Insect Relationships As Guides to Medicinally-Active Plants

TROPICAL HOST PLANT-INSECT RELATIONSHIPS AS GUIDES TO MEDICINALLY-ACTIVE PLANTS

Julie Elizabeth Helson Department of Plant Science - NEO option Mc Gill University, Montreal Submitted August, 2005

A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Masters of Science

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ABSTRACT

Previous studies have shown that: (1) plant defensive compounds may have medicinal properties; and (2) defensive compounds present in aposematic insects are often sequestered from their host-plantes). This study addresses whether aposematic insects can be used as guides to detect plants containing medicinally-active compounds. First, ten tropical medicinally-active plants and ten non-active plants, selected using previous ICBG bioassay results, were observed regularly to determine their insect populations. Aposematic insects were found more frequently on active than non-active plants (X2=8.l67, P=O.Ol). Second, three aposematic insects feeding on Tithonia diversifolia were examined chemically to determine the fate of the plant's pharmaceutically-active compounds. They were not found to sequester or excrete these compounds. Therefore, using aposematic insects could increase the likelihood of finding plants with medicinally-active compounds; however, these insects may not necessarily utilize these compounds for defensive purposes. The underlying basis for this significant association between aposematic insects and medicinally-active plants requires further investigation. 3

RESUMÉ

Des études précédentes ont montré que: (1) certains composés défensifs utilisés par les plantes peuvent également avoir des propriétés médicinales; et (2) les composés défensifs présents chez les insectes aposématiques sont souvent séquestrés de leur plantees) hôte(s). Cette étude évalue si les insectes aposématiques peuvent être utilisés comme indices pour la détection de plantes contenant des substances potentiellement actives médicinalement. Premièrement, dix plantes panaméennes médicinalement actives et dix plantes médicinalement inactives ont été observées régulièrement afin de caractériser leur population d'insectes. Des insectes aposématiques ont été trouvés plus fréquemment sur les plantes médicinalement actives qu'inactives (X2=8.l67, P=O.Ol). Deuxièmement, trois èspeces d'insectes aposématiques se nourisssant sur Tithonia diversifolia ont été examinés chimiquement afin de déterminer s'il séquestrent les composés actifs de la plante. Ils ne séquestrent ni n'excrètent pas ces composés. L'utilisation d'insectes aposématiques pourrait donc augmenter la probabilité de trouver des plantes avec des composés médicinalement actifs; cependant, ces insectes n'utiliseraient pas nécessairement ces composés pour leur défense. Les causes sous-jacentes de l'association significative entre les insects aposématiques et les plantes médicinalement actives requièrent des recherches plus approfondies. 4

ACKNOWLEDGEMENTS l thank my supervisors Todd Capson and Tim Johns for guidance and suggestions during the design of the study and subsequent analysis of the data. l also thank my supervisors for help during the editing process. l also thank my supervisor Todd Capson for finanical support for the fieldwork portion ofmy study, as weIl as for access to materials, equipment, and facilities for performing the laboratory experiments. l thank Jacquie Bede who served on my thesis advisory committee and provided me with many helpful comments on my thesis. l thank Nilka Tejeria for administrative support at STRI, Erika Garibaldo, and Maria HelIer for administrative and technical support at STRI and within the ICBG, and Carolyn Bowes for administrative support from the department of Plant Science at McGill. l thank Donald Windsor, Annette AieIlo, Henry StockweIl, and Fred Vend for their comments concerning my study, for sharing with me their knowledge on insect ecology, for providing me with information regarding the collection, rearing, and mounting of insects, as weIl as for help with the identification of numerous insect species. l thank Jason Hall and Matthew Barnes for help with Lepidoptera identifications. l thank Rafael Aizprua for helping me locate aIl of my study plants, accompanying me in the field, as weIl as for helping me with plant identifications and voucher specimens. l thank Irma Alvarez for advice on making voucher specimens. l thank Johant Lakey, Carlos Rios, and Carlos Jimenez for aIl the instructions and assistance in the chemistry labo l also thank them for helping me determine which chemical procedures and chemical conditions should be used for analyzing my samples. l thank Johant Lakey for the instructions and assistance on HPLC techniques and helping me determine what chemical conditions should be used to run my samples. l thank Roger Linington for guidance on chemistry techniques. l thank everyone in Luis Cubilla's chemistry laboratory at the University of Panama for technical support. l thank the laboratories of Eduardo Ortega and Luz Romero for running the disease bioassays. l am extremely grateful to Erick Sarmiento for assistance and for accompanying me in the field. 5

1 am extremely grateful to Blair Helson and Susan Helson for their constant support and helping to revise my thesis.

1 thank Oscar Puebla for helping with the translation of my abstract to French.

1 thank Oscar Gabriel Lopez Chong for the help collecting insects in the field.

1 am extremely grateful to Catherine Potvin, the NEO students, and the ICBG members.

This project was supported financially by a NSERC PGS-A scholarship, as well as a Levinson Fellowship from the NEO Program. 6

TABLE OF CONTENTS

ABSTRACT ...... 2

RESUMÉ ...... 3

ACKNOWLEDGEMENTS ...... 4

TABLE OF CONTENTS ...... 6

LIST OF FIGURES ...... 7

LIST OF TABLES ...... 8

1. INTRODUCTION AND LITERATURE REVIEW ...... 9 1.1. Introduction ...... 9 1.2. Bioprospecting in tropical rainforests ...... 10 1.3. Primary and secondary plant metabolism ...... 11 1.4. Insect-plant relationships ...... 13 1.5. Insects and their use of plant secondary metabolites ...... 14 1.6. Aposematic and other mechanisms to display unpalatability ...... 16 1.7. Improving the bioprospecting process ...... 18 1.8. Previous studies on aposematic insects and medicinally-active plants ... .21 1.9. Relevance to science, health, and conservation ...... 21 1.10. Introduction to the chapters ...... 23

2. CHAPTER 1 ...... 25 ABSTRACT ...... 26 2.1. Introduction ...... 27 2.2. Methods ...... 30 2.3. Results ...... 35 2.4. Discussion ...... 52 2.5. Conclusion ...... 61

3. LINKING STATEMENT ...... 62

4. CHAPTER 2 ...... 63 ABSTRACT ...... 64 4.1. Introduction ...... 65 4.2. Methods ...... 69 4.3. Results ...... 72 4.4. Discussion ...... 78 4.5. Conclusion ...... 88

5. CONCLUSIONS ...... 90

6. REFERENCES ...... 94 7

LIST OF FIGURES

Figure 2.1. Percentages of aposematic insect species found on tropical study plants, using all insect species observed feeding on the study plants. Comparisons were done using paired study plants. Plants were paired using phylogenetic relationships ...... 47

Figure 2.2. Percentages of aposematic insect species found on tropical study plants, using only those insects where more than one individual was observed feeding on the study plants species. Comparisons were done using paired study plants. Plants were paired using phylogenetic relationships ...... 50

Figure 4.1. HPLC chromatograms of the crude extracts of both young and mature leaves from Tithonia diversifolia (Asteraceae) comparing the results from the wavelengths 210.50 nm and 254.00 nm, as well as HPLC chromatograms of the active fractions from T diversifolia at wavelength 254.00 nm ...... 74

Figure 4.2. HPLC results at wavelength 254.00 nm comparing retenti on times of the crude young leaf extract, and anti-Chagas' fractions of the plant Tithonia diversifolia (Asteraceae), and the extract of Platyphora ligata (Coleoptera: Chrysomelidae), an insect which feeds on the plant ...... 76

Figure 4.3. HPLC results at wavelength 254.00 nm comparing retenti on times of the crude young leaf extract and anti-Chagas' fractions of the plant Tithonia diversifolia (Asteraceae), and the insect and excrement extracts of Chlosyne hippodrome (Lepidoptera: Nymphalidae), an insect which feeds on the plant ...... 79

Figure 4.4. HPLC results at wavelength 254.00 nm comparing retention times of the crude young leaf extract and anti-Chagas' fractions of the plant Tithonia diversifolia (Asteraceae), and the insect and excrement extracts of Dysschema magdala (Lepidoptera: Arctiidae), an insect which feeds on the plant ...... 81 8

LIST OF TABLES

Table 2.1. Selected pairs ofmedicinally-active and medicinally-non-active plants. For the medicinally-active plants their bioactivity in the ICBG screens is also indictated ...... 31

Table 2.2. Number of aposematic and non-aposematic insect species collected on the ten active and ten non-active tropical plants studied ...... 36

Table 2.3. Number of aposematic and non-aposematic insect species collected on the ten active and ten non-active tropical plants studied, using only those insects where more than one individual was observed feeding on the study plants species ...... 38

Table 2.4a. The associations between ten biologically-active tropical plants and their insect herbivores. The lowest taxonomic level of identification determined for insects collected on the study plants, whether these insects were aposematic or non-aposematic, whether they had previously been documented to feed on a study plant, as weIl as general characteristics of each insect species ...... 39

Table 2.4b. The associations between ten biologically-non-active tropical plants and their insect herbivores. The lowest taxonomic level of identification determined for insects collected on the study plants, whether these insects were aposematic or non-aposematic, whether they had previously been documented to feed on a study plant, as weIl as general characteristics of each insect species ...... 43

Table 2.5. Search times to find aposematic insect species that were found to feed on the tropical study plants ...... 53 9

INTRODUCTION AND LITERATURE REVIEW

1.1. INTRODUCTION

This study addresses the general question of whether ecological criteria can be used to facilitate the discovery of plants containing compounds which have active properties against human disease. Specifically, the objective ofthis study is to detennine whether insects with aposematic colouration, that is to say insects that employ warning col ours to avoid predation, can be used as guides to detect plants containing potentially medicinally-active compounds. The hypotheses investigated during this research are that: (1) plants with biological activity in a range of disease bioassays will be more likely to be associated with aposematic insects than non-active plants; and (2) aposematic insects feeding on plants that were active in a range of disease bioassays will have the ability to concentrate the biologically-active compounds, presumably to be used in their own defense. The rationale for this study is based on the knowledge that secondary metabolites used by plants to defend themselves against herbivory may also have medicinal properties (Dutta et al. 1986, Dutta et al. 1993, Goffin et al. 2002, Gu et al. 2002, Goffin et al. 2003, Arnason et al. 2004). In addition, these secondary plant compounds are often distasteful or toxic and may be sequestered by insects feeding on the plant to be used in their own defense strategies (Bowers and Collinge 1992, Despland and Simpson 2005). Furthennore, insects which are defended with plant secondary compounds are often brightly coloured or aposematic to warn potential predators of their noxious nature (Sillén-Tullberg 1988, Salazar and Whitman 2001). Thus, theoretically, aposematic insects can be used as guides to biologically-active plants, narrowing down the plant species to be collected and tested, instead of simply choosing plant species at random. The first part of this study examined whether plants with biological activity in bioassays against tumor celllines and tropical parasites were more likely to have associations with aposematic insects, than non-active plants. This is based on the idea that plants with medicinal activity will have a higher probability of containing biologically-active compounds, which insects can make use of for their own defensive purposes. These insects would then benefit from having aposematic colouration as it is believed to serve as a signal to predators, waming them of the distastefulness or toxicity of a potential prey item (Sillén Tullberg 1988, Salazar and Whitman 2001, Krebs 2001). To investigate this, 10 plant species which showed biological activity in the Panamanian International Cooperative Biodiversity 10

Group (ICBG) project bioassays and 10 plant species without activity were chosen, in accordance with a set of predetermined criteria, which are outlined in Chapter 1. The biological assays, conducted by the Panamanian ICBG, investigate the activity of plant extracts against three cancer celllines (breast (MCF-7), lung (H-460), and central nervous system (SF-268)), malaria, American trypanosomiasis (Chagas' disease), and leishmaniasis. Subsequently, in four Panamanian National parks, representatives ofthese plant species were monitored for insect herbivores for 16 weeks. Active and non-active plants were monitored for approximately the same amount oftime (index of sampling effort). The second part of this study examined whether aposematic insects found on biologically-active plants have the ability to concentrate and sequester the plants' active compound(s). Again, this is based on the idea that aposematic colouration serves as a signal to predators, warning them of the distastefulness or toxicity of a potential prey item (Sillén TuIlberg 1988, Salazar and Whitman 200 1, Krebs 2001). Hence, there is a greater possibility that aposematic insects feeding on these plants have developed the ability to sequester the active plant compounds. To test this hypothesis, the active plant Tithonia diversifolia (Hemsl.) A. Gray and its associated aposematic insects were subjected to chemical analyses. First, the biologically-active plant fraction was isolated using bioassay-guided fractionation. Next, extracts were made of aIl available insect life-stages and substances associated with the insect (e.g. frass) to determine if the same active plant compound(s) was present. Separation of these compounds was performed by high performance liquid chromatography (HPLC) and retenti on times and spectral peaks of the plant active compound(s) were compared to the insect chromatograms.

1.2. BIOPROSPECTING IN TROPICAL RAINFORESTS

Although tropical rainforests occupy less than 7% of the world' s land area, they are believed to contain over half the world' s species (Primack 1998). This includes an estimated one-halfto two-thirds of the world's flowering plant species (Capson et al. 1996). However, many of these plant species have not been examined for their pharmaceutical potential (Capson et al. 1996, Roth and Lindorf 2002). In comparison to temperate plants, tropical rainforest plants are subject to greater levels of both herbivory and disease, resulting in both higher levels and a greater diversity of 11 defensive compounds (Martin 1995, Capson et al. 1996, Coley 1998). Additionally, tropical rainforests have a higher ratio of specialist species to generalist species than temperate forests. Consequently, herbivores tend to have more specialized diets, as well as more tightly co-evolved and ecologically-linked interactions in the tropical rainforests (Coley 1998). Traditionally, plants have been an extremely important source for medicines. For example, the two most effective treatments for malaria are derived from the plant species Chincona ledgeriana and Artemisia annua (Capson 2005). However, it is becoming increasingly difficult to find new lead compounds from plants, because many of the chemical structural types have already been isolated and evaluated and because of the frequent occurrence of compounds with moderate levels of activity and structural types that are not suitable for pharmaceutical purposes, such as tannins (Koehn and Carter 2005). Therefore, an emphasis is now being placed on the intelligent search for plants that are likely to yield novel structural types and greater potency against disease, using ecologically-guided bioprospecting (Coley et al. 2003).

1.3. PRIMARY AND SECONDARY PLANT METABOLISM

Primary and secondary metabolism cannot always be distinguished on the basis of precursor molecules, chemical structure, or biosynthetic origins (Cotton 1996); thus, a functional definition is required to distinguish between the two. Primary metabolism performs metabolic roles that are essential to survival, such as the production of phytosterols, lipids, nucleotides, amino acids, and organic acids (Cotton 1996, Croteau et al. 2000). In contrast, secondary metabolism does not appear to be directly involved in growth and development and different plant species often produce different secondary metabolites (Cotton 1996). Secondary metabolites are known to play important roles in ecological interactions, acting as protectors against nonadapted herbivores and microbial infections (Rhoades 1977, Dreyer and Jones 1981, Croteau et al. 2000), as allelopathic agents (Muller 1970), and as attractants for pollinators and seed-dispersers (Hamamura et al. 1962, Ndiege et al. 1996). Therefore, they are important for both the plants' survival and reproductive fitness (Wink 2003). The three major groups of secondary compounds are the alkaloids, terpenoids, and phenyl propanoids (allied phenolic compounds) (Croteau et al. 2000). Secondary metabolites have been a source 12 of many drugs (e.g. quinine, digitoxin, taxol, vincristine and vinblastine), pesticides, and chemicals important to the food industry (Newman 1990, Eisner and Meinwald 1995). Secondary metabolites are produced at a metabolic cost and, it is believed, exist because they confer a selective advantage to the plant species (Krebs 2001). From this plant defense hypothesis, four general predictions can be made: (1) plants will evolve more defenses ifthey are exposed to higher levels ofherbivory/disease and fewer if the cost of defense is high; (2) plants will allocate higher levels of chemical defense to more valuable tissues that are at risk; (3) defensive mechanisms should be less when enemies are absent and increase when plants are under attack; and (4) costly defense mechanisms are difficult to maintain if the plant is severely stressed by environmental factors (Krebs 2001). Another interesting aspect of defensive secondary metabolites is that although many are constitutive, sorne are induced, being synthesized in response to plant tissue damage. This is not surprising, if anti-herbivore defenses are costly to the plant (Edwards and Wratten 1985). For example, tobacco plants (Nicotiana sylvestris) constitutively produce the alkaloid nicotine, which has anti-insecticidal properties (Baldwin 1988). Larvae of the tobacco homworm, Manduca sexta, are fairly insensitive to this alkaloid; however, they are susceptible to N-acylnomicotine which accumulates rapidly in plant leaves subject to wounding (Laue et al. 2000). Tobacco homworm larvae fed leaves from a wounded plant consume significantly less, attain smaller masses, and have significantly reduced survival rates (72%) compared to those fed leaves from an unwounded plant (Baldwin 1988). Plant species often do not rely on only one class of secondary metabolites, but may contain five or six biosynthetically-derived groups of secondary metabolites, and within each group, there may be many structurally related analogs and derivatives (Arnas on et al. 2004). For example, the neem tree Azadirachta indica (Meliaceae) contains more than 50 limonoids and related compounds (e.g. azadirachtin) which have anti-feedant and growth-reducing effects on insects (Amason et al. 2004). A plant may bene fit from having a large number of analogs by lowering the rate that insects can metabolize the defensive plant compounds and by slowing the rate that insects can adapt to such compounds (Amason et al. 2004). 13

1.4. INSECT -PLANT RELATIONSHIPS

The theory of co-evolution put forward by Ehrlich and Raven (1964) is the most accepted theory to explain the evolution of insect-host plant relationships (Jermy 1984). It proposes that the selection pressure of insect herbivores enhances the development of plant defense mechanisms. These defenses can provide plants with protection from phytophagous insects, potentially allowing for plant diversification. However, sorne insect species may then develop adaptations to the plants' defenses, largely freeing these insects from competition with other phytophagous insects, and allowing for their diversification (Ehrlich and Raven 1964, Jermy 1984). This type of prey-predator evolution has also been referred to as an "arms-race" (Dawkins and Krebs 1979). Ultimately, this reciprocal selective response between insects and plants can cause an increase in the species diversity of both groups (Jermy 1984). One potential example of co-evolution is between the Heliconian butterflies and the plant family Passifloraceae (Benson et al. 1975). However, this the ory cannot explain the relationships where closely-related insects feed on distantly-related plant species (Jermy 1984). Other theories have also been proposed. Jones and Fim (1991) suggested that plants produce and maintain a high diversity ofmostly "inactive" secondary compounds as part oftheir defensive strategy. With this range of compounds, plants can cope with multiple stresses and, through mutations, may pro duce novel compounds for protective purposes (Jones and Fim 1991). Insect-plant relationships range from monophagous, to oligophagous, to polyphagous, with insects feeding on one plant species, a few closely-related plants, or numerous plants, respectively. Generally, monophagous and oligophagous insects, with their narrow host plant range, are considered to be specialists, whereas, polyphagous insects are generalists. In many cases, specialist herbivores have evolved mechanisms to benefit from plant defensive compounds by sequestering or modifying these compounds to use for their own protection. In contrast, generalist herbivores feeding on the same plant often have not developed the ability to sequester these compounds, but may have evolved mechanisms to cope with these chemicals (Johnson and Bentley 1988, Krebs 2001, Nishida 2002). For example, the specialist sawfly Rhadinoceraea nodicornis is able to concentrate veratrum alkaloids from its host plant Veratrum album, but the generalist Aglaostigma spp. cannot (Schaffner et al. 1994). In comparison, Raudsepp-Heame (2003) found that both the generalist (Periphoba 14 arcaei) and the specialist (Pyrrhopyge pseudophidias) Lepidopteran larvae, as weIl as the specialist Chrysomelid beetle (subfamily Eumolpinae) were able to sequester the highly cytotoxic plant secondary compound vismione B from their host plant Vismia baccifera. The huge amount of variability in the relationships between plants and phytophagous insects has been the subject ofmuch evolutionary speculation (Jenny 1984). Even today, there is an incomplete understanding of the mechanisms regulating plant-herbivore interactions (Coley 1998).

