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Natural products derived from plants, marine organ-isms, and microbes continue to play an important

role in drug discovery for a broad range of diseases(Koehn and Carter 2005). In the case of plants, a varietyof strategies have been employed to select candidates fordrug discovery. A random screening of extracts fromapproximately 12 000 plant species by the US NationalCancer Institute yielded a number of active chemotypes,two of which advanced to commercial products for thetreatment of cancer (Cragg and Newman 2005).Ethnobotanically guided collections are responsible fornearly three-quarters of the 119 plant-derived drugs incurrent use, but the use of traditional knowledge to guide

plant collections has proven highly controversial due tothe difficulty of linking traditional knowledge with intel-lectual property ownership and benefit sharing (Craggand Newman 2005; Rosendal 2006).

While the field is still in its infancy, ecological andevolutionary theories based on the chemical defense pat-terns of rainforest plants have been used to directsearches for active plant compounds by the PanamaInternational Cooperative Biodiversity Groups Program(ICBG), a program involved in drug discovery for cancerand tropical disease, scientific capacity building, and bio-diversity conservation (Coley et al. 2003; Kursar et al.2006; Capson in press). Young leaves have been preferen-

tially collected and tested in the drug discovery bioassaysof the Panama ICBG program, because young leaves typ-ically have higher concentrations and a greater diversityof chemical defense compounds than do mature leaves.This strategy has proven successful for discovering novel

compounds with activity against tumor cell line(Hussein et al. 2005), and protozoan agents of cutaneouleishmaniasis (Leishmania mexicana and Leishmania panamensis) and Chagas’ disease (Trypanosoma cruzi; JiméneRomero et al. 2007). To broaden the collecting strategiebased on ecological and evolutionary criteria used by thPanama ICBG program, and to provide additional insighinto the nature of plant–insect chemical ecology, wexamined associations between tropical insect herbivoreand their host plants.

It has been proposed that the co-evolution or “armrace” between insect herbivores and plants has encouraged the development of defense mechanisms (eg plan

secondary metabolites) and fostered the diversification oboth groups (Ehrlich and Raven 1964; Kareiva 1999Thompson 1999; Rausher 2001). Similar interactionbetween insects and their predators may also have led tthe development of conspicuous aposematic (“warning”coloration in insects, to advertise their distastefulness otoxicity to visually oriented predators (Guilford 1988Ruxton et al. 2007). Aposematic coloration typically consists of bright and contrasting colors, such as redoranges, yellows, blues, and purples, alternating witblack, dark brown, or dark gray (Salazar and Whitma2001). Moreover, aposematic insects often feed on toxihost plants that possess potent plant secondary com

pounds, which insects can use for defense against predators (Prieto et al. 2007). Some of these plant secondarcompounds also have medicinal properties (Nishid2002). However, because many edible insects mimiaposematically colored, distasteful species (Batesian mimicry; Brower et al. 1967), we cannot expect all aposematcally colored, herbivorous species to be reliable indicatoof plants with high levels of secondary compounds. Athe presence of Batesian mimics in insect populationmay obscure possibly useful linkages between aposematiherbivores and toxic host plants, our study investigate

RESEARCH COMMUNICATIONS RESEARCH COMMUNICATIONS

Ecological and evolutionary bioprospecting:using aposematic insects as guides torainforest plants active against disease Julie E Helson1,2, Todd L Capson1,3*, Timothy Johns1, Annette Aiello3, and Donald M Windsor3

We examined Coleoptera and Lepidoptera assemblages feeding on two different groups of plants: one i

which plants were active against cancer cell lines and/or protozoan parasites responsible for tropical para

sitic diseases, and a second group that was inactive in the same bioassays. Aposematic species were found o

nine of the ten active plant species, but on only four of the ten inactive plant species. Non-aposematic

insects did not show a significant difference in their association with active versus inactive plants. Ou

results suggest that the presence of aposematic, herbivorous insects can be used to facilitate the identifica

tion of plants with compounds active against important human diseases.

Front Ecol Environ 2009; 7(3): 130–134, doi:10.1890/070189 (published online 7 Jul 2008)

1Department of Plant Science, McGill University, Ste Anne de

Bellevue, Quebec, Canada; 2current address: University of Toronto at

Scarborough, Scarborough, Ontario, Canada; 3Smithsonian Tropical

Research Institute, Balboa, Ancón, Panama, Republic of Panama*([email protected])



JE Helson et al. Aposematic insects and bioprospecting

whether there is a net positive benefit to using the pres-ence of aposematic insects to guide the search for plantswith biologically active secondary compounds.

