BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 19 pdf

19
Phytochemical Modification of Taste:
An Insect Model
J. Alan A. Renwick
CONTENTS
19.1 Introduction
19.2 Repellents, Antifeedants, and Toxins
19.3 Cabbage Butterfly, Pieris rapae
19.4 Diet-Dependent Sensitivity
19.5 Conclusions
References
19.1 Introduction
Plant life in its various forms is widely recognized as the most readily available, abundant
source of new chemicals or leads for synthetic chemicals to meet the growing needs of the
agricultural and pharmaceutical industries. The array of chemicals produced by plants is
enormous, but the number of known compounds represents only a fraction of the total. Fur-
thermore, the diversity of biological activities exhibited by different groups of compounds
continues to offer countless opportunities for practical applications. While we have become
more aware of this chemical treasure and we increasingly exploit the unique properties of
individual phytochemicals, especially as pharmaceuticals, the adaptive significance of these
natural chemicals in the evolution of plants tends to be either ignored or forgotten.
Although some controversy exists over the relative importance of pathogens and herbivores
in the evolution of secondary metabolite production by plants, it is clear that the selective pres-
sure exerted by invading organisms has played a major role.
1
Most chemicals that we now
value for their biological activity are, therefore, likely to be products of the evolution of chem-
ical defenses against such attacks. The properties that cause a specific phytochemical to deter
oviposition by an insect pest, to function as an antibiotic, or to exhibit anticancer activity, may
be considered as side benefits of this evolutionary process of self-protection. Compounds that
defend a plant against invertebrates or vertebrates may now be used to protect crop plants in

an agricultural setting. Other compounds that are highly toxic to vertebrates have proved to
have valuable pharmacological properties, and phytochemicals that combat pathogen attack
are often active against human pathogens (Figure 19.1). In many cases, common biochemical
or physiological pathways may be involved in the activity of a chemical in different organ-
isms. Such similarities point to the possible value of using one organism as a model for study-
ing the reaction of another organism, especially humans, to phytochemicals.
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19.2 Repellents, Antifeedants, and Toxins
Many studies designed to develop crop plants that are resistant to insects or to find new con-
trol agents have utilized basic information about the mechanisms used by plants to protect
themselves against herbivores.
2
Chemical defenses of a plant against insects can be roughly
categorized as repellents, antifeedants, or toxins. Repellents generally prevent landing by
adult insects or movement onto plant surfaces by larvae. Antifeedants inhibit or deter feed-
ing by those insects that venture onto the plant, whereas toxins either kill or immobilize
those insects that do feed. However, the production of these chemicals by a plant may be
dramatically influenced by many environmental factors, both abiotic and biotic.
3
Nutrition,
particularly, nitrogen, sulfur, and phosphorus levels, can affect the biosynthesis of com-
pounds that are rich in any of these elements.
4,5
Allelochemical production may be induced
by herbivory, pathogens, or mechanical damage,
6
and exposure to UV-B radiation or air pol-
lutants may have profound effects on some biosynthetic pathways (Figure 19.2).
When we examine the way that plant chemistry may influence the selection of hosts by
phytophagous insects, we are dealing with basic principles of sensory evaluation and pal-

atability of potential foods. As humans, our discriminatory eating habits depend on percep-
tion of both olfactory and gustatory chemical signals associated with the food, and personal
preferences or individual reactions depend on the way that this chemical information is pro-
cessed by the brain. Similarly, insects are often guided by the volatiles from a potential food
plant for their initial approach (or avoidance), and then by nonvolatile gustatory stimuli for
acceptance or rejection of the plant. The sense of taste, therefore, plays a major role in deter-
mining host ranges, or dietary discrimination, of insects as well as higher animals.
19.3 Cabbage Butterfly, Pierus rapae
The cabbage butterfly, Pieris rapae, has been used as a model for studying the interplay of
gustatory cues that affect the host selection behavior of a specialist insect. The female but-
terfly has contact chemoreceptors on its tarsi that are responsible for perceiving both posi-
tive and negative gustatory stimuli at the plant surface.
7,8
Before accepting a plant for
FIGURE 19.1
Evolution and human benefits of secondary metabolite production in plants.
© 1999 by CRC Press LLC
oviposition, the female often exhibits rapid movement or tapping with the forelegs. This
behavior has been called “drumming”, and presumably allows more receptor hairs to
come in contact with the chemicals that stimulate oviposition.
9,10
These receptors, or sen-
silla, are peg-like structures which are arranged in rows between spines on the foretarsi
(Figure 19.3). A single pore at the tip of the sensillum is typical of contact chemoreceptors,
and electrophysiological recordings have been used to confirm that receptor cells associ-
ated with these sensory hairs respond to active plant constituents.
7,8
FIGURE 19.2
Dynamics of allelochemical production in the defense of plants against insects. (Adapted from Renwick, J.A.A.,
in Phytochemical Diversity and Redundancy in Ecological Interations, Plenum Press, New York, 1996.)