1.5. INSECTS AND THEIR USE OF PLANT SECONDARY METABOLITES

Numerous insect groups (8 orders) are known to sequester plant secondary compounds, including Lepidoptera, Coleoptera, Orthoptera, Hymenoptera, Hemiptera, and Homoptera, with this ability being particularly prevalent in the Lepidopteran families Saturniidae, Ithominae, Arctiidae, and the Coleopteran families Chrysomelidae and Curculionidae (Bowers and Collinge 1992, Rothschild 1993). Only a fraction of arthropods have been examined for their ability to excrete, sequester, or metabolize plant defensive compounds (Fordyce 2000). Secondary metabolites that have been found to be sequestered by insects include: flavonoids (Wiesen et al. 1994), quinolizidine alkaloids (MontlIor et al. 1990, Szentesi and Wink 1991), veratrum alkaloids (Schaffner et al. 1994), tropane alkaloids (Blum et al. 1981), pyrrolizidine alkaloids (von Nickisch-Rosenegk and Wink 1993, Cardoso 1997), iridoid glycosides (Boros et al. 1991, Bowers and Collinge 1992), cardenolides (Brower and Glazier 1975), and numerous other alkaloids. However, this represents only a portion ofthe plant defensive compounds that can deter insect herbivores (Nishida 2002). Consequently, there is a need to study insects and their defensive compounds. Insects are known to have the capabilities of direct sequestration, metabolism folIowed by sequestration, excretion of intact secondary compounds, and degradation folIowed by excretion (Szentesi and Wink 1991, Schaffner et al. 1994, Nishida 2002). Sorne in sect species selectively accumulate only one or more of the derivatives of a specific secondary compound type (e.g. flavonoids, quinolizidine alkaloids, iridoid glycosides, cardenolides), while metabolizing and/or excreting other derivatives (Brower and Glazier 1975, Szentesi and Wink 1991, Bowers and Collinge 1992, Wiesen et al. 1994, Geuder et al. 1997). AIso, insects may alter a plant secondary compound and then sequester its metabolite(s), for example, the 15 glucosylation of flavonoids (Wiesen et al. 1994, Geuder et al. 1997), the acetylation of veratrum alkaloids (Schaffner et al. 1994), and the biotransformation ofpyrrolizidine alkaloid bases into pyrrolizidine alkaloid N-oxides (von Nickisch-Rosenegk and Wink 1993). Secondary compounds may be sequestered and/or concentrated by insects for defensive purposes against generalist predators and/or parasitoids (von Nickisch-Rosenegk and Wink 1993, Schaffner et al. 1994), to aid in visual mate recognition and sexual selection (Geuder et al. 1997, Jordan et al. 2005), and/or to produce sex pheromones (Wunderer et al. 1986). Sorne insect species have displayed sex-related differences in the sequestration of plant secondary compound concentrations, such as in the lycaenid butterfly, Polyommatus bel/argus, where females have significantly higher flavonoid concentrations than males (Geuder et al. 1997) and in the monarch butterfly, Danaus plexippus, where females contain higher concentrations of cardenolides, as well as cardenolides that are more emetic (agents that induce vomiting) than males (Brower and Glazier 1975). Insects must also be studied throughout their lifecycle as their ability to sequester toxic secondary metabolites may change throughout development. For example, the aposematic larvae of Junonia coenia sequester iridoid glycosides; however, these iridoid glycosides are metabolized during the pupal stage leaving the cryptic adult butterfly (cryptic underside and bright eyespots on upperside) without these compounds (Bowers and Collinge 1992). This appears to be the case for many other Lepidopteran species feeding on plants with iridoid glycosides, which have aposematic larvae with iridoid glycosides, but cryptic adults without iridoid glycosides (Boros et al. 1991). In contrast, secondary metabolite concentrations may remain relatively stable throughout the life-stages of sorne insect species, such as the lycaenid butterfly (P. bel/argus) (Geuder et al. 1997). Furthermore, secondary compounds are not always equally distributed throughout an insect. For example, 80% offlavonoids in P. bel/argus adults are concentrated in the wings, with the rest throughout the body (Geuder et al. 1997). R. nodicornis sawfly larvae concentrate veratrum alkaloids in their hemolymph, but not in their integuments, fat bodies, or salivary glands (Schaffner et al. 1994). The pyralid moth larvae, Uresiphita reversalis, concentrate quinolizidine alkaloids primarily in their integument (Montllor et al. 1990), and several arctiid moth species also accumulate pyrrolizidine alkaloids in the integument (von Nickisch Rosenegk and Wink 1993). Monarch butterfty adults also have higher cardenolide concentrations in their wings compared to their abdomen and thorax; however, the 16 cardenolides in the thorax have a higher emetic potency than those in the wings or abdomen (Brower and Glazier 1975).

1.6. APOSEMATISM & OTHER MECHANISMS TO DISPLAY UNPALATABILITY

Bright colouration (e.g. reds, oranges, yellows, and white, alternating with black/dark grey/dark brown, and sometimes blues and purples) is believed to serve as a signal to predators warning of the distastefulness or toxicity of their potential prey (Sillén-Tullberg 1988, Salazar and Whitman 2001, Krebs 2001). This correlation is usually explained through the theory of warning colouration or aposematism, which suggests that prey species prosper by advertising their adverse qualities through conspicuous colouration (Guilford 1988). The protective value of warning colouration is also often enhanced by increasing both the size of the signal pattern elements and pattern symmetry. This follows both theoretical and empirical evidence suggesting that a good warning display should be easy to detect, learn, and recall, as well as be associated with defense (Forsman and Merilaita 1999). Numerous examples exist where aposematic insects specialize on toxic host plants (Reichstein et al. 1968, Montllor et al. 1990, Boros et al. 1991, Bowers and Collinge 1992, F ordyce 2000). This specialization offers the insect the possibility of making use of the se potent plant secondary compounds in their defensive strategies (Bernays and Graham 1988, Nishida 2002, Despland and Simpson 2005). Defense success becomes a function of the host plants' chemical content, the insects' ability to select plants or plant parts rich in defensive chemicals and the insects' sequestrative efficiency (Bowers and Collinge 1992, Mebs and Schneider 2002). In contrast, sorne insect species use cryptic colourations often consisting of greenish or brownish colours, typically to mimic the background, as a defensive strategy (Gamberale and S-Tullberg 1998). Importantly, it has been found that predators can quickly learn to avoid distasteful insects as a result of aposematic colouration (Brower 1988, Cardoso 1997). Roper (1990) concluded that the most important aspect to explain naïve predator aversion to a prey item is specifie colours. Increasing prey size in aposematic insects is aiso believed to increase prey protection, as the amount of visible warning colouration is increased (Gamberale and S Tullberg 1998). Additionally, for sorne predators, aggregated aposematic prey produce a greater unconditioned aversion than prey presented alone. This advantage was not found for aggregations of cryptic species. It is likely that aggregated aposematic prey generate a more 17 effective aposematic signal because of the increased amount of visible warning colouration (Gamberale and S-Tullberg 1996, 1998). Therefore, unpalatable insects not only advertise through conspicuous colouration, but also through gregarious and sedentary behaviour (Brower 1984, Hatle and Whitman 2001). Generally, gregariousness is found more often in aposematic species than in cryptic species (Sillén-Tullberg 1988). There are several different views regarding the evolution of aposematic colouration and gregarious behaviour. When prey are gregarious and nearby individuals are closely-related, kin selection may favour aposematism, since a predator would only need to sample a few individuals from the group before learning to avoid others. Thus, the presence of an allele(s) to impart distastefulness (e.g. ability to sequester plant secondary compounds) could increase in frequency via kin selection (Krebs 2001). In a contrasting study, Sillén-Tullberg (1988) evaluated the evolution of aposematism and gregariousness by phylogenetic analysis of buttertly larvae and found that aposematism as a criterion for unpalatability always preceded the development of gregariousness. This follows the idea that gregariousness should be disadvantageous for palatable species, as they would be more easily seen by a potential predator (Sillén-Tullberg 1988). Therefore, kin selection should be of little significance for the evolution of aposematic colouration, assuming that the degree to which kin selection occurs is proportional to the degree of larval gregariousness (Sillén-Tullberg 1988). Instead, individual selection appears to be of greater importance for the evolution of aposematic colouration than kin selection (Sillén-Tullberg 1988). Following this, Thomas et al. (2004) found experimentally that conspicuous prey can gain protection from wild birds simply by their being novel (different colour from regular prey items provided), which can act as a deterrent. Another view from Alatalo and Mappes (1996) is that unpalatability alone has selected for gregariousness which subsequently could facilitate the evolution of warning signaIs. Sedentary behaviour is also viewed as a mechanism for signalling unpalatability. As discussed by Haefner (2003) in reference to marine invertebrates, organisms with a sedentary life-style and especially those that are soft-bodied have the greatest need for chemical defense mechanisms. Insect larvae and pupae also fall into this category. The existence of mimi cry complexes, where palatable species have evolved to physically resemble unpalatable species, is another important consideration when studying 18 aposematic insects. Examples of mimi cry include the viceroy butterfly (Limenitis archippuis), a Batesian mimic of the unpalatable monarch butterfly (Danais plexippus), as weIl as many swallowtail butterfly species that mimic the unpalatable pipevine swaIlowtail butterflies, which feed exclusively on plants of the genus Aristolochia, concentrating toxic alkaloids known as aristolochic acids (Fordyce 2000). Another type ofmimicry, called MüIlerian mimicry, refers to the convergence in appearance oftwo or more unpalatahle species, such as has occurred in numerous tropical butterflies (Purves et al. 1998). Not surprisingly, mimi cry is an effective defensive strategy (Fordyce 2000). It also must be recognized that many unpalatable insects are not aposematic, especially ifvisual predation is not ofprimary importance (Schaffner et al. 1994) and that insects may be able to synthesize defensive chemicals de nova (Plasman et al. 2000, Nishida 2002). Therefore, in this study we must be aware that not all insect species will sequester defensive compounds from their host plant (Bowers and Collinge 1992) and that the existence of mimicry complexes means that sorne brightly-coloured insects are not toxic (Fordyce 2000). Consequently, aIl available evolutionary, life history, and ecological data about both the insect and the plant must be taken into account.

1.7. IMPROVING THE BIOPROSPECTING PROCESS

The sale ofpharmaceuticals is a US$330 billion a year industry (Artuso 2002). Today, a large proportion ofpharmaceuticals, 25% in developed and 75% in developing nations still originate from natural products (Moran et al. 200 1, Fim 2003). In addition, between 1981 and 2002, approximately half of aIl the small-molecule New Chemical Entities were of natural product origin (Koehn and Carter 2005). AIso, approximately 60% of the anti-tumour and anti-infective agents that are currently available or are in later stages of clinical trials are of natural product origin (Eldridge et al. 2002). Natural products have historically been invaluable as therapeutic agents; however, during the last decade research on natural products has dec1ined because of issues such as the lack of compatibility of natural-product extract libraries with high-throughput screening, the development of combinational chemistry, which allows for the synthetic development of compounds and their analogues, and the increasing difficulty of accessing biological samples in developing countries (Koehn and Carter 2005). Recently-developed technologies are addressing these incompatibility issues, bringing back a 19 renewed interest in research on natural products (Raskin et al. 2002, Koehn and Carter 2005). Compared to synthetic compounds, natural products have a greater chemical diversity, chemical complexity, biochemical specificity, as well as other molecular properties (e.g. capacity to modulate or inhibit protein-protein interactions), making them favourable for lead structures in drug discovery (Firn 2003, Haefner 2003, Koehn and Carter 2005). These features provide justification to develop more efficient methods to search for novel pharmaceutically-relevant compounds from natural products. Bioprospecting, which is the search for new biologically-active chemicals in organisms (Firn 2003), has focused primarily on plants and microbes (Hamann 2003). Recently, there has been an extension to other taxonomic groups, including marine organisms (Capon 2001, Farrier and Tucker 2001, Haug et al. 2002a, Haug et al. 2002b, Haefner 2003, Hamann 2003), and endophytic microflora (StrobeI2002). Surprisingly, even though known insect diversity is approximately 16 times greater than plant diversity, insects are still an understudied group. In fact, plant chemistry has been studied 7000 times more intensively than insect chemistry on a research-per-species basis (Trowe1l2003). Potential reasons for this include the lack of accurate documentation on the native uses ofinsects compared to plants (Trowe1l2003), as well as the ease of collection, as insects are mobile and population sizes can be variable. Only a few bioprospecting groups have intensively investigated insects. A project sponsored by the International Cooperative Biodiversity Group Pro gram (ICBG) examined insects for potentially active compounds in Costa Rica, but had difficulties collecting sufficient quantities of insects for analyses (Sittenfeld et al. 1999). In contrast, another pro gram being pursued by Entocosm Pty. Ltd., an offshoot of the Entomology Division ofCSIRO (Australia's Commonwealth Scientific and Industrial Research Organization), has discovered potentially useful compounds from numerous insect species, and are still only in their early stages of study, as they were founded in July 2002 (Trowe1l2003). Because defensive compounds present in insects are often sequestered from their host plantes) (Bowers and Collinge 1992), there exists the possibility ofusing insects as guides to plants that may contain pharmacologically-interesting compounds. Using this method eliminates the problem ofnot being able to collect enough insects for analysis. Theoretically, insect ecology can be used to narrow down the plant species to be collected and tested, instead of simply choosing plants species at random. When bioprospecting is done at random, 20 a diverse array of plant species are examined but the chances of finding a biologically-active compound(s) is small (Day-Rubenstein and Frisvold 2001, Koehn and Carter 2005). Cragg et al. (1998) estimated from the National Cancer Institute's (NCI) screening ofplants for anti cancer activity, that 1 in 4000 plants randomly screened would yield activity against a specific disease category. However, this success rate can be increased. For example, Andrade-Neto et al. (2003), searching for anti-malarial activity, and Matsuse et al. (1999), for anti-HIV activity, used ethnobotanical knowledge to chose plants used against fevers or viruses, respectively, and achieved success rates of 1 in 3, and 7 in 39, respectively. Ecologically-guided bioprospecting will no doubt increase the efficiency and the success rate, just as ethnobotanical- or ethnomedical-bioprospecting have (Matsuse et al. 1999, Andrade Neto et al. 2003); however, for ecologically-guided bioprospecting to be successful, the selection criteria must be easy to implement in the field and broad enough to generate a reasonable number of samples (Coley et al. 2003). The Panamanian ICBO Pro gram already successfully utilizes ecological theory, based on the chemical defense patterns of rainforest plants, to direct the search for plant compounds active against the tropical diseases, malaria, American trypanosomiasis (Chagas' disease), and leishmaniasis, as well as three cancer celllines. For example, young leaves are preferentially collected because they typically have higher concentrations and a greater diversity of chemical defenses than mature leaves that rely more on tannins and toughness for defense (Montllor et al. 1990, Capson et al. 1996, Coley et al. 2003). In a study of tropical woody species, 10 of the 18 plants had unique alkaloids present in only the young leaves, whereas, only 3 species had unique alkaloids in the mature leaves (Coley et al. 2003). A potential explanation is that most herbivory in the tropics occurs on ephemeral young leaves (>70%), in contrast to temperate regions where approximately 75% ofherbivory occurs on mature leaves (Coley 1998). The work proposed herein will examine a different ecological criterion that may be employed to facilitate the drug discovery process, namely, whether aposematic insects can be used as guides to plants with potentially medicinally-active compounds. This in the long run will add value to the two independently relevant aspects of the ICBO: (1) finding novel treatments for economically and clinically important diseases; and (2) enhancing the efficiency of the drug discovery process. 21

1.8. PREVIOUS STUDIES ON APOSEMATIC INSECTS AND MEDICINALLY ACTIVE PLANTS

The work of Raudsepp-Hearne (2003) was also designed to explore the potential of using aposematic insects as guides to plants containing potentially medicinally-active compounds. Bioassay-guided fractionation of methanolic extracts from the plant Mikania guaco (Asteraceae) led to the isolation of the novel sesquiterpene lactone named Guaconone, which proved to be active against the amastigote form of Trypanosoma cruzi, the causative agent ofChagas' disease. Three Coleopteran species (Chrysomelidae) that use Mikania guaco as a host plant were investigated for their ability to sequester Guaconone. Guaconone was sequestered by the two aposematic (red and black) specialist species, Eugenysa coscaroni and Echoma anaglyptoides, but not by the visually duller generalist species Platyphora ligata (Raudsepp-Hearne 2003). Raudsepp-Hearne (2003) also found that both the generalist (Periphoba arcaei) and the specialist (Pyrrhopyge pseudophidias) Lepidopteran larvae, as well as the specialist Chrysomelid beetle adult, were able to sequester the plant secondary compound vismione B from their host plant Vismia baccifera (Clusiaceae). AlI three ofthese species were aposematic. Vismione B has significant anti-feedant activity against other Lepidopteran species and also has cytotoxic properties against the ICBG cancer celllines. These insects' ability to tolerate vismione B may be important to their adaptation to the host plant. These data support the hypothesis that aposematic insects have the potential to direct researchers to plants that may contain compounds with biological activity against cancer cell lines and/or parasites.

