usually insects, are involved in this methodology.
Identifying Antifeedants with Feeding Arenas A far simpler and thus much more common technique for testing putative chemicals and plant extracts for antifeedant properties is the choice arena (Figure 1(a)). This technique uses dishes or cages in which a single herbivore (insect, rabbit, etc.) is presented with unadulterated host plant tissue, as well as host tissue that has been sprayed with the test compound. The antifeedant index is calculated by measuring for each arena the relative amount of each type of disk consumed. In general, an antifeedant is considered effective if, on average, 95% of the treated leaf is left uneaten. As mentioned above, the calculated effect might include strict antifeedant effects (those due to the activation of antifeedant receptors), as well as reduced feeding due to the repellent qualities of the tested compound or extract. If the experimenter wishes to
(a)
365
(b)
Figure 1 Choice (a) and nonchoice (b) feeding arenas can be used to estimate the antifeedant effect of test compounds (hatched circles) relative to untreated host material (shaded circles).
separate these effects, the herbivore’s olfactory function must be impaired in some way so that only gustatory contributions to the experimental outcome are measured. A popular variant of this design is to replace the host plant tissue with disks fashioned out of stem pith, artificial diet, filter paper, agar, or glass fiber mat that have been soaked in a feeding stimulant, although this methodology is considerably less meaningful. In particular, using artificial disks probably overvalues the deterrent effect of tested compound because the strength of the applied feeding stimulant can rarely be as strong as that provided by unadulterated, preferred host tissue. Despite these drawbacks associated with artificial disks, use of living material in bioassays has a very important and often overlooked handicap of its own. When living host material is used in bioassays, the phytotoxic effects of putative antifeedants must also be considered. Because many antifeedants are toxins, it is not surprising that some of them are toxic to plants, and thus treated plants may develop an antifeedant quality that is only indirectly caused by the applied compound. For example, plants often produce gamma-aminobutyrate in response to various types of stress, and this compound has antifeedant properties against a variety of tested insects. An additional indirect mechanism of action is via a simple reduction in nutritional content, such as that resulting from a cessation of photosynthesis caused by a test compound that is lethal to plant cells, and which could therefore easily change repellent, antifeedant, and stimulant qualities of plant tissues. For the reasons just described, many of the more careful studies on antifeedants also include control tissue that has been treated with the solvent used to extract or resuspend the antifeedant compound, because many common solvents can stress plant tissue and so decrease their acceptability to insects. Another common experimental methodology is to provide the herbivore with the control and test options singly, thus creating a nonchoice arena (Figure 1(b)). Nonchoice assays are particularly useful in assessing whether an antifeedant is an absolute deterrent, evident when an herbivore’s feeding is completely and permanently inhibited when presented with antifeedant-treated host tissue or artificial disks. Although the nonchoice scenario is often accused of being biologically irrelevant, it is very appropriate when testing the efficacy of antifeedant compounds for use in monocultures, where the pests might not have alternative hosts for miles. Similarly, if the pest of interest does not display significant mobility and host selection in nature, it is experimentally inappropriate to facilitate such movement within a test arena with choices available.
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In this scenario, data from choice arenas would overestimate the true effectiveness of the putative antifeedant substance. As might be expected, collection of data from both choice and nonchoice arenas yields a much more satisfying conclusion about the effectiveness of an antifeedant. Specifically, if the antifeedant effect tends to diminish when assessed with a nonchoice design, the compound might be viewed as a rather poor candidate for use in agricultural settings. The more careful studies will also typically include arenas with disks that have different concentrations of the ‘stimulant’ and the ‘antifeedant,’ because manipulation of these amounts can have very important effects on the antifeedant index. An important complication in interpreting data from both choice and nonchoice arenas is that toxicity effects (antibiosis) can inflate the antifeedant index, which should strictly be a measure of the chemical’s effects on the feeding behavior of the insect. For example, if ingestion of the test disk causes rapid and irreversible muscle paralysis, failure to continue feeding might appear, incorrectly, to be due to antifeedant effects. A useful methodology for estimating the antibiosis component of antifeedant action is to deliver the compound directly to the herbivore’s gut, and then estimate growth rate or survivorship. Of course, many compounds (most notably azadirachtin, which is discussed below) have both antifeedant and toxic activities, so researchers should, ideally, report the LD50 (dosage causing 50% mortality) for compounds together with an index of antifeedancy. Another important consideration in antifeedant research is whether herbivores respond differently to antifeedant compounds over time. One such effect, called habituation or desensitization, occurs when the deterrent effect of a compound on a test insect diminishes over time. Therefore, choice and nonchoice trials of short duration might dramatically overestimate the true antifeedant properties of a compound. For herbivores that are polyphagous (i.e., not specializing on a given plant taxa), habituation to antifeedants is particularly rapid. Because much antifeedant research is directed at developing crop protection chemicals, the prevalence of habituation in such trials has diminished the early hopes that antifeedants would become popular commercial products.