FIGURE 19.3
(Left) SEM of ventral side of the distal segment of a female Pieris rapae foretarsus showing a cluster of contact
chemosensory sensilla (arrow). (Right) Enlarged view of a sensillum showing the single pore at the tip, typical
of contact chemoreceptors.
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Tasting by larvae of the cabbage butterfly also depends on the use of contact chemorecep-
tors that perceive the active chemicals at the surface of a plant. These receptors are located
on the galea within the mouth of the caterpillar. Two pairs of sensilla styloconica, medial and
lateral, are generally involved in the process of food recognition and discrimination, resulting
in either stimulation or inhibition of feeding.
11
As in the case of tarsal receptors, electrophys-
iological recordings can be performed to show good correlations between the responses of
sensory receptor cells and behavioral responses to specific compounds.
12
Several compounds responsible for acceptance of suitable host plants and for rejection of
unsuitable plants by adults and larvae of P. rapae have now been identified. For example,
wormseed mustard, Erysimum cheiranthoides, is protected from attack by several specific
cardenolides.
13
However, the most effective oviposition deterrents are not the same as the
most effective feeding deterrents. The best oviposition deterrents are strophanthidin glyco-
sides, whereas the strongest feeding deterrents are glycosides of digitoxigenin.
14,15
When
extracts of E. cheiranthoides are subjected to solvent partitioning to remove the deterrents,
aqueous extracts actually become stimulatory to ovipositing females of P. rapae.
16
This has
served to demonstrate the fact that plants may contain both stimulants and deterrents and

that the balance of these positive and negative chemical messages determines whether a
plant is accepted or rejected.
17
This balance is likely to be influenced by the physiological
state of an individual insect, perception of the compounds, and processing of the informa-
tion that reaches the central nervous system (Figure 19.4).
The possibility of manipulating the balance of positive and negative sensory cues to
cause an insect to reject plants that they would normally accept has been suggested as a
means to reduce crop losses to insect pests. Oviposition or feeding deterrents could con-
ceivably be applied to protect the plants or they might be introduced through plant breed-
ing or genetic engineering to produce resistant plants. Alternatively, we might attempt to
interfere with the ability of an insect to taste chemical constituents of plants that they
encounter. If an insect loses its sensitivity to stimulants in a plant, it would no longer be
capable of recognizing a good host on the basis of secondary plant chemistry. On the other
hand, a loss of sensitivity to deterrents might result in feeding on unsuitable food plants
that would have a deleterious effect.
19.4 Diet-Dependent Sensitivity
Recent studies on larvae of P. rapae have resulted in the discovery of diet-dependent sensi-
tivity to an antifeedant in a normally acceptable host plant. The butterflies readily lay eggs
on garden nasturtium, Tropaeolum majus, and the hatching larvae feed and develop nor-
mally. However, if larvae that have fed and developed on cabbage plants are transferred to
nasturtium, they refuse to feed. The effect is so extreme that the larvae will starve to death
rather than feed on the nasturtium.
18
Similar results were obtained when larvae were trans-
ferred from a range of other host plants, including both crucifers and noncrucifers, to nas-
turtium. This rejection behavior was explained by the presence of chlorogenic acid and some
additional, unidentified constituents of T. majus.
19
Chlorogenic acid was strongly deterrent