1.9. REL EVANCE TO SCIENCE, HEAL TH, AND CONSERVATION

Little research has focused on the relationship between aposematic insects and plant derived medicines, with the previous masters thesis by Raudsepp-Hearne (2003) being one of the only studies to touch on the subject. Consequently, this project will add to the limited amount of knowledge which may eventually be used to accept or reject the hypothesis that aposematic insects can be used as guides to plants with medicinally-active compounds. This project will also add to the information on chemical ecology of insects and their host plants in the tropics, as weIl as will provide information on insect-host plant relationships. 22

Overall, the development of new drugs is increasingly important to stem the tide of drug-resistant microbes, to control tropical diseases of the developing world, and to control diseases ofboth the developed and developing worlds, including cancer, cardiovascular, neurological, and metabolic diseases (Hamann 2003). In terms ofhealth benefits, there is the potential for isolating novel compounds that are active against cancer and the tropical parasitic diseases. Tropical diseases are a huge burden, with the three above-mentioned parasitic diseases directly affecting approximately 320 million people annually (Gelb and Hoi 2002). Malaria accounts for approximately 3% of the worldwide disease burden measured by disability-adjusted life years (Sachs 2002). Recently, drug-resistant strains of Plasmodium falciparum, one of the parasites that cause malaria, have begun to appear in many parts of the world, including the Amazon region (Andrade-Neto et al. 2003). Consequently, drugs such as chloroquine, which is derived from the compound quinine that is extracted from Cinchona spp., have lost sorne oftheir effectiveness, making the need to find new drugs increasingly urgent (Sachs 2002, Andrade-Neto et al. 2003). Sadly, recent estimates show that drug and vaccine research on malaria is less than $100 million of the $70 billion spent on annual worldwide biomedical research and development (Sachs 2002). Chagas' disease, caused by the protozoan Trypanosoma cruzi, affects between 16 and 18 million people annually in tropical and sub-tropical America, killing approximately lOto 20% of infected people (Alves et al. 1995, Gelb and Hoi 2002). In addition, leishmaniasis, caused by the protozoan Leishmania spp., affects between 1.5 and 2 million people annually. For Chagas' disease and leishmaniasis, as well as most other tropical protozoan diseases, few, if any, safe efficacious drugs are available. Another potential offshoot from this research is that there may be the potential for the promotion of forest conservation through bioprospecting, but this will only occur if there are explicit benefits for the countries that have the biodiversity resources (Capson et al. 1996, Coley et al. 2003). Traditionally, little, if any, of the revenues derived from the sale of drugs discovered in tropical regions have returned to the source countries. Fortunately, the attitude of the "biological commons" is becoming a thing of the past (Brush 1999); with this recent change being in large part due to the Convention on Biological Diversity (Moran et al. 2001). Bioprospecting organizations are now expected to provide benefits to host-country partners in exchange for access to biochemical resources, such as a share in milestone payments or long- 23 term royalty benefits derived from the sale of drugs, technology-transfer, capacity building, development of infrastructure in host-country institutions, job opportunities, joint research programs, and the training of students (Day-Rubenstein and Frisvold 200 1, Moran et al. 200 1, Artuso 2002, Davalos et al. 2003). Therefore, bioprospecting has the potential to contribute to sustainable development by providing economic incentives for conservation, while developing technological capabilities that enhance the opportunity for long-term growth (Rausser and SmaIl2000, Artuso 2002). Moreover, since only a small fraction of the natural chemicals from plants and microbes have been assessed, there is real economic and social value in retaining biodiversity to preserve chemical diversity (Garrity and Hunter-Cevera 1999, Barrett and Lybbert 2000, Fim 2003).

1.10. INTRODUCTION TO THE CHAPTERS

In both studies by Raudsepp-Heame (2003), aposematic insects were associated with plants containing medicinally-active compounds. However, these studies are limited by focusing on two plants which is insufficient to determine whether there is a significant correlation between aposematism and the presence ofbiologically-active compounds. It may be that the majority or even aIl plant species have at least one aposematic insect associated with them, for example, as a result of Batesian mimicry complexes. If this were the case, then aposematic insects would not be effective guides to biologically-active plants. Therefore, to circumvent this problem, both active and non-active plants need to be investigated for their relationship with aposematic insects. The non-active plants serve as controls when analysing the insect associations. Furthermore, a greater number of insect-host plant pairs need to be examined to either reject or accept the hypothesis that aposematic insects can be used as guides to plants containing potentially medicinally-active compounds. The objective ofthis study is to determine whether aposematic insects can be used as guides to detect plants containing potentially medicinally-active compounds. 1 hypothesize that: (1) plants having biological activity in a range of disease bioassays will be more likely to have associations with aposematic insects than plants that did not show biological activity; and (2) aposematic insects feeding on these plants that were active in a range of disease bioassays will have the ability to sequester these biologically-active compounds. 24

The first chapter "Can Aposematic Insects Guide us to Medicinally-active Plants in the Tropics?" examines the hypothesis that plants showing activity in the ICBG bioassays will be more likely to have associations with aposematic insects, than those plants that have not shown activity in the bioassays. Using existing data on plant species that have been previously examined in the Panamanian ICBG bioassays; ten biologically-active plant species and ten biologically-non-active species were chosen based on the following criteria: (1) biological activity, (2) accessibility in the field, and (3) abundance in the field. In addition, an attempt was made to pair active and non-active plants by plant family to increase the similarity of general characteristics between the plant pairs. Following this, plants were examined 16 times, on a weekly to bi-weekly basis, to ascertain their associated insect populations. Subsequently, insects found feeding on both the active plants and the non-active plants were categorized as aposematic or non-aposematic. Finally, the literature supporting the hypothesis that aposematic insects can be used as guides to biologically-active plants, as weIl as the potential problems with this hypothesis and future research needs are discussed. The second chapter "Do Aposematic Insects Feeding on Tithonia diversifolia Concentrate its Medicinally-active Compound(s)?" tests the hypothesis ofwhether aposematic insects feeding on a biologically-active plant have the ability to sequester its pharmacologically-active compound(s). After studying the insect populations found on the active plants in the first chapter, an interesting plant-insect association was chosen for further chemical study. The following criteria were used to determine the plant-insect association to be studied: (1) an aposematic insectes) had a c1ear association with the plant species; (2) a sufficient amount of insect material was available to do chemical analysis; and (3) the degree to which a plant species had previously been studied. Using these criteria, the plant Tithonia diversifolia and its three associated insect species Platyphora ligata, Chlosyne hippodrome, and Dysschema magdala were chosen. Next, bioassay-guided fractionation was performed, fractionating the crude extract of T diversifolia, using its activity against Chagas' disease as a guide. The chemical fraction of T. diversifolia that was found to be active against Chagas' disease was related to the chemical composition ofits three aposematic insects. FinaIly, both the pharmacological and anti-feedant activities of T diversifolia, as weIl as the abilities of the insects to ingest and sequester the active compound(s) are discussed. 25

CHAPTERl

Can aposematic insects guide us to medicinally-active plants in the tropics?

JULIE E. HELSON1,2*, TODD L. CAPSON1,2, and TIM JOHNS2

ISmithsonian Tropical Research Institute Apartado 2072, Balboa, Anc6n, Panama City Republic of Panama

2Department of Plant Science McGill University 21,111 Lakeshore Road Ste. Anne de Bellevue, Quebec H9X 3V9 Canada

*To whom correspondence should be addressed. E-mail: [email protected]

CONTRIBUTIONS OF AUTHORS

For the first manuscript the first author designed the study, as well as did the fieldwork and the labwork involving the rearing of insects. In addition, the first author analysed the data and wrote the manuscript. The second author provided financial support for the fieldwork, as weIl as suggestions for the design of the study and subsequent analysis of the data. The second author also aided in editing the first manuscript. The third author made a substantial contribution by providing guidance and suggestions to the first author during the experimental design stage. In addition, the third author aiso heiped in the editing process. 26

ABSTRACT

Aposematic insects, which are insects with warning colouration, could be used as guides to plants with potentially medicinally-active compounds. This is based on the fact that plant defense compounds, which have been found to have pharmacological activities, are often sequestered and utilized by aposematic insects for their own defensive purposes. This study addresses this question by examining Coleopteran and Lepidopteran populations feeding on ten biologically-active plants and ten non-active plants, chosen from plants previously tested in the Panamanian International Biodiversity Group (lCBG) bioassays, which test for activity against cancer celllines and tropical parasitic diseases. Plants with activity in Panamanian ICBG bioassays were more likely to have associations with aposematic insects, than those plants that did not have activity in the bioassays, using both the chi-square test (df = 1, X2 = 8.167, P = 0.01) and the t-test paired two sample for means (df= 9, t = 1.973, and p = 0.08). Overall, it was found that a bio10gically-active plant was four times more 1ikely to have an association with an aposematic insect than a non-active plant. This study supports the idea that aposematic insects can potentially lead us to medicinally-active plants. 27

2.1. INTRODUCTION

It is becoming increasingly difficult to find new lead pharmacologically-active compounds from plants, because many of the chemical structural types are already known and because of the frequent occurrence of compounds with moderate levels of activity and structural types that are not suitable for pharmaceutical purposes, such as tannins (Koehn and Carter 2005). Therefore, there is a need to search intelligently for plants that are likely to yield novel structural types and provide greater potency against disease, using ecologically guided bioprospecting (Coley et al. 2003). In addition, in diverse ecosystems, such as tropical rainforests, it is advantageous to have an effective strategy for finding plant materials that are likely to have biologically-active secondary metabolites (Firn 2003). This follows the rationale that when bioprospecting is performed at random, a diverse array of plant species are examined but the chances of finding a given species with a biologically-active compound are small (Koehn and Carter 2005). Ecologically-guided hioprospecting, which utilizes ecological knowledge or theories to direct the selection of plants, is a potential means of enhancing the efficiency of the process of selecting plants for drug discovery research (Coley et al. 2003). Ecological theory, based on the chemical defense patterns of rainforest plants, has been used to direct searches for active plant compounds (Coley et al. 2003). For instance, young leaves are preferentially collected because they typically have higher concentrations and a greater diversity of chemical defenses than mature leaves that rely more on tannins and toughness for defense (Montllor et al. 1990, Capson et al. 1996, Coley et al. 2003). This ecological theory has proven successful in encountering novel compounds active against the parasites that cause Chagas' disease (Mendoza et al. 2003) and leishmaniasis (Montenegro et al. 2003); however, it has not been successful against aIl ofthe disease targets. For example, this theory has only lead to anti-malarial compounds with moderate activity that are non specific inhibitors (e.g. tannins), whereas what are really needed are compounds with high activity and biological specificity. Therefore, there is a need to explore other ecological criteria that may also expedite the drug discovery process. This study examines a different ecological criterion that may he employed to facilitate the drug discovery process; namely, whether aposematic insects, insects with waming colouration, can be used as guides to plants with potentially medicinally-active compounds. 28

This is based on the knowledge that defensive plant secondary metabolites may also have medicinal properties (Dutta et al. 1986, Dutta et al. 1993, Goffin et al. 2002, Gu et al. 2002, Goffin et al. 2003, Amason et al. 2004) and that host plant defensive compounds may be sequestered by aposematic insect herbivores and used in their own defensive strategies (Bowers and Collinge 1992, Despland and Simpson 2005). Thus, theoretically, aposematic insects could serve as guides to medicinally-active plants, narrowing down the plant species to be collected and tested to enhance the efficiency of the bioprospecting process. For this ecological criterion to work: (1) there should to be a close ecological and evolutionary relationship between the host plant and its associate insect herbivores; (2) the insect herbivores should have the ability to sequester the plant secondary compounds, presumably for their own protection; and (3) there should be a relationship between aposematic colouration and predator deterrence. The theory of co-evolution put forward by Ehrlich and Raven (1964) proposes that the selection pressure ofinsects enhances the evolution of plant defense mechanisms. In tum, these defenses can provide plants with protection from phytophagous insects, potentially allowing for plant diversification. However, sorne insect species may then develop adaptations to the plant defenses, largely freeing these insects from competition with other phytophagous insects, and allowing for their diversification (Ehrlich and Raven 1964, Jermy 1984). This type of prey-predator evolution has also been referred to as an "arms-race", in this case a "biochemical arms-race" (Dawkins and Krebs 1979). Insect species within numerous orders deal with noxious plant metabolites by sequestrating these plant secondary compounds, either directly or following metabolism, or by metabolizing and/or excreting the plant compounds (Szentesi and Wink 1991, Rothschild 1993, Schaffner et al. 1994, Nishida 2002). In many cases, specialist herbivores, those with a narrow host plant range, have evolved mechanisms to benefit from noxious plant chemicals by sequestering the plant compounds or related derivatives. In contrast, generalist herbivores, those that feed on numerous plant species, often have evolved "more general" defensive strategies that involve the metabolism and excretion of noxious chemicals, rather than sequestration (Johnson and Bentley 1988, Krebs 200 1, Nishida 2002). Therefore, specialist insects may be more likely to sequester active plant compounds and have aposematic 29 colouration than generalist insects, which will be more likely to have cryptic colouration patterns. Bright colouration (e.g. reds, oranges, yellows, alternating with black (dark brown, dark grey), and sometimes blues and purples) is believed to serve as a signal to predators, waming them of the distastefulness or toxicity of a potential prey item (Sillén-Tullberg 1988, Salazar and Whitman 200 1, Krebs 2001). The theory of warning colouration or aposematism suggests that it is advantageous for a prey species to advertise their adverse qualities through conspicuous colouration (Guilford 1988). Often, these aposematic insects specialize on toxic host plants (Reichstein et al. 1968, Montllor et al. 1990, Boros et al. 1991, Bowers and Collinge 1992, Fordyce 2000, Despland and Simpson 2005), and this specialization offers them the possibility of using potent plant secondary compounds for defense against predators (Bernays and Graham 1988, Nishida 2002, Despland and Simpson 2005). Importantly, it has been found that predators quickly learn to avoid distasteful insects as a result of aposematic colouration (Gittleman et al. 1980, Roper and Redstone 1987, Brower 1988, Cardoso 1997). Experiments have shown that it takes less time for predators to leam to avoid conspicuous compared to less conspicuous distasteful prey (Gittleman and Harvey 1980, Sillen-Tullberg 1985). AIso, it is known that unpalatable insects not only advertise through conspicuous colouration, but also through gregarious and sedentary behaviour (Brower 1984, Hatle and Whitman 2001). Raudsepp-Heame (2003) found that aposematic insects feeding on the tropical plants Vismia baccifera and Mikania guaco concentrated the medicinally-active compounds present in these plants. However, the study oftwo plants is insufficient to determine whether there is a significant correlation between aposematism and the presence of biologically-active compounds. It could be the case that the majority or even all plant species have at least one aposematic insect associated with them, for example, because of Batesian mimi cry complexes. Therefore, to circumvent this problem, both biologically-active and non-active plants need to be investigated for their relationship with aposematic insects. Furthermore, a greater number ofinsect-host plant pairs need to be examined to determine whether aposematic insects can be used as guides to plants containing potentially medicinally-active compounds. 30

To investigate whether aposematic insects can be used as guides to enhance the efficiency of finding plants containing potentially medicinally-active compound s, ten plant species with activity and ten plant species without activity in the Panamanian ICBG bioassays (three cancer celllines; malaria, American trypanosomiasis (Chagas' disease), and leishmaniasis) were chosen and their associated insect herbivore populations studied. In four Panamanian national parks, active plant and non-active plant populations were monitored for approximately the same amount oftime; for 16 . weeks (weekly to biweekly) over a period from late May 2004 to early November 2004, to determine their associated insect herbivores. In this study, we test the hypothesis that plants containing compounds which have medicinal activity in the Panama ICBG bioassays will be more likely to have associations with aposematic insects than non active plants.

2.2. METHODS

Criteria for the Choice of Plant Study Species

Ten plant species containing compounds which were biologically-active in the Panamanian International Cooperative Biodiversity Group (ICBG) anti-cancer and anti parasitic screens and ten plants which were non-active were chosen from the approximately 1380 species ofplants tested by the ICBG to date. Plants were deemed biologically-active if they had activity in one or more of the bioassays tested, which inc1uded three cancer bioassays (lung, breast, and central nervous system), as well as bioassays testing against malaria, American trypanosomiasis (Chagas' disease), and leishmaniasis (Table 2.1). Plants categorized as non-active did not show activity in any of the above bioassays. Plants from six plant families (Asteraceae, Boraginaceae, Bignoniaceae, Convolvulaceae, Rubiaceae, and Solanaceae) were chosen on the basis of: (1) consistent activity or inactivity in the bioassays; (2) accessibility in the field; and (3) abundance in the field. Active and non-active plants were paired by family whenever possible in order to increase the similarity of general characteristics between the plant pairs (Table 2.1). The only two cases where pairs were not from the same plant family were Baccharis trinervis 31

Table 2.1. Selected pairs of medicinally-active and medicinally-non-active plants. For the medicinally-active plants their bioactivity in the ICBG screens is also indicated.

MEDICINALLY-ACTIVE MEDICINALLY-NON-ACTIVE Tithonia diversifolia (Asteraceae) Tilesia baccata (Asteraceae) (cancer, malaria, Chagas') Neuro/aena /obata (Asteraceae) Wedelia ca/ycina (Asteraceae) (cancer, Chagas') Baccharis trinervis (Asteraceae) Alibertia edulis (Rubiaceae) (cancer, malaria, Chagas') Me/ampodium divaricatum (Asteraceae) Brugmansia candida (Solanaceae) (cancer, Chagas') Phryganocydia corymobosa (Bignoniaceae) Jacaranda copaia (Bignoniaceae) (cancer, malaria, Chagas') Cordia curassavica (Boraginaceae) Tournefortia hirsutissima (Boraginaceae) (cancer) Bonamia trichantha (Convolvulaceae) Maripa panamensis (Convolvulaceae) (cancer, Chagas') Chomelia recordii (Rubiaceae) Chiococca a/ba (Rubiaceae) (malaria, Chagas', leishmaniasis) Hamelia axillaries (Rubiaceae) Isertia haekeana (Rubiaceae) (cancer, malaria) Witheringia so/anacea (Solanaceae) So/anum jamaicense (Solanaceae) (cancer, malaria, Chagas') 32

(Asteraceae) and Alibertia edulis (Rubiaceae), and Melampodium divaricatum (Asteraceae) and Brugmansia candida (Solanaceae). In cases where there was more than one potential plant pair per plant family, phylogenetic relationships were investigated to pair those active and non-active plants that were most related. Phylogenies for Asteraceae were obtained from Bremer (1994), for Solanaceae from Hunziker (1979), and for Rubiaceae from Pereira and Barbosa (2004) and Andreasen and Bremer (2000). A literature search was also preformed to see if non-active species had shown medicinal activity in other independent studies. It was found that Chiococca alba roots have shown anti microbial activity (Borges-Argaez et al. 1997); however, only foliage was important in this study. Extracts of Tilesia baccata (previously called Wulffia baccata) were found to have casein kinase II inhibition activity (lshibashi et al. 1999). In traditional medicine, Tournefortia hirsutissima is used to make a cooling drink for the bladder (Roth and Lindorf 2002). Most importantly, Jacaranda copaia was found to have leishmanicidal activity (Sauvain et al. 1993). In traditional medicine, J copaia bark is used to treat colds, pneumonia, and leishmaniasis, and is also emetic and cathartic. Moreover, its bark and leaves are used to treat skin infections, and leaves are placed on wounds to quicken healing (Roth and Lindorf 2002). However, in bioassays performed by the ICBG in 1998, J copaia leaf extracts did not have leishmanicidal activity and it is known that leishmanicidal activity is much more variable than many other types of activity. Recollection permits (SC/P-17-03, SCIP-22-03, SCIP-23-03, SCIP-24-03, SCIP-25-03, SCIP-26-03, SCIP-27-03, SCIP-28-03) were obtained from the Panamanian National Authority ofthe Environment (ANAM) for the collection of plant species. Plant species identities were confirmed by Rafael Aizprua of the Smithsonian Tropical Research Institute (STRI). Voucher specimens of each of the plant species studied have been place in the Herbaria at both the University of Panama and STRI: Tithonia diversifolia (Hemsl.) A. Gray (2855-RAJ); Neurolaena lobata (L.) R. Br. ex Casso (2902-RA); Baccharis trinervis Pers. (2853-RAJ); Melampodium divaricatum (Rich.) De. (2848-RA), Tilesia baccata (L.) Pruski (2903-RA), Wedelia calycina Rich. (2852-RAJ); Phryganocydia corymobosa (Vent.) Buereau ex K. Schum. (2947-RA); Jacaranda copaia (Aubl.) D. Don (2906-RA); Cordia curassavica (Jacq.) Roem. & Schult. (2842-RA and 2851-RAJ); Tournefortia hirsutissima L. (2846-RAJ); Bonamia trichantha Hallier F. (2845-RA); Maripa panamensis Hemsl. (2897- 33

RA); Chomelia recordii Standl. (2844-RA); Hamelia axillaries Sw. (2849-RA); Chiococca alba (L.) Hitchc. (2850-RAJ); Isertia haekeana DC. (2905-RA); Alibertia edulis (Rich.) A. Rich. ex DC. (2901-RA); Witheringia solanacea L'Hér. (2854-RAJ); Solanumjamaicense Mill. (2856-RAJ); and Brugmansia candida Pers. (2843-RA).