We test the hypothesis that plants with compoundsthat have activity against cancer cell lines and/or tropicaldisease-causing parasites are more likely to have feedingassociations with aposematic insects than are inactiveplants. To test this hypothesis, we selected ten plantspecies that demonstrated activity in one or more bioas-says against protozoan agents responsible for cutaneousleishmaniasis (L mexicana or L panamensis), Chagas’ dis-ease (T cruzi), and malaria (Plasmodium falciparum), orbreast, lung, and central nervous system cancer cell lines,and ten plant species that showed no activity in the samebioassays. Each of the species was then surveyed for itsassociated coleopteran and lepidopteran herbivoreassemblages in four Panamanian Protected Areas.

Methods

Criteria for the choice of plant study species

Plant species included in this study were chosen fromamong 1380 species tested by the Panama ICBG pro-gram in bioassays for activity against breast, lung, andcentral nervous system cancer cell lines, and the para-sites P falciparum, T cruzi, and L mexicana or L panamen-sis, from January 1999 through January 2004. Plantspecies were chosen from six plant families (Asteraceae,Boraginaceae, Bignoniaceae, Convolvulaceae, Rubiaceae,and Solanaceae) on the basis of: (1) consistent activityor inactivity in the bioassays, (2) accessibility in thefield, and (3) abundance in the field. Plants were

deemed “active” if they displayed activity in one or moreof the bioassays (Table 1). Plants categorized as “inac-tive” yielded extracts that showed no activity in thebioassays. Plant extracts were prepared using the proto-col described by Montenegro et al. (2003).

Active and inactive plant species were paired by familywhenever possible, in order to optimize the similarity of general characteristics within the plant pairs (Table 1).Phylogenetic relationships were used to pair those activeand inactive plants in cases where there was more thanone potential plant pair per plant family. Phylogenies forAsteraceae were obtained from Bremer (1994),Solanaceae from Hunziker (1979), and Rubiaceae from

Pereira and Barbosa (2004) and Andreasen and Bremer(2000). Rafael Aizprúa of the Smithsonian TropicalResearch Institute (STRI) collected and subsequently con-firmed plant species identities. Voucher specimens of eachplant species studied have been deposited in the herbariaof the University of Panama and STRI (WebTable 1).

Monitoring and collection of insects

Research was carried out in Panamanian ProtectedAreas, two of which are located in areas of lowland moist

tropical forest (Soberania National Park [9˚7’ N, 79˚42’W, 60 m above sea level (asl)] and Barro Colorado NatureMonument [9˚9’ N, 79˚51’ W, 80 m asl], and two of whichare located in mountainous areas of humid tropical forest(Chagres National Park [9˚13’ N, 79˚22’ W, 950 m asl]and Altos de Campana National Park [8˚13’ N, 79˚22’ W,800 m asl]).

Sampling time – the time actively spent searching aplant for herbivores – was equalized between active andinactive plants to the greatest practical degree.Individuals of active and inactive plant species weresearched for approximately 210 and 190 minutes,respectively, during each census. Time was used as ameasure of sampling effort, because the study plantswere of diverse types (including herbs, lianas, shrubs,and trees) and because the abundance of the studyspecies varied greatly, both within and among studysites. The minimum number of individuals of eachspecies searched during each census is given in Table 1.Each plant included in the study was marked and

observed a total of 16 times, at weekly or biweekly inter-vals, between May 2004 and November 2004, duringthe Panamanian rainy season (typically April throughDecember), which is when insects are most abundant(Windsor et al. 1992).

Monitoring for insect herbivores consisted of thor-oughly checking all leaf surfaces of the plant, paying par-ticular attention to whether insects were actually feeding.When necessary, a hooked pole was used to carefullybring branches within reach. All adult and larvalColeoptera and larval Lepidoptera found on study plantswere brought to the laboratory. Each insect was placed ina container with an uneaten leaf of the plant species upon

which it was found, in order to test whether it had beeneating the plant or had simply been resting on it. Onlyinsect species that ate the plant sample in the laboratorywere included in the analyses.

Larvae of Coleoptera and Lepidoptera found on studyplants were reared to the adult stage, mounted, and iden-tified. All adult Coleoptera found on the study plantswere also mounted and identified. Voucher specimens of all collected insect species have been deposited in theFairchild Museum of Invertebrates, University of Panama, and the STRI Synoptic Insect Collection.