to cabbage-reared larvae, but only slightly deterrent to nasturtium-reared individuals. Fur-
thermore, larvae that were reared on a wheat germ artificial diet were almost completely
insensitive to chlorogenic acid. It appears, therefore, that P. rapae larvae are insensitive to
deterrents at the time of hatching and will feed on a wide range of plants or artificial diet.
However, as they feed on cabbage or other crucifers, they develop sensitivity that results in
their refusal of food that contains deterrents. We have concluded that continuous exposure
© 1999 by CRC Press LLC
FIGURE 19.4
Factors affecting the balance of positive and negative chemical cues that influence acceptance or rejection of a potential host plant by an insect.
(Adapted from Renwick, J.A.A. and Huang, X., in Functional Dynamics of Phytophagous Insects, Oxford & IBH Publishing, New Delhi, 1994.)
© 1999 by CRC Press LLC
to and/or consumption of food containing deterrents results in a type of habituation, or
more precisely, suppression of sensitivity development.
19
Since habituation is usually defined as a “waning of response to a repeatedly presented
stimulus over time,” the term is not precisely applicable to the lack of sensitivity develop-
ment seen in this insect. However, experiments conducted to test the effects of dietary
experience with one or more compounds on responses to unrelated compounds seem to
indicate that a type of “cross-habituation” is possible. When larvae were reared on nastur-
tium, they were less sensitive than cabbage-reared larvae to a range of feeding deterrents.
20
Furthermore, larvae reared on wheat germ diet were almost completely insensitive to the
same compounds. When larvae fed and developed on cabbage leaves that had been treated
with selected deterrents, including cardenolides and cucurbitacin glycosides, they
remained less sensitive to the nasturtium deterrents.
20
The development of sensitivity to feeding deterrents can be followed at any larval stage.
When larvae were transferred from nasturtium to cabbage, they became progressively
more sensitive to the nasturtium deterrents. Although all instars show the same general
effect, second instars of P. rapae appear to be most plastic in their development of sensitivity

as they feed on cabbage.
21
The possible involvement of an “inducer” in cabbage has been
discounted, since larvae that were reared on cabbage foliage treated with nasturtium
extract remained insensitive to the deterrents from that plant. It is likely, therefore, that sen-
sitivity develops naturally as larvae feed on host plants that lack deterrents or other com-
pounds that suppress this development. This phenomenon of increased sensitivity within
an instar after removal from plants containing suppressors has prompted the proposal to
use P. rapae as a model organism for future analysis of the physiological and biochemical
processes involved in chemosensory development.
22
The idea of feeding experience changing the characteristics of taste receptors is not com-
pletely new. Already in 1969, Schoonhoven
23
found that the sensitivity of a so-called deter-
rent receptor in Manduca sexta larvae to salicin was considerably lower for larvae reared on
artificial diet that contained this compound than for larvae reared on control diet . Similar
electrophysiological results have since been obtained for larvae of Pieris brassicae in
response to chlorogenic acid, proline, and cyanin chloride,
24
and for larvae of Spodoptera
species in response to azadirachtin, nicotine, or sinigrin.
25
The effect of wheat germ diet on the ability of insects to taste and respond to deterrents
is particularly interesting. Artifical diets containing wheat germ have long been used by
entomologists for rearing a wide range of insects for various purposes. However, many
insects that are normally reared on wheat germ diets will refuse this artificial food if they
have previously fed on a host plant. Extracts of an artificial diet containing wheat germ
were found to be deterrent to Manduca sexta larvae, and wheat germ itself was thought to
be the source of deterrent components, as omission of the wheat germ increased the accept-