Monitoring and Collection of Insects

AH study plants were present in four Panamanian National Parks. Two were located in areas ofmainly lowland moist tropical forest, Soberania National Park (9° 7' N; 79° 42' W) and Barro Colorado National Monument (9° 9' N; 79° 51' W), and the other two were located in mountainous areas ofhumid tropical forest, Chagres National Park (Cerro Azul) (9° 13' N; 79° 22' W) and Altos de Campana National Park (8° 13' N; 79° 22' W). The actual time (sampling effort) that active plants and non-active plants were checked for insects was equalized, as best as possible. In general, active plants were examined for approximately 210 minutes per week and non-active plants for approximately 190 minutes per week. It was not possible to simply use the number of plants for equalizing the time active and non-active plants were checked for insects since the types of study plants were diverse, including herbs, lianas, shrubs, and trees. AIso, sorne of the species were more abundant that other study plant species. Each study plant was observed a total of 16 times between May 2004 and November 2004, with the majority of field work occurring from June to September. Plants were typically checked once every week, sometimes with intervals of approximately two weeks occurring between monitoring dates. This period of study was chosen because insects are most abundant during the rainy season in Panama, which begins in April and ends in December (Windsor et al. 1992). Monitoring plants for insects involved thoroughly checking allleaf surfaces of the plant. First, aIl areas that could be checked without touching the plant were examined and then leaveslbranches were carefully moved to examine non-visible areas. In cases where the leaveslbranches were out of arms-reach, a pole with hooks at the end was used to carefuHy pull the branches down. All insect species, in the orders Coleoptera (beetles) and Lepidoptera (butterflies and moths), which were found on study plants were collected in small vials and brought back to the labo In the lab, the insects were kept enclosed in a container with an 34 uneaten leaf of the plant species they were found on to determine if they did indeed eat the foliage or were simply found resting on the plant. Those that did not eat the leaf after three days were killed and when possible mounted for reference purposes.

Rearing of Insects

Insects were kept in plastic containers that had their bottoms covered with paper towel. Insects were provided with fresh plant material and containers were cleaned when necessary. Humidity was provided by plant foliage, as well as by added water droplets when required. To prevent moisture lost, no holes were put in the containers. Adult Coleopterans that were found to feed on non-active plants were killed and mounted for reference purposes. To confirm feeding practices, the same Coleopteran species were collected on at least two separate occasions and observed in the labo If the same species was subsequently observed on the same host plant its presence was simply recorded. Lepidopteran larvae that were found feeding on non-active plants were reared to adults. Species were collected on at least two more occasions or until adults were successfully reared. On active plants, the first adult Coleopteran specimen found to feed on a plant was killed and mounted for reference purposes. Subsequently, all further specimens were reared and eventually killed by deep freezing at -80°C, where they were kept until chemical analysis. On active plants, the first specimen of a Lepidopteran larva found feeding on a plant was reared to the adult stage and then was mounted for reference. For subsequent collections larvae were reared to reach the desired life stage for reference purposes or chemical analysis. When large numbers of Lepidopteran larval specimens were obtained, sorne were stored in ethanol for future reference. A recollection permit (SCI AP-I-05) was obtained from ANAM for the collection of insect species. Voucher specimens of insect species collected have been place in the Fairchild Museum, University of Panama and the Smithsonian Tropical Research Institute Synoptic Insect Collection. Digital photographs were taken of the majority of insect species collected. 35

Identification of Insects

Insect identifications were made using the specimens in the Insect Collection Room at the Smithsonian Tropical Research Institute, as weIl as websites specializing in tropical insect species (e.g. Janzen and Hallwachs 2005). Dr. Annette Aiello (STRI) confirmed and made Lepidopteran identifications. Dr. Henry Stockwell (STRI) and Dr. Don Windsor (STRI) confirmed and made Coleopteran identifications. In rare cases, photos of insect specimens were sent to experts in a particular field.

Aposematic Evaluation and Insect Life-History

All insects were categorized as either aposematic or non-aposematic. Insects were considered to be aposematic ifthey were red, orange, or yellow, often altemating with black (dark grey, dark brown), and sometimes blue and purple (Salazar & Whitman 2001). Photographs of the insects were sent to 15 individuals, including both scientists and non scientists, who determined if they considered the insect aposematic or not. The most frequent response decided the category in which the insect was placed. Literature searches, web searches, consultation with specialists, and examinations in STRI's Insect Collection Room were performed to determine whether an insect had previously been documented to feed on a study plant, as well as to determine its classification of generalist or specialist.

Statistics

All statistical analyses were performed using the pro gram Systat (Systat Software Inc.). The chi square test is used to test a distribution observed in the field against another distribution determined by a null hypothesis.

2.3. RESUL TS

Forty-six insect species were collected and found to feed on active plants, with 41.3% (19) of these being considered aposematic. In contrast, 25 insect species were collected and found to feed on non-active plants, with only 20.0% (5) ofthese being considered aposematic (Table 2.2). Aposematic insect species were found on 9 of the 10 active plants, in contrast to 36

Table 2.2. Number of aposematic and non-aposematic insect species collected on the ten active and ten non-active tropical plants studied.

Number of Insect Herbivore Species Aposematic Non-aposematic Active Plants 19 27 Non-active Plants 5 20 37 only 4 of the 10 non-active plants. Therefore, approximately 1.9 aposematic insect species were found on each active plant, compared to only 0.5 on each non-active plant. In comparison, non-aposematic insect species were found on 8 of the 10 active plants and on aIl 10 of the non-active plants. Thus, approximately 2.7 non-aposematic insect species were found on each active plant and 2.0 on each non-active plant. When considering insect species where more than one individual was collected, 34 insect species were collected and found to feed on active plants, 47.1 % (16) of which were considered aposematic (Table 2.3). Thus, on active plants 73.9% of total insects and 84.2% of aposematic insects were collected more than once. On non-active plants, 15 insect species were collected and found to feed on the plants more than once, with 26.7% (4) ofthese being aposematic. Therefore, on non-active plants 60.0% of total insects and 80.0% ofaposematic insects were collected more than once. Overall, 41.3% of all insect species collected on active plants were considered aposematic, whereas only 20.0% of insect species found on non-active plants were considered aposematic. Of those insect species where more than one individual was collected 47.1 % on active plants were considered aposematic, in contrast to only 26.7% on non-active plants. On biologically-active plants, a total of 22 Coleopteran species and 25 Lepidopteran species were collected, of which 13 Coleopterans and 6 Lepidopterans were considered aposematic. Hence, 59.1 % of Coleopteran species found on active plants were aposematic, compared to only 24% of larval Lepidopteran species. On non-active plants, there were a total of 12 Coleopteran species and 13 Lepidopteran larval species identified, ofwhich 3 Coleopteran and 2 Lepidopteran species were considered aposematic. Therefore, 25% of Coleoptera found on non-active plants were aposematic, compared to only 15.4% of Lepidopteran larval species. Interestingly, the total number of Lepidopteran larval species found on both active and non-active plants was greater than the total number of Coleopteran species; however, there were higher numbers of aposematic Coleopteran than Lepidopteran larval species on both active and non-active plants. Tables 2.4a and 2.4b show the lowest level of taxon identification for each of the insect species collected. These tables also indicate whether these insects species were considered aposematic or non-aposematic, whether they had previously been documented to feed on the study plant on which they were found, as weIl 38

Number of Insect Herbivore Species Aposematic Non-aposematic Active Plants 16 18 Non-active Plants 4 10 39

Table 2.4a. The associations between ten biologically-active tropical plants and their insect herbivores. The lowest taxonomic level of identification determined for insects collected on the study plants, whether these insects were aposematic or non-aposematic, whether they had previously been documented to feed on a study plant, as well as general characteristics of each insect species.

ACTIVE INSECT SPECIES APOSEMATIC PREVIOUS GENERAL NOTES: PLANTS OR NON- RECORD SPECIALIST OR APOSEMATIC ON PLANT GENERALIST, SIZE, SP. HAIRS, BEHAVIOUR RESPONSES Baccharis Alticinae sp. 1 Aposematic Small trinervis Fast-moving (Coleoptera: Chrys:>meloidea: (orange) (Asteraceae) Chrysomelidae) Pachybrachis Aposematic NO Small

reticulata Fabr. (yellow & dark brown) (Coleoptera: Chrys:>meloidea: Chrysomelidae: Cryptocephalinae) Diabrotica sp. 1 Aposematic Small Fast-moving (Coleoptera: Chrys:>meloidea: (yellow & black) Chrysomelidae: Galerucinae) Myrmex vicinus Ch. Non-aposematic NO Medium Drop from leaf (Coleoptera: Curculioridae) (black) Geometridae sp. 1 Non-aposematic Medium Inchworm (Lepidoptera: Geometroidea) (black) Semiothisa sp. 1? Non-aposematic Medium Inchworm (Lepidoptera: Geometroidea: (brownish-green) Geometridae) Noctuidae sp. 1 Non-aposematic Medium

(Lepidoptera: Noctuoidea) (green) Neurolaena Platyphora ligata Aposematic YES SPECIALIST 2 (Asteraceae) lobata Stal. (yellow & dark Large (Asteraceae) brown) (Coleoptera: Chrys:>meloidea: Defensive secretions Chrysomelidae: Chrysomelinae) Calephelis Sp. Non-aposematic SPECIALIST 2 (Asteraceae) laverna? (white hairs, with Medium yellow and brown (Lepidoptera: Papilionoidae: marking) Fine hairs Riodinidae) Noctuidae sp. 2 Non-aposematic Medium

(Lepidoptera: Noctuoidea) (green) Torticidae sp. 1 Non-aposematic Small-medium Leafroller (Lepidoptera: Tortricoidea) (green) Pyralidae sp.l Non-aposematic Small-medium Leafroller {Lepidoptera: Pyraloidea) (green) 40

Tithonia Melitaeinae sp. 1 Aposematic Medium diversifolia Thick spines (Lepidoptera: Papilionoidea: (orange and black) (Asteraceae) Nymphalidae) Platyphora ligata Aposematic NO SPECIALIST 2 Stal. (Asteraceae) (yellow & dark Large brown) (Coleoptera: Chry~meloidea: Defensive secretions Chrysomelidae: Chrvsomelinae) Dysschema Aposematic NO Large Long, fine hairs magdala Bois. (orange and dark grey) (Lepidoptera: Noctuoidea: Arctiidae: Pericopinae) Chlosyne Aposematic NO Small-Medium Thick spines hippodrome Gey. (orange and black)

(Lepidoptera: Papilionoidea: Nymphalidae Gelichiidae sp. 1 Non-aposematic Small Leafroller (Lepidoptera: Gelechioidea) (green and black) Melampodium Systena s-littera L. Aposematic GENERA LI ST divaricatum Small (Coleoptera: Chrysomeloidea: (yellow and black) Fast-moving (Asteraceae) Chrvsomelidae: A1ticinae) Rhabdopterus sp. 1 Non-aposematic GENERALIST Medium (brown) (Coleoptera: Chry~meloidea: Chrysomelidae: Eumolpinae) Noctuidae Sp. 3? Non-aposematic Medium Leafroller (Lepidoptera: Noctuoidea) (green) Phryganocydia Unknown Aposematic Medium corymobosa Lepidopteran sp. 1 (yellow and black) (Bignoniaceae) Cordia Physonota alutacea Aposematic YES SPECIALIST 2 currassavica Boh. (larvae) Cordia sp. (Boraginaceae) (yellow, white and Medium (Boraginaceae) black) (Coleoptera: Chrysomeloidea: Furcal appendages Chrysomelidae: Cassidinae) No shield Lebia sp. 1 Aposematic Small

(Coleoptera: Carabidae: (yellow and dark Harpalinae) brown) Galerucian sp. 1 Non-aposematic Small Fast-moving (black) (Coleoptera: Chry~meloidea: Drop from leaf Chrysomelidae) Polydacrys Non-aposematic GENERA LIST Small-medium depressifrons Boh. (Iight and dark grey) (Coleoptera: Curculioridae) Polychalma N on-aposematic YES SPECIALIST 3 multicava Latr. Cordia curassavica (black) (Boraginaceae) Helicteres guagumaefolia (Coleoptera: Chry~meloidea: Chrysomelidae: Cassidinae) (Sterculiaceae) 41

Large Drop from leaf Physonota alutacea Non-aposematic YES SPECIALIST 2 Boh. (adult) Cordia sp. (Boraginaceae) (greenlsilver) Large (Coleoptera: Chrysomeloidea: Drop from leaf Chrysomelidae: Cassidinae) Noctuidae Sp. 4? Non-aposematic SmaU Leafroller or Pyralidae Sp. 2? (green)

(Lepidoptera) Alticinae sp. 2 N on-aposematic Small Fast-moving (Coleoptera: Chrysomeloidea: (brown) Chrysomelidae) Bonamia Chersinellina Aposematic YES SPECIALIST 1 trichantha heteropunctata B. trichantha (gold) SmaU-medium (Convolvulaceae) Boh.

(Coleoptera: Chrysomeloidea: Chrysomelidae: Cassidinae) Chomelia Charidotis coccinea Aposematic YES SPECIALIST 1 C. recordii recordii Boh. (red) Small (Rubiaceae) (Coleoptera: Chrysomeloidea: Drop from leaf Chrysomelidae: Cassidinae) Charidotis Aposematic YES SPECIALIST 2 erythrostigmata (Rubiaceae sp.) (red and black) Small Champ. Drop from leaf

(Coleoptera: Chrysomeloidea: Chrysomelidae: Cassidinae) Phanaeta ruficollis Aposematic YES SmaU Drop from leaf Lef. (orange and blue)

(Coleoptera: Chrysomeloidea: Chrysomelidae: Eumolpinae) Unknown Aposematic Small Lepidopteran sp. 2 (orange) Omiodes sp. 1? Non-aposematic Medium-large

(Lepidoptera: Pyraloidea: (green) Pyralidae: Pyraustinae) Geometridae sp. 2 Non-aposematic Small

. (Lepidoptera: Geometroidea) (Iight green) Coelocephalapion N on-aposematic GENERALIST nodicorne Shp. Very small (grey) Fall from leaf (Coleoptera: Curculioridae) Unknown Non-aposematic Small Lepidopteran sp. 3 (brown) Unknown Non-aposematic Lepidopteran sp. 4 Unknown Non-aposematic 42

Lepidopteran sp. 5 Unknown Non-aposematic Small Leafroller Lepidopteran sp. 6 (green) Hamelia Pyralidae sp. 3 Non-aposematic Medium axillaries Leafroller (Lepidoptera: Pyraloidea) (green) (Rubiaceae)

Witheringia Lema bitaeniata Aposematic YES SPECIALIST 2 solanacea (Solanaceae) (Coleoptera: Chry&:lmeloidea: (yellow, red, and Small (Solanaceae) Chrysomelidae: Criocerinae) black) Fast-moving Noctuidae sp. 5 Aposematic Medium

(LeQidoptera: Noctuoide!l) (orange and black) Ithomia iphianassa Non-aposematic Small panamensis Bates (grey) (Lepidoptera: Papilionoidea: Nymphalidae: Ithomiinae) Plagiometriona Non-aposematic YES SPECIALIST 2 gibbifera Champ. (Solanaceae sp.) (c1ear, black and W. solanacea green) (Coleoptera: Chry&:lmeloidea: Aureliana lucida Chrysomelidae: Cassidinae) Small-medium Geometridae sp. 3 Non-aposematic Medium Imitates a stick (Lepidoptera: Geometroidea) (Iight green) Note: SPECIALIST 1 = known to feed on a single plant species; SPECIALIST 2 = known to feed on species within a single plant family; SPECIALIST 3 = known to feed on a small number of species with more than one plant family; GENERALIST = known to feed on many plant species within more than one family. For the column 'Previous record on plant sp.' there are many empty cells, which reflect the lack of information on tropical host plant-insect interactions. 43

Table 2.4b. The associations between ten biologically-non-active tropical plants and their insect herbivores. The lowest taxonomie level of identification determined for insects collected on the study plants, whether these insects were aposematic or non-aposematic, whether they had previously been documented to feed on a study plant, as weIl as general characteristics of each in sect species.

