Ecological biochemistry and its development

E. Arthur Bell *
Neurodegenerative Diseases Research Centre, Hodgkin Building, King's College London,
Guy's Campus, London SE1 1UL, UK
Received in revised form 8 August 2000
Abstract
The concept that chemistry and biochemistry may play signicant roles in ecological relationships is not a new one. It is only
within the last quarter of a century, however, that this concept has gained general acceptance and ecological biochemistry has
become a well dened interdisciplinary subject in its own right. Examples are given of dierent types of biochemical relationships
involving plants and animals and reference to other areas of the subject are made. #2001 Elsevier Science Ltd. All rights reserved.
Keywords: Danaus plexippus; Asclepias species; Cardenolides; Pyrrolizidine alkaloids; Flavonol glycosides; 3-Hydroxy-2-butanone; Caryedes
brasiliensis; Dioclea megacarpa; Canavanine; Eumaeus atala orida; Zamia oridana; Cycasin; Feeding deterrents; Polymorphism in plants;
Cyanogenic glycosides; Polymorphism in herbivores; Adaptation to monouoracetate
1. Introduction (with acknowledgement to the monarch
buttery)
As previously pointed out (Rothschild, 1972) it is
more than a century since early pioneers in the eld of
insect mimicry, such as Bates, Muller and Wallace,
postulated that certain aposematic insects may be pro-
tected from potential predators by sequestered toxins
obtained directly from their host plants. Haase indeed
claimed specically that brilliantly coloured danaid
butteries whose larvae feed on poisonous Asclepias
species make use of the plant toxins in their own
defense. More than 50 years were to pass, however
before it was shown (Reichstein et al., 1968) that cardiac
glycosides (cardenolides) synthesized by milkweeds
(Asclepias species) on which the larvae of the monarch
buttery Danaus plexippus feed could also be detected in
the insect. This important conrmation that insects
can sequester toxins originating in their food plants
was obtained through the joint eorts of chemists and
biologists and such collaboration must be seen as the
sine qua non of ecological biochemistry. The conr-
mation also made the monarch buttery something
of an ecological celebrity and a brief account of
further studies relating to this insect will serve in this
introduction to illustrate some of the various, complex
and subtle ways to which chemistry can be put by even
a single organism. Evidence that the sequestered
toxins do actually deter potential avian predators was
obtained by Brower et al. (1968). They demonstrated
that toxin-free monarch butteries whose larvae had
been raised on a cardenolide free diet were eaten with
impunity by the inexperienced young of the blue jay
Cyanocitta cristata. In contrast butteries containing
cardenolides whose larvae had fed on Asclepias species,
their usual host plants, caused the jays to vomit and
avoid future encounters with butteries of similar
appearance.
In addition to its need for cardenolides as defense
compounds the monarch buttery has a requirement for
pyrrolizidine alkaloids from which it synthesizes sex
pheromones. These compounds, which are liberated
from hair pencils on the wings of the males during
courtship, encourage the female to mate. Unlike the
cardenolides, the pyrrolizidine alkaloids are not ingested
from food plants by the larvae of the buttery but are
obtained by the adult male which visits species of the
Boraginaceae and Compositae containing these alka-
loids. The insect typically sucks in alkaloid-containing
exudates, from sources such as withered leaves, through
its proboscis. The more recent identication of avonol
glycosides as oviposition stimulants in the leaves of
Asclepias curassavica (Haribal and Renwick, 1996)
emphasizes again the importance to this buttery of
plant secondary compounds.
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PI I : S0031- 9422( 00) 00448- 9
Phytochemistry 56 (2001) 223227
www.elsevier.com/locate/phytochem
* Tel.: +44-20-78148-6021.
E-mail address: e_arthur.bell@kcl.ac.uk
Not all of the monarch's requirements for ecochemi-
cals are met by plants however, and its ability to syn-
thesize its own defense compounds is seen in the
response of its larvae to the potential threat posed by
other insects. In a fascinating paper, Rothschild and
Bergstrom (1997) have described the vigorous waving
induced in the larvae of this buttery by the buzzing of
bees and wasps in their vicinity and the even greater
activity induced by low ying planes of the Royal Air
Force. This physical response to the proximity of wasps
(or war planes) is also accompanied by a chemical
response. The larvae liberate a mixture of over 16 vola-
tile compounds into the atmosphere immediately sur-
rounding them. The compound occurring in highest
concentration (ca 30% of the mixture) is 3-hydroxy-2-
butanone [1], a ketone which did not occur in the
Asclepias species on which the larvae had fed.