ability of the diet.
26
Similarly, larvae of the cabbage butterfly refuse to feed on wheat germ
diet when transferred from a cabbage plant, and the presence of deterrents has been dem-
onstrated to explain this behavior. Both hexane extracts and butanol-soluble material from
aqueous extracts of the whole wheat germ diet were highly deterrent to cabbage-reared lar-
vae of P. rapae.
27
Wheat germ diet for insect rearing generally consists of seven components in addition to
wheat germ itself and water. These include agar, aureomycin, casein, methylparaben, salt
mix, sorbic acid, and a vitamin mix. When tested in a feeding deterrent assay using fourth
instars of P. rapae, only the sorbic acid was slightly deterrent. However, extracts of the
wheat germ itself were highly deterrent. Since strong deterrent activity was found in hex-
ane as well as butanol extracts, a separation scheme was developed to examine both active
fractions (Figure 19.5). Seven compounds were isolated by HPLC separation of the butanol
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fraction of the aqueous material. The UV spectra suggested that most of these are apigenin-
based flavones. Fractionation of the hexane-soluble material by sequential solvent parti-
tioning revealed that most of the active compounds were methanol-soluble, and HPLC of
the methanol fraction showed that some compounds were common to both the butanol
and hexane fractions, but others were present only in one of the fractions. When neonate
larvae were allowed to feed on cabbage leaves that were treated with individual fractions
collected from the HPLC, they remained insensitive to deterrents. Thus, individual constit-
uents of wheat germ could account for the “cross-habituation” that is necessary for larval
acceptance of nasturtium.
27
The sensitivity suppressing activity of specific compounds in wheat germ would suggest
that these phytochemicals are acting as taste modifiers. This could have considerable prac-
tical significance, since modification of taste in humans is an effect that is often desired and
is poorly understood. The value of using insects as models for the investigation of higher

animal taste mechanisms has already been suggested, and the validity of comparing the
two biological systems is supported by the fact that bitter taste to humans has been used as
a guide in the search for insect feeding deterrents in plants.
28
Insects are stimulated to feed
by sugars that taste sweet to humans, and the interaction of sweet and bitter tastes in
humans may be compared with the interactions between stimulants and deterrents in
insects (Figure 19.6). Selected compounds that are bitter tasting to humans have been
found to deter both oviposition and feeding by the tobacco budworm, Heliothis virescens,
and preliminary electrophysiology has suggested that responses of a sucrose-sensitive neu-
ron in the gustatory sensilla of the ovipositor are inhibited by these compounds.
29
In addi-
tion, both insects and vertebrates exhibit a rather general phagostimulatory response to
low molecular weight amino acids such as glycine, β-alanine, α-aminobutyric acid, γ-amino-
butyric acid (GABA), L-arginine, and L-proline.
30
19.5 Conclusions
Modification of taste is of interest for several reasons. Specific inhibitors of bitter taste are
continually sought for pharmaceutical or food science applications, whereas bitter taste
might be considered desirable in other products, such as beer. Suppression of bitterness may
FIGURE 19.5
Extraction and isolation scheme for feeding deterrents (sensitivity suppressors) in wheat germ. *Denotes deter-
rent activity.
© 1999 by CRC Press LLC
be accomplished simply by the addition of salt,
31
or the perception of bitter compounds may
be modified by the presence of citrate or malate that have a sour taste.
32

However, most
studies in this area have focused on modification of sweet taste. Many plant-derived chem-
icals have been shown to act as either sweetness inhibitors or sweetness inducers or enhanc-
ers. As a result, much of the work on taste modification has capitalized on this knowledge
to examine mechanisms that govern sweet taste, and considerable progress has been made
in explaining how sweetness modifiers might work using an insect model system.
33
Although information about the biochemistry of taste receptors in insects as well as
mammals is still rather fragmentary, significant advances in the molecular biology of taste
transduction in mammals have recently been made.
34
Some key component G proteins
involved in taste transduction in mammals have been cloned. Electrophysiological, bio-
chemical, and molecular biological studies have suggested that bitter as well as sweet taste
is transduced by second messenger-mediated pathways involving cyclic adenosine mono-
phosphate (AMP).
35
However, G proteins have yet to be isolated and identified from insect
taste receptors. As soon as taste cell proteins can be cloned from insects, the restriction
imposed by the palate of human tasters will be removed to allow for the design and screen-
ing of novel taste agents.
34
The development of taste sensitivity in mammals or invertebrates is a phenomenon that
still remains a mystery. Observations with human newborns have indicated that responses
to a bitter compound increase within a period of 14 to 180 days.
36
These results appear to
parallel the gradual development of sensitivity to deterrents found in newly hatched larvae
of P. rapae. These caterpillars, therefore, represent an ideal model for detecting taste recep-
tor proteins as the sensory system develops. Such critical information about the processes

involved in taste modification may then provide a starting point for the needed isolation
and cloning of taste receptor proteins. The identities and exact role of phytochemicals in
the modification of taste will be key components of this puzzle that now seems possible to
solve in the foreseeable future.
FIGURE 19.6
The role of taste in food selection and discrimination by insects and humans.
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