NON- INSECT SPECIES APOSEMATIC PREVIOUS GENERAL NOTES: ACTIVE OR NON- RECORD SPECIALIST OR APOSEMATIC ON PLANT PLANTS GENERALIST, SIZE, SP. HAIRS, BEHA VI OUR RESPONSES Wulffia Microctenochira Aposematic YES SPECIALIST 2 baccata flavonotata Boh. (Asteraceae) (black and Wedelia trilobata (Asteraceae) yellow/gold) (Coleoptera: Chrys:>meloidea: Small Chrysomelidae: Cassidinae) Fast-moving Brachypnoea sp. 1 Non-aposematic Small

(Coleoptera: (black) Chrysomeloidea: Chrysomelidae: Eumolpinae) Wedelia Colaspis sp. 1 Aposematic Small calycina (Coleoptera: Chrys:>meloidea: (orange and black) (Asteraceae) Chrysomelidae: Eumolpinae) Brachypnoea sp. 2 Non-aposematic Small

(Coleoptera: Chrys:>meloidea: (brown) Chrysomelidae: Eumolpinae) Alibertia Oxytenis modestia Non-aposematic YES Large Imitates bird dropping & edulis Cram. (brown and green) snake depending on instar (Rubiaceae) (Lepidoptera: Bombycoidea: Saturniidae: Oxyteninae) Pyralidae sp.4 Non-aposematic Medium Leafroller (Lepidoptera: Pyraloidea) (green) Brugmansia Percopinae sp. 1 Aposematic Medium candida Fine hairs (Lepidoptera: Noctuoidea: (red and black) (Solanaceae) Arctiidae) Torticidae sp. 2 Non-aposematic Medium Leafroller (Lepidoptera: Tortricoidea) (green) Gegurgitates when disturbed Geometridae sp. 4 Non-aposematic Medium Imitates a stick (Lepidoptera: Geometroidea) (brown) Rutelinae sp. 1 Non-aposernatic GENERA LIST Large (Coleoptera: Scarabaeilae) (brown) Unknown Non-aposematic Medium-large Thick hairs Lepidopteran sp. 7 (brownlmaroon) Jacaranda Geometridae sp. 5 Non-aposematic Medium copaia Imitates a stick (Lepidoptera: Geometroidea) (grey/green) (Bignoniaceae) Tournefortia Ischnocodia Aposematic YES SPECIALIST 2 44

hirsutissima annulus Fabr. Cardia sp. (yellow and black) (Boraginaceae) T hirsutissima (Coleoptera: Chryromeloidea: (Boraginaceae) Chrysomelidae: Cassidinae) Ocatea veraguensis (Lauraceae) Small Dysschema Aposematic Medium Fast-moving But!. jansonis (orange and dark Fine hairs blue) (Lepidoptera: NocbJOidea: Arctiidae: Pericopinae) Charidotis vitreata Non-aposematic YES SPECIALIST 2 Cardia sp. Perty (brown and black) T hirsutissima (Coleoptera: Chryromeloidea: (Boraginaceae) Chrysomelidae: Cassidinae) Small Maripa Microctenochira Non-aposematic YES Phryganocydia corymobosa Champ. panamensis cruxjlava (black and (Bignoniaceae) (Convolvulaceae) greenlgold) Small (Coleoptera: Chryromeloidea: Chrysomelidae: Cassidinae)

Unknown Non-aposematic Small Leafroller Lepidopteran sp. 8 (green) Chiococca Geometridae sp. 6 Non-aposematic Medium alba Imitates a stick (Lepidoptera: Geometroidea) (brownlblack) (Rubiaceae) Noctuidae Sp. 6? Non-aposematic Small Fine hairs (Lepidoptera: Noctuoidea) (green) Isertia Perigonia lusca Non-aposematic Large

haekeana Fabr. (green) (Rubiaceae) (Lepidoptera: Sphingoidea: Sphingidae) Alticinae sp. 3 Non-aposematic Small Fast-moving (Coleoptera: Chryromeloidea: (green) Chrysomelidae) Eumolpinae sp. 1 N on-aposematic Small

(Coleoptera: Chryromeloidea: (black) Chrysomelidae) Solanum Colaspis Non-aposematic Medium jamaicense sanjoseana (morphs: green (Solanaceae) Bechyné and brown)

(Coleoptera: Chryromeloidea: Chrysomelidae: EumolDinae) Colaspis nr. Non-aposematic Small jlavipes (dark blue)

(Coleoptera: Chryromeloidea: Cllrysomelidae: Eumoloinae) Unknown N on-aposematic Small Leafroller Lepidopteran sp. 9 (green) 45

Note: SPECIALIST 1 = known to feed on a single plant species; SPECIALIST 2 = known to feed on species within a single plant family; SPECIALIST 3 = known to feed on a sm ail number of species with more than one plant family; GENERALIST = known to feed on many plant species within more than one family. For the column 'Previous record on plant sp.' there are many empty cells, which reflect the lack of information on tropical host plant-insect interactions. 46 as general characteristics of each species (e.g. generalist or specialist, size, and other defensive mechanisms and responses).

Chi Square Distributions

For testing the distribution of aposematic insects the nun hypothesis states that an equal number of aposematic insect species will be found on the active plants and non-active plants studied, since the sampling efforts were similar. This nun hypothesls was rejected and it was found that significantly more (df= 1,X2 = 8.167, P = 0.01) aposematic insect species were found on active than non-active plants. The distribution of the total number of insect species found on the study plants was also tested, with the nun hypothesis being that active and non-active plants should have similar insect populations because the sampling efforts were similar. Again, the nun hypothesis was rejected and it was found that there were significantly more (df = 1, X2 = 6.58, P = 0.05) insect species on active plants than on non-active plants. Finany, the distribution of non-aposematic insects was tested, with the nun hypothesis being that active and non-active plants should have similar numbers of non-aposematic insect species, since the sampling efforts were similar. In this case the nun hypothesis was supported and the difference in the number of non-aposematic insect specifie found on active and non-active plants was not significant (df= 1, X2 = 2.041, p> 0.05).

Percentages of Aposematic Insects found on Study Plants

The percentages of aposematic insects found on study plants pairs listed in Table 2.1, were examined. First, an insect species that were found to feed on the study plants were analysed (Figure 2.1), using at-test paired two sample for means. A greater percentage of aposematic insect species were found on biologicany-active plant species compared to non active plant species, with the results being close to significant (df = 9, t = 1.973, and p = 0.08). For seven plant pairs, the plant species with biologically-active properties had a higher percentage of aposematic insect species associated with it than the non-active plant species (Figure 2.1); for example, 80% of the insect species on the active plant Tithonia diversifolia were aposematic, and 50% of the insect species on the non-active plant Tilesia baccata were aposematic. The numbers in the brackets show the percentage of aposematic insect species 47

Figure 2.1. Percentages of aposematic insect species found on tropical study plants, using aIl insect species observed feeding on the study plants. Comparisons were do ne using paired study plants. Plants were paired using phylogenetic relationships. 48

120

() +" 100 ê Q) U) 0 80 ca-c..U) -o ()Q) 60 Q) ~ 0)'- ca c 40 -Q) ....() Q) a.. 20

o~------~ Non-active Active plants plants 49 found on each plant: Baccharis trinervis (42.9%) - Alibertia edulis (0%); Melampodium divaricatum (33.3%) - Brugmansia candida (20%); Phryganocydia corymobosa (100%) Jacaranda copaia (0%); Bonamia trichantha (100%) - Maripa panamensis (0%); Chomelia recordii (40%) - Chiococca alba (0%); and Witheringia solanacea (40%) - Solanum jamaicense (0%). For one plant pair, no aposematic insects were found on either the active or non-active plants: Hamelia axil/aries (0%) - Isertia haekeana (0%). In two cases, the non biologically-active was found to have a higher percentage of aposematic insects than its active plant pair: Neurolaena lobata (20%) - Wedelia calycina (50%); and Cordia curassavica (25%) - Tournefortia hirsutissima (66.6%). Next, only those insect species where more than one individual was observed feeding on a study plant were used for further analysis (Figure 2.2). These results were also analyzed using at-test paired two sample for means. Again, a greater percentage of aposematic insects were found on active plant pairs compared to non-active plant pairs, with the results being close to significant (df= 9, t = 2.024, and p = 0.074). In this analysis, there were six plant pairs where the active plant had a higher percentage of aposematic insects than the non-active plant, including, the active plant Tithonia diversifolia where 80% of its associated insect species were aposematic, and the non-active plant Tilesia baccata where 50% of its insect species were aposematic. The numbers in the brackets show the percentage of aposematic insect species found on each plant: Baccharis trinervis (50%) - Alibertia edulis (0%); Melampodium divaricatum (50%) - Brugmansia candida (0%); Bonamia trichantha (100%) - Maripa panamensis (0%); Chomelia recordii (57.1 %) - Chiococca alba (0%); and Witheringia solanacea (33.3%) - Solanumjamaicense (0%). For two plant pairs, aposematic insects were not found on either the active or non-active plants: Hamelia axil/aries (0%) - Isertia haekeana (0%) and Phryganocydia corymobosa (0%) - Jacaranda copaia (0%). In two cases, the non-active plant species had a higher percentage of aposematic insects feeding on them than their biologically-active plant pair: Neurolaena lobata (25%) - Wedelia calycina (50%); and Cordia curassavica (33.3%) - Tournefortia hirsutissima (66.6%). 50

120.------~

(J ê 100 Q) 1/)o 80 0.1/) _ctl (J o Q) 60 Q) ~ 0)'- CU ë 40 Q) ~ a..Q) 20

n~ ______~

Non-active Active plants plants 52

Herbivory Assessment

Based on the qualitative observations of the study plants, plant species which had biological activity in the ICBG bioassays generally had more herbivore damage than non active plants. This was observed in six of the plant pairs (Neurolaena lobata - Wedelia calycina, Baccharis trinervis - Alibertia edulis, Phryganocydia corymobosa - Jacaranda copaia, Bonamia trichantha - Maripa panamensis, Chomelia recordii - Chiococca alba, and Witheringa solanacea - Solanumjamaicense). In contrast, in only one plant pair (Hamelia axillaries - Isertia haekeana) did the non-active plant species appear to have and/or accumulate more herbivory damage than the active plant over the study period. The other three plant pairs (Tithonia diversifolia -Tilesia baccata, Melampodium divaricatum Brugmansia candida, and Cordia curassavica - Tournefortia hirsutissima) appeared to have relatively equallevels of herbivore damage.

Search Times to find Aposematic Insects

The approximate search times spent analyzing each plant to find associated aposematic insect herbivores are displayed in Table 2.5. Overall, eleven of the aposematic insects were found in less than 100 minutes, eight aposematic insects were found in between 100 and 200 minutes, and four took over 200 minutes of search time to find (Table 2.5).

2.4. DISCUSSION

More Aposematic Insects on Active Plants

The hypothesis that more aposematic insects would be found on plants with activity in the ICBG bioassays was supported with significant (Chi-square test) and close to significant results (t-test paired two sample for means), as the percentage of aposematic insects on active plants was found to be approximately twice that on non-active plants. This trend was found when considering all insects that were observed to feed on the study plant species, as well as when considering only those insects where more than one individual was observed feeding on the study plants species. This suggests that the plant metabolites which had activity in the ICBG bioassays may be exploited by insect herbivores that have evolved the ability to feed 53

Table 2.5. Search times to find aposematic insect species that were found to feed on the tropical study plants.

PLANT SPECIES INSECT SPECIES SEARCHTIME (minutes) Baccharis trinervis Alticinae sp. 60-90 Pachybrachis reticulata 150-180 Diabrotica sp. 390-420 Neurolaena lobata Playtphora ligata 15-30 Tithonia diversifolia Melitaeinae 1-30 (Nymphalidae) Playtphora ligata 30-60 Dysschema magdala 390-420 Chlosyne hippodrome 420-450 Melampodium Systena s-littera 60-80 divaricatum Phryganocydia Unknown sp. (L32) 120-135 corymbosa Cordia curassavica Physonota alutacea 120-150 (larvae) Lebia sp. 270-300 Bonamia trichantha Chersinellina 45-60 Heteropunctata Chomelia recordii Charidotis coccinea 1-25 Charidotis 1-25 Erythrostigmata Phanaeta ruficollis 1-25 Unknown sp. (L17) 125-150 Witheringia Lema bitaeniata 30-45 solanacea Noctuidae (L40) 105-120 Wedelia calycina Colaspis sp. 120-140 Brugmansia candida Percopinae sp. 120-130 Tournefortia Ischnocodia annulus 60-75 hirsutissima Dysschema jansonis 105-120 54

on these plants. If the se compounds are concentrated or sequestered by the insect, they may provide the insect with protection against potential predators and these insect herbivores may then evolve bright colouration as a waming signal. Support exists for this reasoning. First, there are numerous examples where insects feeding on plants with secondary plant metabolites are known to concentrate and sequester them (Brower and Glazier 1975, Blum et al. 1981, Montllor et al. 1990, Boros et al. 1991, Szentesi and Wink 1991, Bowers and Collinge 1992, von Nickisch-Rosenegk and Wink 1993, Schaffner et al. 1994, Wiesen et al. 1994, Geuder et al. 1997, Nishida 2002). Second, there are many examples where insects specializing on toxic host plants have aposematic colouration (Reichstein et al. 1968, Montllor et al. 1990, Boros et al. 1991, Bowers and Collinge 1992, Fordyce 2000, Despland and Simpson 2005), which is likely to advertise their distastefulness/toxicity to potential predators (Brower and Glazier 1975, Bemays and Graham 1988, Nishida 2002). Further support cornes from a study by Raudsepp-Heame (2003), who found that the biologically-active plant compounds were also found in aposematic insects feeding on these plants. Raudsepp-Heame (2003) found that two aposematically-coloured (red and black), specialist beetle species Eugenysa coscaroni and Echoma anaglyptoides which fed on the plant Mikania guaco (Asteraceae), sequestered the sesquiterpene lactone, Guaconone, which was active against the amastigote form of Trypanosoma cruzi, the causative agent ofChagas' disease. Raudsepp-Heame (2003) also found that the anti-cancer compound from Vismia baccifera (Clusiaceae), called vismione B, was sequestered by two aposematically coloured larval Lepidopteran species, the generalist, Periphoba arcaei and the specialist, Pyrrhopyge pseudophidias, as well as an aposematically coloured specialist Chrysomelid species. These findings by Raudsepp-Heame (2003) revealed the potential ofusing aposematic insects as guides to plants containing potentially medicinally-active compounds. This study further supports this idea, as a higher percentage of aposematic insects were found feeding on active plants than on non-active plants. Therefore, promise exists to use these insect-plant relationships as guides to plants containing potentially medicinally-active compounds. 55

Using Aposematic Insects as Guides to Medicinally-Active Plants

This study provides information on whether ecologically-guided bioprospecting, which utilizes ecological knowledge or theories to direct the selection of plant species for testing, can help to increase the efficiency and the success rate of the bioprospecting process. The percentage of aposematic insects found on plants with biological activity was found to be almost twice the percentage found on non-active plants. Moreover, the chance that an aposematic insect would lead a researcher to an active plant was almost four times greater than the chance an aposematic insect would lead a researcher to a non-active plant. This is based on the finding that an average of 1.9 aposematic insect species were found on each of the 10 active plant species, whereas only 0.5 aposematic insect species were found on each of the 10 non-active plant species. The next question is how can aposematic insects practically be used as guides to plants with potentially biologically-active metabolites? There are two approaches that could be utilized. First, one could simply search for aposematic insects in the field and then do laboratory experiments to determine their host plants. Second, one could use the literature/scientific resources to find aposematic insects that feed on specific plants, which could then be collected. Without a doubt, the second approach would be more time efficient; however, the ability to use this approach is limited by the amount of research that has been done on the insects of a specific geographic area. The first approach would likely be more time consuming, but even in this experiment where the insect-host plant relationships were not well known, for almost half of the aposematic insects found it took less than 100 minutes of search time to encounter them. The first approach would also help to increase the limited amount of information on tropical insect-host plant interactions. Even though there was a significantly greater number of aposematic insects found feeding on biologically-active plants, aposematic insects were still found to feed on non active plants. One possible reason for this is that these aposematic insects are mimics of other toxic/distasteful aposematic insects, which do, in fact, feed on plants with biologically-active compounds. A second possibility is that these aposematic insects are actually sequestering biologically-active compounds, but these were not active in the limited ICBG bioassays tested. A third possibility is that these insects can synthesize defensive compounds de novo. 56

Finally, in the discussion of the use oftoxic compounds by insects to deter predation or parasitoids, it must also be realized that many unpalatable insects are not aposematic, especially ifvisual predation is not ofprimary importance (Schaffner et al. 1994), that the existence of mimi cry complexes means that brightly coloured insects may not be toxic (Fordyce 2000), that insects may be able to synthesize defensive chemicals de nova (Nishida 2000), and that not all insect species feeding on a plant species with defensive compounds will sequester them (Bowers and Collinge 1992). Therefore, not all aposematic insects will be associated with plants containing potentially medicinally-active compounds. Nevertheless, the results of this study suggest that aposematic insects can help direct us to such plants, thus enhancing the productivity of the bioprospecting process.

The 'Theory of Co-Evolution' by Ehrlich and Raven (1964)

When examining all insects that were observed feeding on the study plant species, 46 species were found on biologically-active plants and 25 species on non-active plants. Moreover, greater amounts of herbivore damage were found on the active plants in 6 out of the 10 plant pairs. For example, Chomelia recordii, Neurolaena lobata, Baccharis trinervis, and Witheringia solanacea, all known for being biologically-active and having medicinal properties (Sanchez Palomino et al. 2002, Berger et al. 2001, Caballero-George et al. 200 l, Sharp et al. 2000, Passreiter 1998, Passreiter and Isman 1997, Francois et al. 1996), were found to have at least at one point during this study more than half of their leaf surface consumed. There were even sorne occasions where individuals of these plant species would have nothing but the leaf veins left. There are also other examples where biologically-active or noxious plants sustain extremely high levels ofherbivory; for example, Merremia umbellata (Convolvulaceae) is attacked by the beetles Chelymorpha alternans and Acromis sparsa, and Ipomoea phillomega (Convolvulaceae) is attacked by the beetles Chelymorpha sp. and Stolas plagiata (Fred Vencl pers. comm.). These results are counterintuitive; one would imagine that a plant with biologically-active compounds would have a higher level of defense against herbivores and thus have fewer insect species feeding on them, as well as lower levels ofherbivory. However, in this study the biologically-active plants had higher numbers ofboth aposematic and non-aposematic insects feeding on them, resulting in a significantly greater number of total insect species feeding on biologically-active plants 57 compared to the non-active plants. In addition, many of the active plants had more herbivore damage than the non-active plants. Ehrlich and Raven's (1964) theory of co-evolution proposes that the selection pressure ofinsects enhances the development ofplant's defensive mechanisms, which could inc1ude the biologically-active compounds found in medicinally-active plants. Insect herbivores often cause decreases in plant growth rate, survivorship, and reproductive success; hence, insects exert selection pressure on plants to defend themselves (see Rausher 1988 and references therein). In tum, these defenses can provide plants with protection from phytophagous insects, as well as potentially allow for plant diversification. However, sorne insect species may then develop adaptations to the plants defenses, which could largely free these insects from competition with other phytophagous insects, and allow for their diversification (Ehrlich and Raven 1964, Jermy 1984). It appears in this study that the insects have the "upper hand", as more insect species and often more insects were found to feed on the biologically-active plants. Therefore, the assumption that plants will bene fit from their investment in secondary metabolites and be at a selective advantage, in terms of decreased levels of herbivory (Ehrlich and Raven 1964, Krebs 2001), does not appear to be supported by this study. Consequently, the question arises, if these biologically-active plants are receiving a higher degree of herbivory then why are these defensive compounds made in the first place? Ultimately, co-evolution involves reciprocal adaptive genetic changes within populations of interacting species that act as selective agents on one another (Kareiva 1999). One good example, by Berenbaum (1998), is the wild parsnip (Pastinaca sativa) an introduced European weed, which is found throughout eastem North America, and its only herbivore, the parsnip webworm (Depressaria pastinacella). This system has shown that the furanocourmarin (plant defensive compounds) profiles from different geographic areas correspond to the types of furanocourmarins that the parsnip webworms are best able to metabolize (Kareiva 1999). In addition, there is the wide range of examples involving pesticide use, where insects have been able to adapt to these chemicals after only a few generations (Hoy 1998). Another example, is the breeding of new crop varieties for resistance attributes, which often only end up selecting for enemy populations (herbivores or pathogens) with counter-adaptations to these defenses (Kareiva 1999). Therefore, it could be the case that insects are often able to adapt rapidly to changes in the chemistry of their host plants. 58

Subsequently, it also seems reasonable to assume that ifthese biologically-active compounds could be used effectively by insects for their own defense against predators that numerous insect species would adapt and take advantage of this. However, it is not always the case that biologically-active plants have higher numbers ofinsects feeding on them and/or greater herbivore damage. For example, the Yew tree (Taxus baccata), a temperate, coniferous species, which contains taxine, an alkaloid that is a precursor to the potent anti-cancer compound Taxol, in almost all of its plant parts, has very few insect predators (Daniewski et al. 1998). Its wood is not attacked by woodworm and its needles are attacked by very few insects. In addition, needle extracts show strong anti-feedent activity against storage pests. This activity is likely due to the compounds, 10- deacetylbaccatin III and 10-deactybaccatin V, which are useful for the semisynthesis of Taxol (Daniewski et al. 1998). Hence, in this case, it appears that plant defenses can provide very effective protection to a plant species. As Dawkin and Krebs (1979) state: in an "arms race" whenever either side produces a new adaptation they may have a grace period before the other side produces a counter-adaptation. Could this be the case with the Yew tree and its associated herbivore population? Is it possible that the four study plants and their insects mentioned above are at a different stage in the co-evolutionary process than the Yew tree with its apparent lack of insect predators? There are many factors that must be taken into account when considering this question inc1uding the overall biological activity of the plants secondary metabolites, the diversity of the plants secondary metabolites, the ease with which insects are able to adapt to certain types of secondary compounds, and the evolutionary age of the plant species. Should we be looking at plants that have extremely little herbivory, as well as plants which are associated with aposematic insects? Evidence exists that both methods have the potential of leading us to plants with medicinally-active compounds. However, more work is necessary, looking both at the insect populations ofknown medicinal plants, and testing plant species with aposematic insect associations for medicinal activity. It is also critical to have more information on the ecology of the insects, such as their predators/parasitoids and whether they are specialists or generalists. 59

The Importance of Predators

Another interesting piece of information would be to know what types of compounds are in insect herbivores to defend themselves against predationlparasitism and whether or not these chemicals are medicinally-active. Therefore, the actual predators of an insect may yield sorne light on whether the insect is concentrating interesting compounds. For example, the Neem tree (Azadirachta indica A. Juss.) has both insect anti-feedant activity and medicinal activity. Even though this tree has a range of insect herbivores, few appear to have aposematic colouration. This lack of aposematic colouration could be because neem compounds are not toxic to the majority ofvertebrate species, many ofwhich have colour vision and could be predators of the insect herbivores (Schmutterer 1995). Ultimately, it must be remembered that most insect herbivores are preyed on by more than one predator species, which could differ in foraging styles, visual (visual acuity, colour vision, etc.), perceptual, and leaming abilities (Endler 1988). For example, Dyer and Floyd (1993) and Dyer (1995) found that caterpillar colouration did not affect predation by the tropical ant Paraponera clavata, which are primarily chemically-orientated predators. Dyer (1995) postulated that this could be because many of the waming coloured caterpillars are palatable mimics, or simply that these species are unpalatable to other predators. Subsequently, Dyer (1997) did find that brightly coloured caterpillars were frequently rejected by the visually orientated wasps (Polistes instabilis), but not by the visually-orientated bugs (Apiomerus pictipes). Many of the ideas ofpalatable prey characteristics have been shaped by research with vertebrate predators; however, it is perhaps necessary to re-examine sorne ofthese assumptions as invertebrate predators, such as ants, and parasitoids produce substantiallevels of mortality (Dyer 1995).