A nal reference to the monarch buttery is anecdo-
tal, but may serve to identify a problem worthy of
solution and possibly a novel way of solving it. Some
years ago the buttery house in Syon Park lost a num-
ber of monarch butteries. Subsequently, eggs and lar-
vae of the same species were found on Asclepias plants
in the Royal Botanic Gardens Kew rather more than a
mile away across the river Thames. Assuming that the
eggs were laid by some of the Syon Park escapees, we
are faced with the intriguing problem as to how these
exotic insects found their equally exotic food supply (and
possibly pyrrolizidine containing plants) while at large in
an alien environment. Did they see them? Did they detect
volatile compounds liberated by the plants? and if so what
were the compounds? Perhaps the controlled release of
exotic insects (or other animals) in botanic gardens
might provide new insights into the ways that animals
perceive plants and the chemicals that they contain.
In this short presentation an arbitrary selection of
subjects has had to be made. The examples chosen are
concerned with plant and animal interrelationships but
it is hoped nevertheless that they will illustrate something
of the diversity and interest of the subject and provide an
insight into the ways in which it has developed.
2. Beetles and seeds
Seeds are important sources of food to animals large
and small. Plants cannot aord to have all their seeds
eaten, however, and adaptations such as hard seed coats
and dispersal by dehiscence can reduce the chances of
this happening. The presence of secondary compounds
that are toxic to, or deter feeding in, potential predators
can serve the same purpose.
The seeds of species belonging to the less advanced
tribes of the Papilionoideae (Leguminosae) character-
istically contain quinolizidine alkaloids. These alkaloids
are toxic even at low concentrations to the larvae of the
cow pea weevil Callosobruchus maculatus. Seeds from
species of the more advanced tribes lack these alkaloids
but frequently contain major concentrations (ca 110%)
of non-protein amino acids such as canavanine [2]
which is a close analogue of the protein amino acid
arginine [3]. Canavanine, though less toxic than the
alkaloids, is nevertheless toxic to the larvae of C. macu-
latus at the concentrations in which it occurs in most
legume seeds. It was suggested, therefore, that canava-
nine might serve the seed both as a nitrogen store (it
contains twice as much nitrogen as storage protein) and
a defense against seed beetles (Bell, 1981). The apparent
freedom of most canavanine-containing seeds from
beetle infestation supported this hypothesis but one
notable exception was known. The seeds of Dioclea
megacarpa, which may contain over 10% of canavanine,
provide food for the larvae of Caryedes brasiliensis a seed
beetle found in Costa Rica. Biochemical studies (Rosen-
thal et al., 1976) showed, however, that C. brasiliensis
224 E.A. Bell / Phytochemistry 56 (2001) 223227
possesses an arginyl tRNA synthetase that is able to
discriminate against canavanine and prevent the toxic
eects which its incorporation into insect protein in
place of arginine would otherwise cause. This nding
has a wider signicance, however. It suggests that one of
the most convincing pieces of evidence for the defensive
role of a secondary compound may be evidence that a
successful predator is specically adapted to circumvent
the potentially adverse eects of that compound.
3. Caterpillars and leaves
Nicotine has a long history of use as an insecticide yet
the larvae of the tobacco hornworm Manduca sexta feed
unharmed on the leaves of the tobacco plant Nicotiana
tabacum. This insect is not poisoned by the alkaloid
because it has evolved a highly ecient means of
excreting it (Self et al., 1964). The monarch buttery, as
mentioned previously, benets rather than otherwise
from the cardenolides ingested by its larvae when feed-
ing on Asclepias species. Two other species of the Lepi-
doptera which make use of a host plant's chemical
defenses, but in rather a dierent way, are the tiger
moth Seirarctia echo and the lycaenid buttery Eumaeus
atala orida whose larvae feed on the young leaves of
the cycad Zamia oridana. The Cycadales character-
istically synthesize and store glycosides of the highly
toxic carcinogen methylazoxymethanol (MAM). When
ingested by animals the glycosides are hydrolysed by
b-glucosidase enzymes of the gut and free MAM is lib-
erated. Although b-glucosidase is present in the gut of
S. echo, the insect is protected from the toxic eects of
liberated MAM by its ability to glycosylate that com-
pound (Teas, 1976). Cycasin [4], the non-toxic b-d-glu-
coside so formed, is stored in the moth and also in the
buttery, which presumably detoxies MAM in the
same way. The brilliantly coloured adults of E. atala
have been found to contain as much as 1.8% (dry
weight) of cycasin while the young leaves on which their
larvae feed contain only 0.2% (Rothschild et al., 1986).