Identification of insects

AA identified the Lepidoptera, and H Stockwell andDMW identified the Coleoptera. In a few cases, insectspecimens were identified from photographs sent to expertsin a particular field. Insects were considered aposematic if their integuments were colored, visibly to the human eye,wholly or in part, in red, orange, yellow, blue, or purple,with or without black, dark gray, or dark brown bands orother markings (Salazar and Whitman 2001). In contrast,insects were considered non-aposematic if they were green,black, brown, or gray, or used “imitation” for defense (eg

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found to be associated with each species are shown iWebTable 2. Forty-six insect species were collected fromplants that were active in tropical parasite or tumor celine bioassays, whereas 25 insect species were collectefrom plants whose extracts were inactive in the sam

bioassays. Given that sampling efforts of active and inactive plants were similar, the total numbers of insect speciecollected from active plants was significantly greater thathat from inactive plants (2 [1] = 6.22, P = 0.013).

Aposematic species were found on nine of the teactive plant species, but on only four of the ten inactivplant species (one-tailed Fisher’s exact test, P = 0.03Two of the aposematic insects found on biologicallactive plants are shown in Figure 1. Aposematic specieconstituted 19 of the 46 insects collected from activplants, whereas aposematic insects constituted only fivof the 25 insects collected from inactive plants (2 [1] 8.167, P = 0.01; Figure 2). On average, 1.9 aposemati

insect species were found on each active plant speciecompared to only 0.5 on each inactive plant specie(Wilcoxon matched-pairs signed rank test, P = 0.03).

Non-aposematic insect species were found on eight othe ten active plants and on all ten of the inactivplants (one-tailed Fisher’s exact test, P = 0.24). No significant difference was found between the number onon-aposematic insects on active plants (27 of 46) versus inactive plants (20 of 25; 2 [1] = 1.043, P = 0.31Figure 2). On average, 2.7 non-aposematic insecspecies were found on each active plant species and 2.

geometrid larvae mimicking “sticks” and Oxytenis modestialarvae mimicking either bird droppings or snakes, depend-ing on the instar). Aposematic insects were scored accord-ing to the feeding stage observed in the field, which werelarval Lepidoptera and adult Coleoptera, with the excep-

tion of the beetle Physonota alutacea, in which both the lar-val and adult stages were observed feeding.

Statistical analyses

Chi-squared goodness of fit tests were used to test distri-butions (numerical) observed in the field againstexpected distributions, determined by the null hypothesis(no difference). A Fisher’s exact test was used in caseswhere the sample size was small (when an expected valuewas less than ten). A Wilcoxon matched-pairs signedrank test was used to compare the number of insectsbetween plant pairs because of non-normal data distribu-

tions and the prevalence of zero values.

Results

Table 1 shows the biologically active and inactive plantpairs that we examined in this study, the bioassays inwhich the extracts of active plants tested positive againstone or more of the tumor cell lines or tropical protozoanparasites used in this study, and the approximate numberof individuals of each plant that we searched for insects.The aposematic and non-aposematic insects that we

Table 1. Pairs of biologically active and biologically inactive plants and the minimum number of individuals of each

plant species searched for insects during each census

Biologically active (type of bioactivity in thePanama International Cooperative Approximate number of Approximate number of Biodiversity Group screens) plants examined Biologically inactive plants examined

Tithonia diversifolia (Asteraceae) 20 Tilesia baccata 15

(cancer, P falciparum,T cruzi) (Asteraceae)Neurolaena lobata (Asteraceae) 15 Wedelia calycina 40

(cancer, T cruzi) (Asteraceae)

Baccharis trinervis (Asteraceae) 50 Alibertia edulis 20

(cancer, P falciparum, T cruzi) (Rubiaceae)

Melampodium divaricatum (Asteraceae) 50 Brugmansia candida 10

(cancer, T cruzi) (Solanaceae)

Phryganocydia corymobosa (Bignoniaceae) 7 Jacaranda copaia 12

(cancer, P falciparum, T cruzi) (Bignoniaceae)

Cordia curassavica (Boraginaceae) 15 Tournefortia hirsutissima 7

(cancer) (Boraginaceae)

Bonamia trichantha (Convolvulaceae) 10 Maripa panamensis 10

(cancer, T cruzi) (Convolvulaceae)

Chomelia recordii (Rubiaceae) 8 Chiococca alba 10

(malaria, T cruzi, L mexicana) (Rubiaceae)