Antifeedants Can Be Feedants to Other Pests For those wishing to make antifeedants commercially viable, a constant source of disappointment is the fact that some antifeedants, despite being very active against many herbivore species, are potent feeding stimulants to a few economically important pests. For example, gossypol, a dimeric sesquiterpene found in cotton (Gossypium spp.), has antifeedant action toward generalist insect pests (e.g., Heliothis spp. and Epicauta spp.) but acts as a feeding stimulant for the boll weevil (Anthonomous grandis), a specialist on this species. In the end, it might be necessary to treat crops with both ‘specialist-targeted’ and ‘generalist-targeted’ antifeedants to achieve full protection of a given crop species. Because the vast majority of research on antifeedants is performed with generalist Lepidopteran larvae (Spodoptera spp., Heliothis spp., and Pieris spp.) and Orthopterans (Locusta migratoria and Schistocerca gregaria), our knowledge of antifeedants affecting specialists is limited.
Evolution of Antifeedants Because most identified antifeedants are toxic, it is not particularly surprising that herbivores have evolved gustatory senses that can perceive such toxins. The response of many insect species to specific antifeedants, therefore, may be explained as an adaptation to avoid further toxicity. In other words, genetic variants in a herbivore population that cannot perceive an existing or new plant toxin tend to have low fitness, and will not contribute many offspring to the next generation. However, there are numerous examples showing that chemicals (e.g., strychnine) can have strong antifeedant effects even though the insect species has probably had no prior encounter with the chemical throughout evolutionary time. In these instances, it is necessary to invoke the evolution of a generalized antifeedant receptor. Most hypotheses on the evolution of antifeedant sensitivity suggest that deterrent sensilla were originally sensitive to a wide array of compounds. Indeed, at first they were not even involved in deterrent activity, but rather in general gustatory identification of food. It is assumed that over evolutionary time these cells gradually lost their sensitivity to certain compounds (e.g., water, sugars, amino acids, etc.) but retained their sensitivity to other classes of compounds. Coupled with this restriction of sensitivity is the development of an antifeeding neural activity for these receptors. For some herbivores, these antifeedant receptors still identify large numbers of compounds as antifeedants. Why do some plants apparently lack strong antifeedants? Even for antifeedants that are active at very low concentrations, it is likely that there is a metabolic cost involved in their production and storage. Therefore, protection comes at some fitness cost, and plant species that lack such antifeedants might enjoy high levels of growth and reproduction, at least in areas where herbivore pressures are absent or minimal. Like other types of chemicals involved in plant resistance, antifeedant content (e.g., phenolics, hydrogen cyanide, cucurbitacins) of some plants can be increased by herbivory. Induction thereby increases resistance when insects are present, but minimizes the fitness costs of producing a resistance phenotype when pests are absent or infrequent. When considering the adaptive benefit of toxic secondary compounds, selection might reasonably favor those compounds that are easily identified by existing deterrent cells in herbivorous species. Plants possessing compounds that are not easily perceived by pests would therefore be subject to the occasional massive loss of tissue that occurs before the herbivore ceases feeding. Therefore, one could make the prediction that natural selection imposed by herbivore sampling (ingestion of small amounts of tissue) could add to the selective advantage of those toxic compounds that can be perceived by common pests.