Like the monarch buttery, S. echo and E. atala have
the warning colouration of aposematic species. The
presence of cycasin in dried museum specimens of
cycad-feeding aposematic species of other genera and its
absence from a cryptic species whose larvae feed on the
same plants is a further indication of the compound's
defensive role (Nash et al., 1992).
A compound acting as a feeding deterrent may be as
important as a toxin in protecting a plant from preda-
tion. The eectiveness of a feeding deterrent, as of a
toxin, will however be related to its concentration
within the plant. This is well illustrated by the reaction
of the larvae of the African leaf worm Spodopteris lit-
toralis to the non-protein amino acid l-2,4-diaminobu-
tanoic acid [5] which occurs in the leaves of Lathyrus
latifolius. At low concentrations the amino acid acts as a
phagostimulant and at high concentrations as a feed-
ing deterrent (Bell et al., 1996). The insect's response
to l-2,4-diaminobutanoic acid cannot but remind us of
our own to mustard or curry powder.
4. Biochemical polymorphism in plants
Individual plants within a population may dier from
one another in their biochemical characteristics. Clover
Trifolium repens and birdsfoot trefoil Lotus corniculatus
both exists in two forms, one of which liberates hydro-
gen cyanide when crushed and the other which does not.
The cyanogenic forms contain both cyanogenic glyco-
sides, and a hydrolytic enzyme which catalyses the
breakdown of the glycosides when brought into contact
with them in the damaged tissue. For example, lina-
marin [6] which occurs in both species is hydrolysed
enzymatically to give acetone cyanohydrin [7] which in
turn breaks down spontaneously to acetone [8] and
hydrogen cyanide [9]. The acyanogenic forms of the
plants may lack either the cyanogenic glycosides, the
enzyme, or both. In Europe, a remarkable correlation
was found to exist between the frequency of occurrence
of the cyanogenic form of T. repens and the mean Jan-
uary temperature (Daday, 1954). The signicance of this
became apparent when Jones (1972) demonstrated that
slugs and snails preferred the acyanogenic forms of both
species. In regions such as the British Isles and the
Iberian peninsular where winter temperatures are rela-
tively mild and the molluscs continue to be active, cyano-
genic forms dominate. In Scandinavia and Russia, lower
winter temperatures ensure that the plants are able to
establish themselves before they are eaten by slugs or
snails and here the acyanogenic forms predominate.
Another example of polymorphism of this type is seen
in bracken Pteridium aquilinum where the acyanogenic
form is grazed by sheep and deer while the cyanogenic
form is avoided (Cooper-Driver and Swain, 1976).
5. Biochemical polymorphism in herbivores
Many indigenous Australian plants are toxic to domes-
tic animals introduced from Europe. These plants include
species of the legume genera Gastrolobiumand Oxylobium
that grow in Western Australia. The toxin that they con-
tain is monouoroacetate [10] which is highly poisonous
to many mammals including man. After ingestion the
compound is converted to uorocitrate which inhibits
the enzyme aconitate hydratase, blocks the tricarboxylic
acid cycle and causes respiratory failure.
Of the native fauna, the western grey kangaroo
Macropus fuliginosus, has evolved a tolerance to this
compound. This tolerance is found in animals from
E.A. Bell / Phytochemistry 56 (2001) 223227 225
both southwestern and southeastern Australia. Similar
tolerance is found in the rat kangaroo Bettongia peni-
cillata and the bush rat Rattus fuscipes but only in those
members of the two species living in southwestern Aus-
tralia where plants containing monouoroacetate grow.
These ndings suggest that the rat kangaroo and the
bush rat originated in eastern Australia and only when
members of the species moved west and encountered
plants containing the toxin was their tolerance to
monouoroacetate developed. In contrast the western
grey kangaroo probably originated in the west and
tolerance was retained in populations as they spread
eastward (Mead et al., 1985).