Specialists versus Generalists

Another aspect that should be considered is whether the insect species feeding on a plant species are generalists or specialists. Unfortunately in tropical areas this type of information is often unknown, as was found to be the case in this study. Less than 30% of the study insects could be confidently classified into specialist or generalist categories (Table 2.4a and 2.4b). Typically, specialists are monophagous or oligophagous, while generalists are 60 polyphagous; however, it is often difficult to draw the lines separating these categories. In many cases, when considering chemically defended host-plants, specialist herbivores have evolved mechanisms to benefit from these chemicals by sequestering the plant compounds or derivatives. In contrast, generalist herbivores feeding on the same plant often have not developed the ability to sequester compounds, but still may be able to cope with these chemicals (Johnson and Bentley 1988, Krebs 2001, Nishida 2002). For example, the specialist sawf1y Rhadinoceraea nodicornis can concentrate veratrum alkaloids from its host plant Veratrum album, but the generalist species Aglaostigma spp. cannot (Schaffner et al. 1994). The potential importance of knowing whether insects are specialist or generalist is illustrated in two studies by Dyer and Floyd (1993) and Dyer (1995) who found that the most important factor determining the probability of Lepidopteran larvae prey rejection by the tropical ant Paraponera clavata was a prey's diet breadth, with specialists being rejected by the ants significantly more than generalists. Dyer (1997) also found that larval chemistry and diet breadth were the most important predictions of Lepidopteran larvae rejection by the assassin bug Apiomerus pictipes and the paper wasp Po/istes instabilis, as larvae with deterrent extracts and specialists were rejected more frequently. Overall, Dyer (1995) found that 25% of Lepidopteran larvae specialists (47 species) offered to ants were completely rejected, while less that 10% of generalists were completely rejected.

The Species of Insects

Unfortunately, because of the higher number of in sect species in the tropics, as well as the limited research and documentation compared with temperate regions, the ability to identify tropical insect species and find information on their ecological characteristics is difficult. In this study, only 26 of the 72 insects (36%) feeding on the study plants were identified to the species level.

Lepidopterans versus Coleopterans

Interestingly, the total number of Lepidopteran larval species found on both biologically-active and non-active plants were greater than the total number of Coleopteran species. In contrast, higher numbers of aposematic Coleopteran species were found on both 61 biologically-active and non-active plants than aposematic Lepidopteran larval species. It is known that insect herbivores in the Lepidopteran families Satumiidae, Ithominae, Arctiidae, and the Coleopteran families Chrysomelidae and Curculionidae are effective at sequestering plant secondary compounds; however, the relative abilities between Lepidopterans and Coleopterans for sequestering plant secondary compounds does not appear to have been studied.

2.5. CONCLUSIONS

This study focused on researching the feasibility of using aposematic insects as guides to plants containing potentially medicinally-active compounds. A significantly greater number of aposematic insect species were found on active plants than on non-active plants, and there was a four-fold greater chance that an aposematic insect would be found to feed on an active plant than on a non-active plant. This supports the idea that aposematic insects can be used to help increase the efficiency of the bioprospecting process. This study also brings up other questions that need to be addressed to increase the success rate of using aposematic insects as guides to biologically-active plants, such as determining the predators/parasitoids of an insect and whether the insect is a specialist or generalist. 62

LINKING STATEMENT

The following chapter "Do Aposematic Insects Feeding on Tithonia diversifolia Concentrate its Medicinally-Active Compound(s)?" augments the first chapter "Can Aposematic Insects Guide us to Medicinally-Active Plants in the Tropics" by investigating the actual abilities of aposematic insects, which were found in association with a medicinally active plant studied in the first chapter, to sequester the plants biologically-active compounds. This second chapter examines the ability of two aposematic Lepidopteran species, Dysschema magdala and Chlosyne hippodrome, and one aposematic Coleopteran species, Platyphora ligata, to sequester the plant secondary compounds from Tithonia diversifolia, which were found to be active against Chagas' disease, malaria, and three cancer celllines. It is hypothesized that the aposematic insects will sequester these active plant secondary compounds, which are used as part oftheir defense strategy. Therefore, their bright colouration is a signal to potential predators oftheir distastefulness or toxicity. 63

CHAPTER2

Do aposematic insects feeding on Tithonia diversifolia (Asteraceae) concentrate its medicinally-active compound(s)?

1 1 1 JULIE E. HELSON ,2*, JOHANT LAKE Y , and TODD L. CAPSON ,2

ISmithsonian Tropical Research Institute Apartado 2072, Balboa, Anc6n, Panama City Republic of Panama

2Department of Plant Science McGill University 21,111 Lakeshore Road Ste. Anne de Bellevue, Quebec H9X 3V9 Canada

*To whom correspondence should be addressed. E-mail: [email protected]

CONTRIBUTIONS OF AUTHORS

For the second manuscript the first author again designed the experiment, chosing both the study subjects and the general methods to be used. The first author also performed the chemical extractions, VLC, TLC, and HPLC experiments. Finally, the first author analysed the data and wrote the manuscript. The second author provided guidance regarding which chemical procedures and chemical conditions should be used for analyzing the samples. The third author provided suggestions for the design of the experiment and also aided in editing the manuscript. In addition, the third author provided the materials, equipment, and facilities needed for performing this experiment. Finally, all bioassays were done by technicians in the laboratories of Eduardo Ortega and Luz Romero at INDICASAT (Institute of Advanced Scientific Research and High Tech Services). 64

ABSTRACT

Aposematic insects, which are insects with warning colouration, can potentially be used as guides to identify plants containing potentially medicinally-active compounds. In this study, three aposematic insects, Platyphora ligata (Stal.), Chlosyne hippodrome (Geyer), and Dysschema magdala (Boisduval), which feed on the medicinally-active plant Tithonia diversifolia (Remsl.) A. Gray, were analyzed to determine whether they sequester the plant's biologicaIly-active compound(s), presumably to use for their own defense against predators and/or parasitoids. In the ICBG bioassays, extracts from T diversifolia leaves were found to have anti-plasmodial (anti-malarial), and anti-trypanosomal (anti-Chagas') properties and activity against three cancer ceIllines. Sesquiterpene lactones found in T diversifolia are the likely candidates responsible for this activity. Anti-trypanosomal activity was used for performing bioassay-guided fractionation. Extracts of aIl insects, insect-related substances, and plant fractions were analyzed using Righ Performance Liquid Chromatography (RPLC), using the same solvent and flow conditions. No evidence was found that any of the three insect species tested sequestered or excreted the plant-derived biologicaIly-active compound(s). Studies have shown that sorne ofthese medicinaIly-active compounds act as feeding deterrents to generalist insect species (Dutta et al. 1986, Dutta et al. 1993). Rence, it appears that the three insects have developed the ability to overcome this aspect of feeding deterrency; however, they do not appear to actively make use ofthese biologicaIly-active compounds for their own defense. These aposematic insects may be sequestering other compounds for defense; however, further research is required. 65

4.1. INTRODUCTION

For defense against insect herbivores, plants have evolved physical and chemical defenses. In the "co-evolutionary" arms race, insect species may evolve the ability to cope with the se defenses. In particular, insect herbivores may deal with potent plant secondary metabolites by metabolism and/or excretion or by sequestration of the compound(s), either directly or modified (Szentesi and Wink 1991, Schaffner et al. 1994, Fordyce 2000, Nishida 2002). Aposematic insects are believed to often use plant defensive compounds by sequestering them and utilizing them for their own protection. Plant secondary metabolites that have been shown to be sequestered by insects (Nishida 2002), include: flavonoids (Wiesen et al. 1994), aristolochic acids (Fordyce 2000), quinolizidine alkaloids (MontlIor et al. 1990, Szentesi and Wink 1991), veratrum alkaloids (Schaffner et al. 1994), tropane alkaloids (Blum et al. 1981), pyrrolizidine alkaloids (von Nickisch-Rosenegk and Wink 1993), iridoid glycosides (Boros et al. 1991, Bowers and Collinge 1992), cardenolides (Brower and Glazier 1975), and numerous other alkaloids. The medicinal plant, Tithonia diversifolia, showed biological activity against Chagas' disease, malaria, and three cancer cell lines in the Panamanian International Cooperative Biodiversity Group (ICBG) bioassays. This study was conducted to determine whether insects feeding on this plant have the ability to sequester these potent biologically-active compounds.

ICBG Bioassays

Malaria accounts for approximately 3% of the worldwide disease burden measured by disability-adjusted life years (Sachs 2002), with an estimated 300-500 million clinical cases and over 1 million deaths each year (Ziemons et al. 2004). The causative agents are four species of parasites of the genus Plasmodium: P. malariae, P. vivax, P. ovale, and P. falciparum, with the final species being the most prevalent and dangerous (Ziemons et al. 2004). Recently, there has been the emergence ofmulti-drug resistant strains of P. falciparum, making the need to find new anti-malarial drugs increasingly urgent (Sachs 2002, Andrade-Neto et al. 2003, Ziemons et al. 2004). However, sadly, recent estimates show that drug and vaccine research on malaria is less than $100 million of the $70 billion spent on annual worldwide biomedical research and development (Sachs 2002). 66

Chagas' disease, caused by the protozoan Trypanosoma cruzi, affects between 16 and 18 million people annually in tropical and sub-tropical America, killing approximately lOto 20% ofthe individuals it infects (Alves et al. 1995, Gelb and HoI2002). For Chagas' disease, as well as most other tropical protozoan diseases, few, if any, safe efficacious drugs are available. In addition, no new classes of anti-parasitic drugs have been developed since the 1960, and few are in the process of development (Capson 2005). Furthermore, of the 1223 new drugs that entered the market between 1975 and 1996 only Il were directed at parasites (Capson 2005). Therefore, the need for new anti-parasitic drugs is enormous. This study, and others (Goffin et al. 2002, Gu et al. 2002, Goffin et al. 2003) have found that leaf extracts of Tithonia diversifolia have potent activity against various cancer celllines (colon (CoI2), breast (MCF-7), lung (H-460), and central nervous system (SF-268)), as well as the tropical diseases malaria and American trypanosomiasis (Chagas' disease).

Tithonia diversifolia

The genus Tithonia (Asteraceae, tribe Heliantheae) is comprised of Il species and 13 taxa, all of which have native ranges from the southwestern United States to Panama (Pereira et al. 1997). Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae), commonly known as the Mexican sunflower is a 2-5 m tall, perennial shrub native to Central America which has become naturalized throughout the tropics (Gu et al. 2002, Goffin et al. 2003). T. diversifolia has several synonyms including Helianthus quinque!obus Sesse & Moc., Mirasolia diversifolia (Hemsl.), Tithonia diversifolia var. g!abriuscu!a (S. F. Blake), Tithonia triloba (Sch. Bip. ex Klatt), Urbaniso! tagetifo!ius fo. grandiflorus (Kuntze), Urbaniso! tagetifolius subfo.flavus (Kuntze), and Urbaniso! tagetifolius var. diversifolius (Kuntze) (Missouri Botanical Gardens 2005). T. diversifolia has been used extensively in traditional medicines, with its extracts being used to treat diarrhea, fever, hematomas, hepatitis, hepatomas, malaria, wounds, and bruises (Ruengeler et al. 1998, Gu et al. 2002, Roth and Lindorf 2002, Goffin et al. 2003). In the bioassays of the ICBG, T. diversifolia extracts were active against malaria, and Chagas' disease, as well as cancers of the lung, breast, and the central nervous system. Several studies (Goffin et al. 2002, Goffin et al. 2003) have illustrated the anti-malarial properties of T. diversifolia, which is primarily because of the presence ofthe sesquiterpene 67 lactone, tagitinin C (Ziemons et al. 2004). T diversifolia also has anti-cancer properties, again the result of sesquiterpene lactones present in the plant (Goffin et al. 2002, Gu et al. 2002). Furthermore, several of its sesquiterpene lactones are also known to have anti-inflammatory and anti-bacterial properties (Ruengeler et al. 1998). T diversifolia also has insect feeding detterent properties (Dutta et al. 1986, Dutta et al. 1993). Compounds from this plant, tagitinin A, and C, and hispidulin, were found to be active against several insect species, in a dose dependent manner, inc1uding 4th instar caterpillars of Eri-silkworm, Philosamia ricini (Lepidoptera: Saturnidae) (Dutta et al. 1986), Diacrisia oblique (Lepidoptera: Arctiidae), Phissama transiens (Lepidoptera: Arctiidae), Trabala vishnu (Lepidoptera: Lasiocampidae) and grubs of Epilachna vigintioctopuncata (Coleoptera: Coccinellidae) (Dutta et al. 1993). Interestingly, tagitinin C was also found to be active against malaria and cancer (Goffin et al. 2002, Goffin et al. 2003).

Insects found Feeding on Tithonia diversifolia

Five insect species were found to feed on Tithonia diversifolia, however, only three of these were examined in this study. Due to an insufficient sample size, the two species that were not analyzed were; (1) a non-aposematic, smaIl, black and white leafroller in the family Gelechiidae, and (2) a medium-sized, aposematic, orange and black caterpillar with thick black spines in the family Nymphalidae, subfamily Melitaeinae. For the other three insect species, Platyphora ligata (Stal.), Chlosyne hippodrome (Geyer) (Nymphalidae), and Dysschema magdala (Boisduval) (Arctiidae) sufficient insect material was collected to perform chemical analyses. The genus Platyphora is common in Central and South America. Platyphora ligata is a relatively large Chrysomelid (leaf-beetle) beetle, which has yeIlow and dark brown longitudinal strips. This species has been found to feed on numerous plant species in the Asteraceae family, inc1uding Mikania guaco (H. & B.), M micrantha (H. B. K.), Ayapana elata (Steetz) (King & H. Robinson), and Neurolaena lobata (L.) (R. Br.) (Plasman et al. 2000), as weIl as T diversifolia from this study. As with many other species in the genus Platyphora, P. ligata has defensive glands. During a disturbance, a secretion is emitted from the gland pores which then accumulates in the marginal grooves of the elytra and pronotum (Plasman et al. 2000). In a study by Plasman et al. (2000) two triterpene saponins, as weIl as a 68 mixture of phosphatidylcholines and chlorogenic acid were found in the defensive secretions; however, these compounds are not directly sequestered from their food plants. Chlosyne hippodrome is found from Mexico to Columbia (DeVries 1987). These medium-sized larvae have a black body, with circular orange markings, and are densely covered with black spines. In addition, this species is known to be semi-gregarious. DeVries (1987) identifies Melanthera aspera (Jacq.) (Asteraceae) as the larval hostplant of C. hippodrome. The adults have a round wing shape. The uppersides of the wings are black with a white forewing (FW) band and hindwing (HW) margin. The undersides are also black with a yellow HW margin and red spots on the HW medial area. Typically, C. hippodrome has been found to occur from sea level up to 1,000 m elevation on the Pacific side, although it is likely also present on the Atlantic side. In addition, C. hippodrome is characteristically found in are as of disturbed deciduous forest and open pastures. In Guanacaste, Costa Rica, this species is found to occur in seasonal populations beginning at the start of the rainy season. In contrast, it is rarely found during the dry season. The adults are often found flying with other Chlosyne species; however, C. hippodrome is considered a somewhat more agile and faster flier (De Vries 1987). To the best of our knowledge, no information has been reported on defensive compounds present in C. hippodrome. Dysschema magdala has been recorded in Central America (Watson and Goodger 1986). These medium- to large-sized larvae have horizontal orange and dark grey strips and are densely covered with fine white and black hairs. These larvae also feed in a semi gregarious fashion. The adult's upperside and underside of the FW is pattemed brown and dark brown; whereas the upperside and underside of the HW is bright orange. The upperside of the thorax is also bright orange, while the underside is a dull brown. D. magdala has been recorded to feed on species in the plant families Araliaceae, Asteraceae, Papaveraceae, Tiliaceae, and Urticacaeae (Walsh 2004, Janzen and Hallwachs 2005). Again, to the best of our knowledge, no information has been reported on defensive compounds present in D. magdala. 69

4.2. METHODS

Insect Material

The insects Platyphora ligata, Chlosyne hippodrome, and Dysschema magdala were all manually collected from Tithonia diversifolia found in Chagres National Park (Cerro Azul), at the GPS coordinates 9° 12' 3" N; 79 ° 23' 31" W and elevation of approximately 700 m. Platyphora ligata was collected periodically between May and November 2004 (wet season), while Chlosyne hippodrome, and Dysschema magdala were both collected in November 2004 (wet season). Insect identifications were made using the specimens in the Insect Collection Room at the Smithsonian Tropical Research Institute (STRI) and were confirmed by the STRI Chrysomelid beetle specialist, Dr. Donald Windsor, and the STRI Lepidopteran specialist, Dr. Annette Aiello. A recollection permit (SC/AP-I-0S) was obtained from the Panamanian National Authority of the Environment (ANAM) for the collection of insect species. Voucher specimens of the insect species collected have been placed in the Fairchild Museum, University of Panama and the Synoptic Insect Collection, STRI.