Commercialization of Plant-Derived Antifeedants The ultimate goal of antifeedant research is to isolate or synthesize a compound that functions as an absolute antifeedant, i.e., one that completely inhibits herbivores from ever attempting to feed again, but is at the same time nontoxic to even the targeted pest species (and therefore unlikely to
Secondary Products j Antifeedant Substances in Plants
O
O
OCH3 C
C
O
O
OH
OH O
O O H
O H3C
C
O
O
OH
H
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O Figure 2 Chemical structure of azadirachtin, a potent antifeedant isolated from the neem tree.
be toxic to other animals such as ourselves). But if that antifeedant is not acutely toxic, evolution of countermeasures in the pest populations are likely to rapidly occur, and fix, as soon as mutations appear that diminish the ability of sensory cells from perceiving the antifeedant. To date, only azadirachtin (Figure 2) derived from the neem tree (Azadirachta indica) has received any commercial success; this is marketed under a variety of trade names, e.g., Margosan-O, Azatin, Bioneem, Neemesis, Neemgard. Azadirachtin probably owes much of its success to its multiple modes of action. In addition to its much-touted antifeedant qualities, it is repellent and also toxic to many insects, in part due to its deleterious effects on insect growth regulation and maintenance of circadian rhythm. Azadirachtin is also an ‘indirect’ antifeedant due to its toxic effects on treated plants, which thus become less advantageous hosts for herbivores. Resistance to azadirachtin, therefore, is regarded as being unlikely to develop rapidly in natural populations because it acts at many different points in the life cycle of affected insects. But despite statements that insects will never be able to mount countermeasures, laboratory selection experiments (with aphids) have shown that the antifeedant and toxic effects on insects can diminish after 40 generations. Interestingly, aphids exposed to a blend of neem compounds, rather than just to azadirachtin, did not develop resistance, indicating that synergistic effects of antifeedants are important in plant resistance. In general, there are several reasons why antifeedant research has not generated successful products. First, as mentioned above, potent antifeedants that naturally occur are likely to be toxins also, and thus are unlikely to be viewed as
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‘safe’ in the eyes of regulatory agencies or the consuming public. Azadirachtin, for example, has toxic effects on certain mollusks, nematodes, fungi, protozoa, and even viruses. Although many of these groups can be pestiferous, the wide range of activity also warrants concerns about additional, untested toxicity against neutral or beneficial organisms in sprayed fields. Second, because toxic secondary compounds often exist in plant vacuoles and other cell macrostructures, spraying extracted antifeedant chemicals on plant leaf surfaces (even species that normally produce the antifeedant) may cause plant stress or mortality. Third, if an antifeedant is to be successfully commercialized, it is important to use choice trials of long duration to rule out the possibility that insects will habituate to its presence. Most trials do not last for the entire period in which feeding takes place, and thus promising antifeedants are often advanced to field tests only to reveal that herbivores habituate rather quickly to their presence. Fourth, attempts at cloning antifeedant genes and incorporating them into transgenic plants is unlikely to be easy, because many of the secondary products involved in antifeedancy are synthesized by the sequential action of multiple genes; to date, there have been few successes of reliably transferring whole biosynthetic pathways into plants that may lack the necessary precursors. Finally, as stated earlier, antifeedants identified to date do not have entirely predictable effects on all herbivores; in contrast, many insect toxins are reliably toxic against the majority of insect targets.
See also: Crop Diseases and Pests: Phytoalexins, Natural Plant Protection.
Further Reading Chapman, R.F., 1974. The chemical inhibition of feeding by phytophagous insects: a review. Bull. Entomol. Res. 64, 339–363. Chyb, S., Eichenseer, H., Hollister, B., Mullin, C.A., Frazier, J.L., 1995. Identification of sensilla involved in taste mediation in adult western corn rootworm (Diabrotica virgifera virgifera LeConte). J. Chem. Ecol. 21, 313–329. Dethier, V.G., 1947. Chemical Insect Attractants and Repellents. The Blakiston Company, Philadelphia. Mordue (Luntz), A.J., Blackwell, A., 1993. Azadirachtin: an update. J. Insect Physiol. 39, 903–924. Norris, D.M., 1986. Anti-feeding compounds. In: Kato, T., Kramer, D.W., Kuck, K.-H., Morris, D.M. (Eds.), Chemistry of Plant Protection: Sterol Biosynthesis, Inhibitors and Anti-feeding Compounds. Springer-Verlag, New York, pp. 97–146. Schoonhoven, L.M., 1982. Biological aspects of antifeedants. Entomol. Exp. Appl. 31, 57–69.