6. Induced response to herbivory
Plants are not only able to accumulate defense com-
pounds prior to attack by a potential predator, they can
also respond to such an attack. Insect feeding, or indeed
mechanical damage, may induce increased synthesis of
pre-existing defense compounds such as alkaloids, ter-
penoids, phenolics, or proteinase inhibitors. Alter-
natively it may activate entirely new biosynthetic
pathways. Pare and Tumlinson (1997) have shown for
example that the beet army worm Spodoptera exigua
feeding on the cotton plant Gossium hirsutum activates
pathways leading to the de novo synthesis of a number
of volatile terpenes and indole. The activation of these
pathways involves both plant and insect, the trigger
being an insect modied fatty acid of plant origin. The
liberated volatiles serve to attract parasitoids, usually
parasitic wasps, that prey on the herbivorous cater-
pillars. It was subsequently shown (De Moraes et al.,
1998) that the composition of the volatile ``distress sig-
nal'' mixture liberated by the plant under attack is
strictly related to the identity of the attacker. Tobacco,
cotton and maize produce dierent volatile blends
depending on whether they are being eaten by Heliothis
virescens or Heliocoverpa zea. The specialist wasp Car-
diochiles nigriceps is able to recognise these dierences
and seek out cotton or tobacco plants suering infesta-
tion by its host H. zea.
7. Conclusion
Examples of plant and animal interactions at the
chemical and biochemical level have been given.
Enough, it is hoped, to indicate how subtle and complex
they can be. Time has not allowed mention of other
important areas such as the biochemical adaptations of
plants and animals to their physical environments, of
higher plants to each other, of higher plants to lower
plants and micro-organisms, or of animals to each
other. As with plant and animal interrelationships our
knowledge of these other areas has greatly increased in
the last quarter of a century due primarily to the skill
and dedication of a small number of enthusiasts. Not
least of these is Jerey Harborne who is now retiring as
Editor of Phytochemistry. He and his colleagues have
made many original contributions in studies ranging
from the response of plants to fungal attack (Harborne
and Ingham, 1978) to the role of sesquiterpene lactones
as feeding deterrents in insects (Rees and Harborne,
1985). As Editor of Phytochemistry he has welcomed
and encouraged the submission of original inter-
disciplinary papers. He has contributed to a great many
symposia and edited proceedings volumes such as Phy-
tochemical Ecology (see Jones, 1972) and Biochemical
Aspects of Plant and Animal Coevolution (Harborne and
Ingham, 1978). He has kept us abreast of current
developments with excellent literature reviews; notably
the Recent Advances in Chemical Ecology series
(Harborne, 1986, 1989, 1993a, 1997, 1999). His most
inuential written contribution may, however, have
been Introduction to Ecological Biochemistry (Harborne,
1993b). This small volume, which is now in its fourth
edition, has awakened the interest of generations of
students (and others) to a fascinating subject and encour-
aged them to enter a eld where the increasingly sophis-
ticated analytical techniques of the physical sciences can
be applied to the solution of biological problems.
References
Bell, E.A., 1981. Non-protein amino acids in the Leguminosae. In:
Polhill, R.M., Raven, P.H. (Eds.), Advances in Legume Systematics
Part 2. Royal Botanic Gardens Kew, London, pp. 489499.
Bell, E.A., Perera, K.P.W.C., Nunn, P.B., Simmonds, M.S.J., Blaney,
W.M., 1996. Non-protein amino acids of Lathyrus latifolius as
feeding deterrents and phagostimulants in Spodoptera littoralis.
Phytochemistry 43, 1003.
Brower, L.P., Ryerson, W.N., Coppinger, L.L., Glazier, S.C., 1968.
Ecological chemistry and the palatability spectrum. Science, N.Y.
161, 1349.
Cooper-Driver, G.A., Swain, T., 1976. Cyanogenic polymorphism in
bracken in relation to herbivore predation. Nature 260, 604.
Daday, H., 1954. Gene frequencies in wild populations of Trifolium
repens 1. Distribution by latitude. Heredity 8, 61.
De Moraes, C.M., Lewis, W.J., Pare, P.W., Alborn, H.T., Tumlinson,
J.H., 1998. Herbivore-infested plants selectively attract parasitoids.
Nature 393, 570.
Harborne, J.B., 1986. Recent advances in chemical ecology. Nat.
Prod. Rep. 3, 523.
Harborne, J.B., 1989. Recent advances in chemical ecology. Nat.
Prod. Rep. 6, 85.
Harborne, J.B., 1993. Recent advances in chemical ecology. Nat.
Prod. Rep 10, 327.
Harborne, J.B., 1993b. Introduction to Biochemical Ecology, 4th.
Edition. Academic Press, London.
Harborne, J.B., 1997. Recent advances in chemical ecology. Nat.
Prod. Rep. 14, 83.
Harborne, J.B., 1999. Recent advances in chemical ecology. Nat.