Insect Rearing

Insects were kept in plastic containers that had their bottoms covered with paper towel. Insects were provided with fresh T diversifolia leaves and their containers were cleaned when necessary. Humidity was provided by the plant foliage, as weIl as by added water droplets when required. No holes were put in the containers, to prevent moisture lost. Insects were reared to the desired lifestage, and were starved for 48 hours to ensure that their guts were voided of plant contents that could confound chemical analysis. They were then killed by deep freezing at -80°C. Observations were also made to ensure that frass was no longer being produced before deep freezing. Insects were then kept frozen at -80°C until they were needed for chemical analysis. During rearing, frass and shed skins were also collected and stored at -80°C.

Plant Material

In November 2004, Tithonia diversifolia leaves were collected in the areas where the insect collections were made (Chagres National Park; Cerro Azul). Approximately 100 grams ofboth young leaves and mature leaves were collected. Leaves were collected that had as 70 little damage from herbivores, fungus, and bacteria as possible. The identity of T diversifolia was confirmed by Rafael Aizprua ofthe Smithsonian Tropical Research Institute (STRI). Recollection permits (SCIP-17-03, SCIP-22-03, SC/P-23-03, SCIP-24-03, SC/P-25-03, SCIP- 26-03, SCIP-27-03, SCIP-28-03) were obtained from ANAM for the collection of plant species. Voucher specimens (2855-RAJ) of T diversifolia have been placed in the Herbariums at both the University of Panama and STRI.

Plant Extractions

Plant leaves were first wiped with paper towel to get rid of extraneous surface material, and then the totalleaf material was weighed. Leaves were cut into small pieces and blended for 10-15 seconds with 100% methanol (MeOH) in a Waring blender. The leaf-MeOH solution was then further mixed in a Polytron (PT 3100 Polytron®, Dispersing and Mixing Technology by Kinematica) for 1 to 2 minutes, depending on the size of the sample. Next, the leaf-MeOH solution was filtered using Whatman paper #4, followed by Whatman paper #1, each time rinsing with ethyl acetate (EtAOc). The MeOH-EtAOc leaf extract was then evaporated in the Rotovap (Büchi Rotavapor R-200 attached to Büchi Heating Bath B-490), followed by further evaporation in the SpeedVac (SpeedVac® SC210A, Savant), connected to Welch 1402 Duo Seel Vacuum Pump (Thomas Industries Inc.) also connected to Refrigerated Vapor Trap RVTI00, Savant) and in the Freeze Dryer (LABCONCO). Subsequently, the dry mass of the extract was recorded and stored at -80°C or -20°C to prevent compound degradation.

Insect Extractions

Before extraction, total insect material or insect excrement was weighed at room temperature. Insect material was then crushed in MeOH using a mortar and pestle, and was then blended in MeOH using a Waring Blender for 15-20 seconds. Subsequently, the volume of Me OH was increased to 50 mL and 50 mL of EtOAc was added. The extract was then filtered using Whatman paper #4 and Whatman paper # 1, each time rinsing the sample with EtOAc. The extract was then evaporated in the Rotavap, Speed Vac, and Freeze Dryer. Finally, the dry mass of the extract was recorded and stored at -80°C and -20°C to prevent compound degradation. 71

Chemical Analysis

Bioassay-guided fractionation was used to determine whether the insects were sequestering the active plant compounds. EssentiaIly, bioassay-guided fractionation involves the fractionation of an active crude plant extract, followed by testing in a bioassay(s), and then further fractionation and bioassay testing, until the active compound(s) has been isolated. The crude plant extracts ofboth the young (B4327) and mature leaves (B4328) were fractionated into nine fractions using Vacuum Liquid Column Chromatography (VLC). Fractionation was done by using 150mL of the following combinations ofsolvents, resulting in the following fractions: (a) 100% Hx; (b) 90% Hx, 10% AcOEt; (c) 80% Hx,20% AcOEt; (d) 70% Hx, 30% AcOEt; (e) 60% Hx, 40% AcOEt; (t) 40% Hx, 60% AcOEt; (g) 20% Hx, 80% AcOEt; (h) 100% AcOEt; and (i) 100% MeOH. The American trypanomiasis (Chagas' disease) bioassay was used to guide the bioassay-guided fractionation process, because the activity of T diversifolia against Chagas' disease had not been reported previously. Bioassay results revealed that fraction B4327-Vl-f had the highest anti- trypanosomal activity. Subsequently, fraction B4327-Vl-fwas separated using semi-preparative HPLC. For this, 8 mg of extract, dissolved in MeOH and MeCN and filtered using a Gelman Glass Acrodisc ®, were injected into a 200 /lL 100p. In each case, 50 vials of 1.3 mL of solvent were collected from the 60 minute run, at a rate of 1 mL/min. The same solvent and flow conditions were used as for the analytical runs, which are stated below. The 50 fractions collected from the HPLC that contained extract were then combined into six fractions to send to the bioassays: (i) 10-14; (ii) 15-16; (iii) 17-19; (iv) 20-23; (v) 24- 30; (vi) 30-40. The crude plant extracts, insect extracts, insect excrement extracts, and aIl active plant fractions were analyzed using the same analytical HPLC flow and solvent conditions, to allow for comparisons. A Waters Delta 600 system (Milford, Massachusetts) equipped with a Waters 2996 photodiode array detector (Milford, Massachusetts) (210 - 800 nm) was the HPLC system used. Extracts were fractionated using a Nova-Pak RP-C18 column (Waters) (4.6 x 250 mm, 4 /lm pore size). For each analytical run, 0.6 mg of extract, dissolved in Me OH and filtered using Gelman Glass Acrodisc ® filters, were injected into a 20 /lL loop. Samples were run using three solvents MeCN (A%), Me OH (B%), and H20 (C%), aIl HPLC 72 grade. The following solvent and flow conditions were utilized: 0-20 min, gradient flow, 10%:90% MeCN:H20 to 100% MeCN; 20-30 min, isocratic flow, 100% MeCN; 30-35 min, gradient flow, 100% MeCN to 100% MeOH; and 35-50 min, isocratic flow, 100% MeOH. Each run was 50 minutes long at a flow rate of 1 mL/min.

Bioassays

Crude extracts, VLC fractions, and further fractions were sent to the ICBO bioassays to test for activity against Chagas' disease, malaria, and cancer. The bioassay testing for activity against Chagas' disease utilizes a recombinant strain of Trypanosoma cruzi, and targets the intracellular amastigote stage. The malaria bioassay uses both chloroquine-sensitive (Sierra Leone clone D6 and Tanzania F32) strains and a chloroquine-resistant (lndochina clone W2) stain of Plasmodiumfalciparum. For the cancer bioassay, three cancer lines are used, including breast tissue (MCF-7), lung (H-460), and central nervous system (SF-268). Refer to Mendoza et al. (2003), Corbett et al. (2004), and Monks et al. (1997) for detailed methods on the Chagas', malaria, and cancer bioassays, respectively. AIl bioassays were done by technicians in the laboratories of Eduardo Ortega and Luz Romero at INDICASAT (Institute of Advanced Scientific Research and High Tech Services).

4.3. RESUL TS

Tithonia diversifolia was found to have activity against cancer, malaria, and Chagas' disease. The crude extracts ofboth young and mature leaves showed activity against the cancer ceIlline SF-268 with %0 (percentage growth) of 7.4 and 14.6, respectively. However, the fractions showed even greater activity and activity in aIl three of the cancer bioassays. For the young leaves the fraction B4327-Vl-fwas the most active with %0 of -49.6 for MCF-7, -21.8 for SF-268, and -50.1 for NCI-H460. Surrounding fractions e and g also showed sorne activity. The mature leaf fractions showed greater activity than the younger leaf fractions. In this case, fraction B4328-Vl-e showed the greatest activity, with %0 of -53.9 for MCF-7, -32.2 for SF-268, and -62.8 for NCI-H460. Fraction B4328-Vl-f also showed a high degree of activity with %G of -48.1 for MCF-7, -32.9 for SF-268, and -51.8 for NCI-H460. Again, the surrounding fractions d and g showed sorne degree of activity. 73

The crude extracts of both young and mature T diversifolia leaves showed activity against malaria having ICso of 17 and 32 ~g/mL, respectively. In this case, the VLC fractions ofthe young and mature leaves showed very similar activities, as both B4327-Vl-f and

B4328-Vl-fhad ICso of 4 ~g/mL. Again the fractions d, e, and g for both the young and mature leaves showed relatively high levels of activity. For Chagas' disease, the crude extracts ofthe young (B4327) and mature (B4328) leaves were either non-active or moderately active in the bioassays with 1C so of >50 and 41.9

~g/mL, respectively. The most active fraction from the VLC fractions was the young leaf fraction B4327-Vl-fwith an ICso of 4.24 ~g/mL. Fraction B4328-V1-fwas also the most active fraction from the mature leaf extract at 7.23 ~g/mL. Fractions d, e and g, which surrounded the most active fractions, had moderate active for both young and mature leaves. Fraction B4327-Vl-fwas subfractioned generating six subfractions. Subfractions B4327-V1- f-i and B4327-Vl-f-ii had the highest activities with ICso of 1.33 and 1.32 ~g/mL, respectively. Fraction B4327-V1-f-iii had an ICso of 5.3 ~g/mL, white the other three fractions had ICso of> 10 ~g/mL. Because the same solvent and flow conditions were used for the HPLC runs of the active plant fractions, as weIl as for the extracts made from insects and insect excrement, comparisons can be made by using retention times and absorption data for each of the peaks. The chromatograms of the crude extracts ofboth the young and mature leaves at 210.50 and 254.00 nm have relatively similar profiles (Figure 4.1). However, the wavelength 254.00 nm gave the c1earest peak of the active compounds. The main peak found in both of the active fractions B4327-V1-fand B4327-Vl-f-i have retenti on times (min) and absorbance values (nm) of 16.19 (249.3) and 16.48 (249.3), respectively. This is believed to be the peak containing the active compound(s). By using both the retention times and absorbance values to identify compounds, it appears that the Platyphora ligata adult is not sequestrating any of the compounds found in the most active plant fractions derived from VLC and semi-preparative HPLC (Figure 4.2). Excrement was not analysed for P. ligata. Again, by using both the retention times and absorbance values it appears that neither the last instar nor the pre-pupation stage of Chlosyne hippodrome are sequestering or 74

Figure 4.1. HPLC ehromatograms of the erude extraets ofboth young and mature leaves from Tithonia diversifolia (Asteraceae) comparing the results from the wavelengths 210.50 nm and 254.00 nm, as well as HPLC chromatograms of the active fractions from T diversifolia at wavelength 254.00 nm. 75

0.30- Crude extract of young leaves (B4327) at 210.50 nm

Mnutes

1.50

1.00 Crude extract of mature leaves 0.50j (B4328) at oooL~-~, . 210.50 nm 10.00 20.00 40~OO 50:00 Mnutes

0.08

0.06 Crude extract of ~ 0.04 young leaves 0.02 (B4327) at 254.00 nm

Mnutes

0.10

0.08 Crude extract of mature leaves (B4328) at 254.00 nm

Mnutes

~------:;2D-J o. :.i! o. Fraction o. B4327-Vl-f at 02~ 254.00 nm 0001:::-.:;:::-:::;::::::;:=:::::::;=;:::~::;=::;:::::;:=;::::;::~~~,=::=:;=:::;: 10~OO 20:00 30~OO 40.00 Mnutes

------

0.400.30- l Fraction ~ 0.20- B4327-Vl-f-i at 0.10- L 254.00 nm o.oo-t+-;::::·-=-;::::-:;:::-=;:=:;=:;:::::::=.~~:::=:::~~=.=~;;;;~=~ 10.00 20.00 30.00 40.00 50.00 Mnutes

AU = Absorbance Units 76

Figure 4.2. HPLC results at wavelength 254.00 nm eomparing retention times of the erude young leafextract, and anti-Chagas' fractions of the plant Tithonia diversifo/ia (Asteraceae), and the extraet of Platyphora ligata (Coleoptera: Chrysomelidae), an insect whieh feeds on the plant. 77

Crude extract of i ore: \ ~ ~ young leaves < o~l \J~ · (B4327)

:~ "j,~~~d10,00 20,00 30,00 40,00 50,00 Mnutes

1.2{}

1.00 0,80- Fraction ~ 0.60- B4327-Vl-f OA{}

0.2{}

O. 10.00 3O~OO Minutes 78 excreting any of the compounds found in the most active plant fractions derived from VLC and preparative HPLC (Figure 4.3). By using both the retention times and absorbance values it appears that Dysschema magdala larvae are also not sequestering or excreting any of the compounds found in the most active plant fractions derived from VLC and semi-preparative HPLC (Figure 4.4). In general, aIl three of the different insects feeding on T diversifolia do have sorne degree of similarity in their chemical composition, with aIl having peaks with retenti on times (min) and absorbance values (nm) at 3.04 (471.6) and again at 30.3 (274.2) (Figures 4.2, 4.3, and 4.4). However, it does appear that the two Lepidopteran larval species have a greater degree of similarity than the beetle, P. ligata, with the Lepidopteran larval species. The excrement of the two Lepidopteran larval species again have sorne degree ofsimilarity, with an interesting peak occurring at 39.85 (268.2) for D. magdala and 39.75 (268.2) for C. hippodrome, which corresponds to peaks in both the crude T diversifolia young and mature leafchromatograms at 39.12 (269.4) and 29.17 (269.4), respectively (Figures 4.1, 4.3, and 4.4). Using the wavelength 210.50 nm a greater variety ofpeaks can be seen for aIl ofthe insect fractions. In terms of T diversifolia, the chemistry of the young leaves appears to be more complex than that of the older leaves; however, both appear to have similar levels of the active plant compound (Figure 4.1).

4.4. DISCUSSION

Anti-Cancer Activity of Tithonia diversifolia

Tithonia diversifolia was found to have moderate activity against the three cancer cell lines tested in this study. Previous studies have also shown T diversifolia to be active against cancer. For example, Goffin et al. (2002) found that tagitinin C also possesses cytotoxic properties (ICso on HTC-166 cells: 0.706 Ilg/mL). Another study by Gu et al. (2002) using an anti-proliferation bioassay performed with human colon cancer (CoI2) cells found that several sesquiterpenoids from T diversifolia had anti-proliferatic and inhibitory properties. This study provides evidence that T diversifolia contains compounds that are active against the cancer ceIllines MCF-7 (breast), SF-268 (central nervous system), and NCI-H460 (lung). 79

Figure 4.3. HPLC results at wavelength 254.00 nm eomparing retention times of the erude young leafextract and anti-Chagas' fractions of the plant Tithonia diversifolia (Asteraceae), and the insect and excrement extracts of Chlosyne hippodrome (Lepidoptera: Nymphalidae), an inseet which feeds on the plant. 80

Crude extract of young leaves (B4327)

10.00 20.00 30.00 40.00 50.00 Mnutes

1.2~ 1.00- Fraction 0.80' B4327-VI-f il 0.60- 0.40,

0.20,

0.00--- 10.00 20:00 30:00 40:00 r..tnutes

0.40, Fraction 0.3Ir B4327-Vl-f-i il 0.20,

0.10, o.oo-~~---~~~~~'~~~~~~ 10.00 20.00 30~OO 40.00 50.00 Mnutes

0.12 0.10, Chlosyne hippodrome il ::1i 004 (Last instar) ::U~. ~~, 1 10.00 20.00 30.00'.'~ 40.00 50.00 Mnutes

Chlosyne hippodrome (Pre-pupation stage)

Chlosyne hippodrome (Excrement)

Moutes

AU = Absorbance Units 81

Figure 4.4. HPLC results at wavelength 254.00 nm comparing retenti on times of the crude young leafextract and anti-Chagas' fractions of the plant Tithonia diversifolia (Asteraceae), and the insect and excrement extracts of Dysschema magdala (Lepidoptera: Arctiidae), an insect which feeds on the plant. 82

0.08 Crude extract of 0.06 VI young leaves (B4327)

'::L, \_lA,"'-_~,,_j-d• l 'i" i ' 'i ' 1 10.00 20.00 30.00 40.00 50.00 Mnutes

1.00 Fraction 0.801.2~ : B4327-Vl-f ~ 0.6 :l~~~J---:=~~=:::::::;=;:=~~d 10.00 20:00 ~ 30:00 40'00 Mnutes

0.4e} Fraction 0.3e} B4327-Vl-f-i ~ 0.2e}

0.1e}

10.00 20.00 30.00 40.00 50.00 Mnutes

Dysschema

010 magdala ~ l (Last and second 005 , last instars) OOOt--) ~~

Moules

0.20 Dysschema 0.15- magdala ~ 0.1e} (Excrement) 0.05

0.0e} V , 10~OO 20.00 30~OO 40.00 Mnutes

AU = Absorbance Units 83

Anti-Plasmodial Activity of Tithonia diversifolia

In this study both the young and mature leaf extracts of T diversifolia were found to have significant activity against malaria. Several studies (Goffin et al. 2002, Goffin et al. 2003, Ziemons et al. 2004) have also illustrated the anti-plasmodial properties of the aerial parts of T diversifolia (ICso on FCA strain: 0.75 llg/mL). Through bioassay-guided fractionation tagitinin C, a sesquiterpene lactone, has been identified as the compound providing these anti-plasmodial properties (ICso on FCA strain: 0.33 llg/mL) (Ziemons et al. 2004). In turn, tagitinin Chas been identified as one of the major compounds of T diversifolia and has been found to have an unsaturated ketone group, a structure which is not often found in plants (Ziemons et al. 2004). Interestingly, tagitinin C concentrations have been found to differ depending on the plant part tested, the solvent used for extraction, and the collection location (Baruah et al. 1979, Schuster et al. 1992, Bordoloi et al. 1996, Pereira et al. 1997, Kuo and Chen 1998, Goffin et al. 2003). This study provides evidence that anti plasmodial activity is present in Central American, specifically Panamanian, T diversifolia plants.

Anti-Trypanosomal Activity of Tithonia diversifolia

Both the young and mature leaf extracts of T diversifolia showed anti-Chagas' disease activity, with this anti-trypanosomal activity becoming greater as the extract was fractionated and the fractions became purer. This activity may be due to one or more sesquiterpene lactones because the VLC fraction containing anti-Chagas activity corresponds to the fractions containing anti-malarial and anti-cancer activity. In previous studies it has been found that different sesquiterpene lactones provide both the anti-malarial (Goffin et al. 2002, Goffin et al. 2003, Ziemons et al. 2004) and anti-cancer activity (Goffin et al. 2002, Gu et al. 2002). This is the first report of Tithonia diversifolia having anti-trypanosomal activity.