Hamelia axillaries (Rubiaceae) 10 Isertia haekeana 10

(cancer, P falciparum) (Rubiaceae)

Witheringia solanacea (Solanaceae) 15 Solanum jamaicense 25

(cancer, P falciparum, T cruzi) (Solanaceae)



JE Helson et al. Aposematic insects and bioprospecting

ally sequestering biologically active defensive com-

pounds, but that these compounds were not active in thebioassays used in this study. Although aposematic insectswere found on inactive plants, they did not affect the netpositive benefit of using aposematic insects as guides toplants containing biologically active compounds. In con-sidering the ecological significance of these results, itshould be recognized that many unpalatable insects arenot aposematic, especially if visual predation is not of pri-mary importance (Schaffner et al. 1994), that not allbrightly colored members of mimicry complexes are nec-essarily toxic (Fordyce 2000), that some insects are ableto synthesize defensive chemicals de novo, and that notall insect species feeding on a plant species with defensive

compounds will sequester them (Nishida 2002).Our results come from only ten plant species pairs.

on each inactive plant species (Wilcoxon matched-pairs

signed rank test, P = 0.38).

Discussion

The results of this study suggest that plants containingbiologically active compounds are more likely to haveassociations with aposematic insects than are inactiveplants. The presence of aposematic insects can thereforeindicate that a particular tropical plant may contain bio-logically active compounds. As non-aposematic insectsare more equally associated with active and inactiveplants, using all the insects collected on plants may notbe as informative as using only aposematic insects. These

observations also suggest that the plant secondarymetabolites that show activity in the bioassays used inthis study may be among those compounds that areexploited by the aposematic insect herbivoresas protection against predation. Insects havealso been examined for their potential toyield biologically active compounds, inde-pendent of their host plants; however, anICBG program based in Costa Rica reporteddifficulties in collecting sufficient quantitiesof insects for the bioassay-guided isolationand characterization of chemical compounds(Sittenfeld et al. 1999; Nielsen et al. 2004).

Although a significantly greater number of aposematic insects were found to eat plantscontaining biologically active compounds,they were also found to feed on inactiveplants. One explanation is that these apose-matic insects do not contain toxic com-pounds and are mimics of other toxic/dis-tasteful aposematic insects, which do in facteat plants containing biologically active com-pounds (Batesian mimicry). Another possi-bility is that the aposematic insects are actu-

Fig ure 2. Number of aposematic and non-aposematic insect species found to feed on the ten biologically active and ten biologically inactive tropical plantsstudied.

Aposematic Non-aposematic

Insect type

N u m b e r o f i n s e c t s p e c i e s 30

25

20

15

10

5

0

Active plants

Inactive plants

Fig ure 1. Two of the aposematic insects found to feed on biologically active plants. (a) The adult stage of the coleopteran, Chari-dotis coccinea, and (b) the larval stage of the lepidopteran, Dysschema jansonis.

(a) (b) 133

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Additional studies of active and inactive plant specieswould increase the generalizability of our findings.

Nevertheless, these results suggest that aposematic insectscan guide researchers to plants that contain biologicallyactive compounds, a finding that provides additional evi-dence that insight from ecology and evolutionary theorycan be used to a practical end, in this case, enhancing thelikelihood of encountering plant species with secondarymetabolites active against human diseases. Our results alsoprovide a demonstration of the benefits to human healthfrom the protection of tropical biodiversity, where thestudy of species from multiple trophic levels can facilitatethe discovery of novel medicines.

Acknowledgements

The authors thank E Ortega, L Romero, and their col-leagues for performing the bioassays reported in this study,R Aizprúa for confirming plant identifications, H Stock-well for helping with insect identifications, the

Panamanian National Authority of the Environment forplant and insect collection permits (permit numbers forplants: SC/P-17-03, SC/P-22-03, SC/P-23-03, SC/P-24-03, SC/P-25-03, SC/P-26-03, SC/P-27-03, and SC/P-28-03; permit numbers for insects: SC/AP-1-05), ICBG par-ticipants P Coley, T Kursar, and M Heller for the use of laboratory facilities, and J Bede for input and support. Wegratefully acknowledge financial support from the US

National Institutes of Health, the US National ScienceFoundation, and the US Department of Agriculturethrough the International Cooperative BiodiversityGroups Program (grant numbers 1U01 TW01021-01 and1U01 TW006634-01), an Engineering Research Council

of Canada (NSERC) PGS-A Fellowship, and the LevinsonFellowship from the STRI–McGill NEO program.

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