Prod. Rep. 16, 509.
Harborne, J.B., Ingham, J.L., 1978. Biochemical aspects of the co-
evolution of higher plants and their fungal parasites. In: Harborne,
226 E.A. Bell / Phytochemistry 56 (2001) 223227
J.B. (Ed.), Biochemical Aspects of Plant and Animal Coevolution.
Academic Press, London, pp. 343405.
Haribal, M., Renwick, J.A.A., 1996. Oviposition stimulants for the
monarch buttery: avonol glycosides from Asclepias curassavica.
Phytochemistry 41, 139.
Jones, D.A., 1972. Cyanogenic glycosides and their function. In: Har-
borne, J.B. (Ed.), Phytochemical Ecology. Academic Press, London,
pp. 103124.
Mead, R.J., Oliver, A.J., King, D.R., Hubach, P.H., 1985. The co-
evolutionary role of uoroacetate in plantanimal interactions in
Australia. Oikos 44, 55.
Nash, R.J., Bell, E.A., Ackery, P.R., 1992. The protective role of
cycasin in cycad-feeding lepidoptera. Phytochemistry 31, 1955.
Pare , P.W., Tumlinson, J.H., 1997. Induced synthesis of plant vola-
tiles. Nature 385, 30.
Rees, S.B., Harborne, J.B., 1985. The role of sesquiterpene lactones
and phenolics in the chemical defence of the chicory plant. Phyto-
chemistry 24, 2225.
Reichstein, T., von Euw, J., Parsons, J.A., .Rothschild, M., 1968.
Heart poisons in the monarch buttery. Science N.Y. 161, 861.
Rosenthal, G.A., Dahlman, D.L., Janzen, D.H., 1976. Anovel means for
dealing with l-canavanine a toxic metabolite. Science N.Y. 192, 256.
Rothschild, M., 1972. Secondary plant substances and warning col-
ouration in insects. In: van Emden, H.F. (Ed.), Insect/Plant Rela-
tionships. Blackwell Scientic Publications, Oxford, pp. 5983.
Rothschild, M., Nash, R.J., Bell, E.A., 1986. Cycasin in the endan-
gered buttery Eumaeus atala orida. Phytochemistry 25, 1853.
Rothschild, M., Bergstrom, G., 1997. The monarch buttery caterpillar
(Danaus plexippus) waves at passing hymenoptera and jet aircraft: are
repellent volatiles released simultaneously. Phytochemistry 45, 1139.
Self, L.S., Guthrie, F.E., Hodgson, E., 1964. Metabolism of nicotine
by tobacco-feeding insects. Nature 204, 300.
Teas, H.J., 1967. Cycasin synthesis in Seirarctia echo (Lepidoptera)
larvae fed on methylazoxymethanol. Biochemical and Biophysical
Research Communications 26, 686.
E. Arthur Bell, after a rst degree in
chemistry at Kings's College New-
castle (University of Durham), spent a
short period with Imperial Chemical
Industries at Billingham. A research
award then took him to Trinity Col-
lege Dublin where he worked for his
PhD on the mode of action of the sul-
phonamide drugs, under the direction
of Professors Cocker and O'Meara.
An assistant lectureship in biochem-
istry at TCD and access to Trinity
College Botanic Garden were keys to a
growing interest in phytochemistry.
From Dublin he moved to King's College London to become Lec-
turer and then Reader in Biochemistry. Here, he continued his
research on non-protein amino acids and related compounds, encour-
aged by Leslie (now Sir Leslie) Fowden, who was at that time a
neighbour in University College. While at King's, he was recruited by
Jerey Harborne as part of a plan to widen the interests of the old
Plant Phenolics Group and create a society concerned with all aspects
of plant chemistry and biochemistry. This was the Phytochemical
Society of which he was later to become chairman.
In 1968, he was appointed Professor of Botany in the University of
Texas at Austin, working with Tom Mabry and Billie Turner. He
returned to King's College London in 1972 as Professor of Biology
and Head of the Department of Plant Sciences.
In 1981, he became Director of the Royal Botanic Gardens Kew and
retired in 1988. For 25 years, he served on the Editorial Board of
Phytochemistry. In retirement, he is Visiting Professor at King's Col-
lege London and Adjunct Professor at the University of Texas at
Austin. CB (1988); FKC; Hon. FTCD; Hon. Mem. Phytochemical
Society of Europe.
E.A. Bell / Phytochemistry 56 (2001) 223227 227