Anti-feedant Activity of Tithonia diversifolia

T diversifolia also contains compounds that are feeding deterrents to insect pests (Dutta et al. 1986, Dutta et al. 1993). The compounds tagitinin A, and C, and hispidulin were found to be potent feeding deterrents, exhibiting dose dependent relationships, against 4th instar caterpillars ofthe Eri-silkwonn, Philosamia ricini (Dutta et al. 1986), Diacrisia oblique, 84

Phissama transiens, Trabala Vishnu, and grubs of Epilachna vigintioctopuncata (Dutta et al. 1993). Fresh leaf extracts were also found to provide higher levels of feeding deterrency, than dried leaf extracts or flower head extractions, and stem extracts did not deter feeding (Dutta et al. 1993). Since T diversifolia is known to contain feeding deterrents, it would suggest that insects able to live on this plant would have to develop special adaptations to be able to feed successfully. Interestingly, tagitinin C, one of the compounds acting as a feeding deterrent, is also active against malaria and cancer (Goffin et al. 2003, Goffin et al. 2002, Ziemons et al. 2004). Amason et al. (2004) also found that the limonoid gedunin, from the common tropical cedar tree of the Americas Cedrela odorata (Meliaceae), has anti-feedant properties, which significantly reduced the growth and delayed the development of Lepidopteran larvae, as well as has potent anti-malarial properties. AIso, the tree Lansium domesticum (Meliaceae), from Bomeo, has nine triterpenes called lansiolides in its bark, which again have both insect anti feedant activity and anti-malaria activity (Amason et al. 2004). Hence, it appears that medicinally-active compounds can have a negative effect on non-adapted insect species. No doubt, insect species feeding on such plants will have to evolve mechanisms to overcome these feeding deterrents. This study examined whether these insects were able to sequester these active plant compounds, presumably for their own defense.

The Fate of Biologically-active Compounds in Tithonia diversifolia that are Ingested by Insect Species

In particular, insect herbivores may deal with potent plant secondary metabolites by metabolism and/or excretion or by sequestration of the compound(s), either directly or modified (Szentesi and Wink 1991, Schaffner et al. 1994, Plasman et al. 2000, Nishida 2002). Interestingly, sorne insect species have the ability to selectively accumulate one or more of the derivatives of a secondary compound type (e.g. flavonoids, quinolizidine alkaloids, iridoid glycosides, cardenolides), while metabolizing and/or excreting other derivatives (Brower and Glazier 1975, Szentesi and Wink 1991, Bowers and Collinge 1992, Wiesen et al. 1994, Geuder et al. 1997). AIso, numerous metabolic pathways have been found where insects alter a plant secondary compound and then sequester its metabolite, for example the glucosylation of flavonoids (Wiesen et al. 1994, Geuder et al. 1997), the acetylation of veratrum alkaloids (Schaffner et al. 1994), the biotransformation of pyrrolizidine alkaloid 85 bases into pyrrolizidine alkaloid N-oxides (von Nickisch-Rosenegk and Wink 1993) and the biotransforrnation of pyrrolizidine alkaloids (epimerization and esterificantion) to retronecine esters (Hartmann et al. 2003, Pasteels et al. 2003). Aposematic insects are known to specialize on biologically-active host plants (Reichstein et al. 1968, Montllor et al. 1990, Boros et al. 1991, Bowers and Collinge 1992, Fordyce 2000), and may use potent plant secondary compounds for defense against predators (Bemays and Graham 1988, von Nickisch-Rosenegk and Wink 1993, Schaffner et al. 1994, Nishida 2002, Despland and Simpson 2005). However, in this study, even though aposematic insects were feeding on a biologically-active host plant, they were not concentrating the intact medicinally-active secondary compounds (Figures 4.2,4.3, and 4.4). In addition, it does not appear that the insects were excreting the intact secondary compounds (Figures 4.3, and 4.4). Therefore, there must be mechanisms that these insect species use to detoxify and metabolize these cytotoxic plant products. The most plausible explanation is that the insects have the ability to degrade and subsequently excrete these metabolized compounds. One example of insects having the ability to degrade secondary plant compounds cornes from the plant Veratrum album (Liliales: Melanthiaceae), which contains a complex ofbiologically-active ceveratrum alkaloids, and its associated insect species, the specialist sawfly Rhadinoceraea nodicornis (Hymenoptera, Tenthredinidae) and a generalist sawfly Aglaostigma sp. It is known that the specialist R. nodicornis sequesters several of the ceveratrum alkaloids for defense, but does not sequester the most active alkaloids, protoveratrine A and B, which are degraded. It is speculated that protoveratrine A and B exceed the tolerance limit of R. nodicornis larvae. Furtherrnore, it appears that the generalist Aglaostigma sp. de grades all of the ceveratrum alkaloids that it ingests (Schaffner et al. 1994). Another example of secondary compound degradation cornes from the geometrid, Meris paradoxa, which feeds on Maurandya antirrhiniflora (Scrophulariaceae) a plant which contains iridoid glycosides. The aposematic larvae are found to have only around half the concentration of iridoid glycosides that are found in the plant, indicating that a proportion of the iridoids are being metabolized (Boros et al. 1991). Another interesting example cornes from the genus Platyphora, which have pyrrolizidine alkaloid adapted species that are known to metabolize and sequester these compounds for their defensive secretions (Pasteels et al. 2003). P. ligata, which is closely related to these pyrrolizidine alkaloid adapted species, can feed on host plants with 86 pyrrolizidine alkaloids but do es not sequester or metabolize them for defense. In contrast, the species P. kollari, which is phylogenetically apart from the pyrrolizidine alkaloid adapted species, refuses to feed on leaves containing pyrrolizidine alkaloids (Pasteels et al. 2003). Therefore, the insects in this study could be at an intermediate stage where they can feed on a plant with active compounds, but do not yet have the ability to utilize these compounds for their own defensive purposes. There is also the possibility that the sesquiterpene lactones are so toxic that the insects can only metabolize them. In addition, the insects could be metabolizing the active plant compounds into related derivatives (less or more toxic) and sequestering those. Finally, there also could be the possibility that they are concentrating sorne other compounds for defensive purposes that were not active in the bioassays tested in this study.

The Insect Species associated with Tithonia diversifolia

The aposematic Platyphora ligata belongs to the Chrysomelid or leaf-beetle family, which contains approximately 25,000 species (Plasman et al. 2000). Their defensive mechanisms range from crypsis, to mechanical devices such as spines or sudden escape, to chemical defensives in many of the brightly coloured, aposematic species (Plasman et al. 2000). The genus Platyphora, which has narrowly oligophagous species, has only recently been getting attention by the scientific community (Plasman et al. 2000, Pasteels et al. 2001). As with many other species in the Platyphora genus, P. ligata has defensive glands, which after a disturbance pro duce a secretion from the gland pores that accumulates in the marginal grooves of the elytra and pronotum (Plasman et al. 2000). Platyphora species are known to have a very efficient transfer of defensive compounds into their defensive glands and secretions with only small amounts of defensive compounds being found in the hemolymph and other body parts (Pasteels et al. 2003). Because an insect has defensive glands and secretions, does not exclude it from being useful in the drug discovery process. However, if the defensive compounds are sequestered directly or derived from the host plant this would make the insect a more useful subject, which was not the case with P. ligata. For instance, Plasman et al. (2000) found two new oleanane triterpene saponins named ligatosides A and B, as well as a mixture ofphosphatidylcholines and chlorogenic acid in P. ligata's defensive secretions. Plasman et al. (2000) also discovered that these compounds were not simply being 87 sequestered by the insect from its host plant Mikania micrantha. First, the se compounds were absent in M micrantha, and second, the TLC patterns of P. ligata secretions that fed on different host plants were identical to those from M micrantha. The biological activities of these defensive compounds (that are stored in defensive glands) against predators are still unknown (Plasman et al. 2000), thus require further study. Therefore, in the case of P. ligata it appears that the insects are neither directly sequestering active plant compounds in their body compartments (HPLC results ofthis study) (Figure 4.2) or in their defensive secretions in their defensive glands. Finally, this study appears to be one of the tirst documenting the ability of P. ligata to feed on T diversifolia. The genera Chlosyne is known to have larvae that are typically gregarious feeders, are dull-coloured, and have short spines on their body (DeVries 1987). Chlosyne species are generally considered to be a palatable group ofbutterflies; however, host plant chemistry and insect behaviour indicate that sorne species may be distasteful to birds (De Vries 1987). For instance, C. lacinia larvae feed on Helianthus (Asteraceae), which contain poisonous compounds, and the adults also tend to fly slowly (DeVries 1987), a trait which is known to signal prey unpalatability. Chlosyne hippodrome larvae are aposematically coloured, having a black body, with orange circular markings, and are densely covered with black spines. Both of these traits could be pro vi ding a waming signal to potential predators of prey distastefulness or toxicity. In addition, this species is known to be semi-gregarious (De Vries 1987) and in this study larvae were observed feeding side by side. Gregariousness again is a trait which is known to signal the unpalatability of an insect species (Brower 1984, Hatle and Whitman 2001). These traits would lead one to believe that perhaps C. hippodrome larvae are sequestering the active compounds from T diversifolia; however, the HPLC results illustrate that this may not be the case (Figure 4.3). Semi-aposematic C. hippodrome adults are often found flying with other Chlosyne species; however they are considered somewhat more agile and faster fliers (DeVries 1987), a trait typical of more palatable species. Again, this study appears to be the tirst documenting the ability of C. hippodrome to feed on T diversifolia. It is known the many Arctiidae larvae are able to sequester defensive compounds from their host plants in their hemolymph, as well as transfer these chemicals to the adult stage (Harvard University 2005, Watson and Goodger 1986). Furthermore, many ofthese Arctiidae moths are day-flying and many display aposematic colour patterns (Harvard University 2005, 88

Watson and Goodger 1986). In addition to this, Dysschema magdala larvae with their aposematic colouration (stripped dark grey and orange) and semi-gregarious feeding behaviour again display traits that indicate the possibility of this species sequestering the active compounds from T diversifolia, for their own defense. However, once again the HPLC chromatograms demonstrate that this may not be the case (Figure 4.4). This could be partly explained by the fact that it is also presumed that sorne Dysschema spp. are mimics of unpalatable butterflies ofvarious groups (Aiello and Brown Jr. 1988); however, whether or not this is the case for D. magdala is not known. Once again this appears to be the first record of D. magdala feeding on T diversifolia. Overall, it appears that P. ligata and C. hippodrome are specialist species, although not ultra specialists (feeding on one plant species), feeding on plants in the family Asteraceae, whereas D. magdala appears to be a generalist species, as it has been recorded to feed on five plant families. Thus, both specialists and a generalist have developed similar mechanisms to overcome the potential feeding deterrent effects of these active compounds.

4.5. CONCLUSION

The insects feeding on Tithonia diversifolia do not appear to be sequestering the biologically-active compounds isolated in this study, thus do not support this studies second hypothesis that aposematic insects will have the ability to concentrate a plant's medicinally active compounds. For an insect such as Platyphora ligata that is known to have the ability to synthesize its own defensive compounds this seems logical. However, for the two Lepidopteran larval species, Chlosyne hippodrome and Dysschema magdala it would appear advantageous for them to make use of the active compounds present in their host plant, if these compounds have properties that could defend them against their predators. Apparently these insects have developed mechanisms to overcome the feeding deterrent effects of these active compounds; however, they do not appear to have developed mechanisms to utilize them. The development of these mechanisms could potentially occur over evolutionary time. Nevertheless, this result helps to strengthen the notion that aposematic insects are good guides for finding medicinally-active plants, since the substances in question are highly active. Furthermore, the crude extracts were relatively inactive which means that had the plant been examined only using the activity of its crude sample and not pre-fractionated it 89 probably would not have been selected as active. The aposematic insects thus did indeed improve the odds of finding active compounds. Finally this study brings up a series of other questions. For example, could the insects be concentrating another compound active from a bioassay not tested in this study or simply a compound that is active against their predators? AIso, do the medicinally-active compounds isolated from this plant act as feeding deterrents to the invertebrate and vertebrate predators ofthese aposematic insects? Feeding experiments need to be done to assess the palatability of the insects feeding on Tithonia diversifolia to their predators, to help explain their aposematic colouration. 90

CONCLUSIONS

Overall Summary

The objective of this study was to determine whether aposematic insects can guide us to plants with potentially medicinally-active compounds. This is based on previous studies that have shown: Cl) plant defensive compounds may have medicinal properties (Dutta et al. 1986, Dutta et al. 1993, Goffin et al. 2002, Gu et al. 2002, Goffin et al. 2003, Amason et al. 2004); (2) these secondary plant compounds are sometimes sequestered by insects feeding on the plant and used in their own defense strategies; and (3) insects which are defended with secondary plant compounds often have aposematic colouration to wam predators of their noxious nature (Brower and Glazier 1975, Boros et al. 1991, Bowers and Collinge 1992, Fordyce 2000, Despland and Simpson 2005). After collecting Coleopteran and Lepidopteran herbivore populations feeding on ten plants with known biological activity in the Panamanian ICBG bioassays and ten plants without activity, it was found that plants with biological activity were significantly more likely to have associations with aposematic insect species, 2 than those plants that had not shown activity in the bioassays: chi-square test (df= 1, X = 8.167, p = 0.01); and t-test paired two sample for means (df= 9, t = 1.973, p = 0.08). AIso, plants which had biological activity had a four-fold greater chance ofbeing associated with aposematic insectes) than non-active plants. In contrast, there was no significant difference seen in the populations of non-aposematic insects between active and non-active plants. These results support the hypothesis that aposematic insects are more likely to have associations with active plants than non-active plants. A second study was conducted to determine whether the aposematic insects feeding on one ofthese previously studied plants, which had biological activity, had the ability to sequester the active compounds found in the plant. Three aposematic insect species, Platyphora ligata, Chlosyne hippodrome, and Dysschema magdala, which fed on the biologically-active plant, Tithonia diversifolia, were chemically analyzed to determine whether they sequestered the anti-Chagas', anti-malarial, and anti-cancer compound(s) found in T diversifolia. Using High Performance Liquid Chromatography (HPLC), there was no evidence that any of these three insect species concentrated or excreted the intact biologically-active compounds. Hence, this study does not support the studies second 91 hypothesis that aposematic insects will have the ability to concentrate a plant's medicinally active compounds. However, it is not known ifthe insects were able to further metabolize the biologically-active compounds from T. diversifolia to, potentially, generate more potent chemicals to be used in insect protection, but which were not identified by HPLC. Moreover, it is possible that the insects are sequestering sorne other biologically-active compound. It is known that sorne of T. diversifolia's medicinally-active compounds (Goffin et al. 2003, Ziemons et al. 2004) act as feeding deterrents to generalist insect species (Dutta et al. 1986, Dutta et al. 1993), thus it does appear that the three insects have developed the ability to overcome this aspect offeeding deterrency.

Study Limitations

In this study, plants were defined as medicinally-active or non-active based on the results of bioassays testing for a limited number of cancer celllines and parasitic diseases. Therefore, there may be potent plant secondary compounds which were not active in these bioassays. The definitions of specialist, generalist, and aposematic were based on those found in the literature plus discussions with other scientists. It should also be noted that sorne aposematic insects could have been mimics. Related to this, non-aposematic insects could have been sequestering active compounds from the plant, especially when non-visual predation is important. Moreover, sorne insect species may be modifying plant compounds to synthesize their own defensive compounds. Furthermore, time limitations dictated the number of active and non-active plants observed for insects and the number of chemical studies on the relationships between host plants and their insects. It also would be interesting to quantitatively measure the amount ofherbivory on each of the study plants over the study period.

Future Studies

The underlying causes for the significant association between aposematic insect species and medicinally-active plants requires further investigation. To obtain a c1earer pattern, it would be useful to investigate plant-insect interactions for a longer period oftime because of the spatial and temporal variability of tropical insect herbivores. In addition, testing an even greater number of plant pairs would be valuable, because of ecological variability and 92 diversity and to increase the sample size. Moreover, testing plant extracts in a wider variety of bioassays would generate a better idea of the plants overall biological activity, providing an increased level of credibility in these studies. Furthermore, a larger number of laboratory studies testing the ability of aposematic insects to sequester or metabolize biologically-active plant compounds need to be done to tease out a clearer pattern. To date, only a few such studies have been performed. Raudsepp-Hearne (2003) has shown that aposematic insects have the ability to concentrate a plants' medicinally-active compound(s), while in this study, it was found that aposematic insects did not sequester the plants compounds. This may reflect different strategies of insect herbivores to cope with plant secondary metabolites. A study examining a second insect-host plant relationship, to investigate the abilities ofthree aposematic and non-aposematic insects to utilize the medicinally-active compounds found in the plant, Cardia curassavica (Jacq., Roem. and Schult), had also been started during the course ofthis masters. However, due to long turnover times for bioassays, changes in methodology, and delays in obtaining necessary equipment, this experiment has not yet been completed. Understanding the ecological basis of plant-insect interactions may help to increase the probability that an aposematic insect willlead us to a medicinally-active plant. First, specialist and generalist insects may deal differently with plant secondary metabolites. Specialist herbivores may have evolved mechanisms not only to deal with, but also to benefit from plant chemicals by sequestering the plant compounds or derivatives, whereas, generalist herbivores often have more generalized strategies to metabolize and excrete plant compounds (Johnson and Bentley 1988, Schaffner et al. 1994, Krebs 2001, Nishida 2002). It is also important to determine whether an aposematic insect is a palatable mimic or is really unpalatable (Brower 1988, Fordyce 2000). Moreover, it could be useful to determine, either from the literature or by conducting field studies, what the predators and or parasitoids of the aposematic insect are since many vertebrates, such as birds, have colour vision, whereas, not aH invertebrates do (Dyer 1995). Related to this, it would be interesting to see if the biologically-active compounds sequestered by an insect act as effective predator-deterrents. This could be tested using purified extracts of a biologically-active compound(s), as well as through insect bioassays. 93

Key Findings To our knowledge, this study is the first that has investigated whether aposematic insects can be used as guides to plants containing potentially medicinally-active compounds. This study has also contributed valuable information on the ecology of tropical plant-insect interactions and on the ability of aposematic insects to concentrate plant secondary compounds with biological properties. Although, significantly more aposematic insects were found associated with plants that had biological activity in the ICBG bioassays, surprisingly, these plants also had greater numbers of insect species feeding on them and higher levels of herbivore damage. The higher levels of herbivore damage may reflect the fact that species had specialization on a food source. It was also surprising that aposematic insects, especially the Lepidopteran larval species, feeding on the biologically-active plant Tithonia diversifolia were not sequestering the compounds that were found to be biologically-active in the ICBG bioassays. This could be because the insects were: (1) metabolizing the active compounds into related derivatives (less or more toxic) and sequestering these; (2) sequestering other compounds; and/or (3) metabolizing compound de novo. Therefore, much research still needs to be done in this field to determine the functioning of plant-insect relationships, particularity in tropical forests. This study has contributed information on the host plant-insect relationships of20 different tropical plants, and 72 different insect species, 26 of which were identified to the species level and has provided valuable information on the chemical ecology of one host plant-insect relationship. The results ofthis study c1early indicate that aposematic insects can increase the likelihood of finding plants with potentially medicinally-active compounds, but that insects may not necessarily be utilizing these particular compounds for defensive purposes. This study supports the use of aposematic insects for enhancing the productivity of the drug discovery process. 94

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