Marine Chemical Ecology

Marine Chemical Ecology: The search for sequestered and bioactive compounds in the sea hares Dolabrifera dolabrifera and Stylocheilus striatus and in their preferred food, the cyanobacterium, Lyngbya majuscula.

Kathryn Elizabeth Clark

Degree of Master of Science

Department of Plant Science/McGill-STRI Neotropical Environmental Option

McGill University Montreal, Quebec, Canada January 11, 2008

DEDICATION

This thesis is dedicated to my Nana Jean Mitchell and to the rest of my family for their love and ongoing support.

ACKNOWLEDGEMENTS Many individuals have provided invaluable assistance to me through their advice and direction.

Professor guidance was given by Dr. Valerie Paul and Dr. Luis Cubilla Rios. Dr. Paul, head scientist of the Smithsonian Marine Station (SMS) in Fort Pierce, provided me with the opportunity to use the lab facilities at SMS. In addition, she provided valuable insight and guidance. Dr. Luis Cubilla Rios, of Associate program three (AP3) of the Panama International Cooperative Biodiversity Groups (ICBG) at the University of Panama provided me with lab space and advice.

Unpaid post-doctoral assistance was given by Dr. Marcy Balunas, Dr. Angela Capper, and Dr. Roger Linington.

Dr. Marcy Balunas, of AP3 of the Panama ICBG and post doctoral student of Dr. William Gerwick from the Scripps Institute of Oceanography (SIO) at the University of California in San Diego (UCSD), provided lab space at the Institute of Advance Scientific Investigations and High Technology Services (INDICASAT). As well, she provided instruction in compound isolation, carried out nuclear magnetic resonance (NMR) analyses and structural elucidation of the isolated peptides, and provided editorial help for chapter three.

Dr. Angela Capper, a marine chemical ecologist and a postdoctoral student of Dr. Valerie Paul, at the Smithsonian Marine Station in Fort Pierce, provided advice in extraction methods, feeding assay design techniques, and statistical analyses. She provided editorial help for chapter three.

Dr. Roger Linington of AP3 of the Panama ICBG and post doctoral student of Dr. William Gerwick, provided instruction for the epidioxysterol purification and carried out the NMR and mass spectral (MS) procedures for this compound.

iii

Bioassays were run by the following Panama ICBG associate program two (AP2) technicians at INDICASAT laboratories in Panama: Liuris Herrera in cancer, Alejandro Almanzo and Laura Maria Pineda in malaria, Michelle Ng and Luis David Ureña in cytotoxicity, Gina Della Togna in leishmania, Michelle Ng in Chagas’ disease and Roldolfo Contreras in dengue fever.

Dr. Alicia Hermosillo, an opisthobranch expert, instructed us where to collect the D. dolabrifera. Dr. Alicia Ibáñez, of associate program four (AP4) of the Panama ICBG provided help to me in the field to collect sea hares and cyanobacteria. Cyanobacterial identification was performed by Cameron Coates at Scripps Institute of Oceanography UCSD and algal identification was performed by Dr. Carlos Frederico Gurgel, a postdoctoral fellow at SMS.

My main supervisor Dr. Todd Capson, an associate scientist at STRI, leader of AP4 Panama ICBG and adjunct professor at McGill in the Department of Plant Science, provided me with office space at STRI, as well as guidance and direction throughout my program. He was the main editor of my thesis. My McGill supervisor, Dr. Tim Johns, provided guidance through committee meetings and assistance through editing my thesis. As well, Dr. Jacqueline Bede provided advice as the third committee member.

This project has been supported financially by: the Natural Sciences and Engineering Research Council of Canada, with a Canadian Graduate Scholarship at the master’s level (NSERC-CGS-M), a STRI-McGill-NEO fellowship, and a Levinson NEO fellowship. Additional funding was provided by Dr. Valerie Paul, through the Smithsonian Marine Station in Fort Pierce Florida and my main supervisor Dr. Todd Capson, through AP4 Panama ICBG and Smithsonian Tropical Research Institute.

PERSONAL THANKS I thank my supervisors Todd Capson and Tim Johns.

I thank Jacquie Bede of the department of Plant Science at McGill as the third committee member.

I thank Marcy Balunas and her students in the ICBG AP3 laboratory at INDICASAT in Panama. I thank Roger Linington and his students in his ICBG AP3 laboratory at INDICASAT in Panama. I thank Albano Díaz and Jose Félix Gómez for their help with HPLC and NMR respectively at INDICASAT.

I thank Valerie Paul and the postdoctoral fellows and students in her laboratory for helping me during my stay at the Smithsonian Marine Station in Florida. I thank Angela Capper for her support and insight. I thank Julie Piraino the lab manager and Raphael Ritson-Williams for teaching me how to use HPLC. I thank Woody Lee for kindly shipping my samples to Panama. I thank Joan Kaminski the administrative officer at the station. I thank Hugh Reichardt for organizing my stay in visiting scientists’ housing at the Tyson House owned by SMS. As well, I thank Debbie and Terry Brisson for providing housing for me in Fort Pierce.

I thank Luis Cubilla Rios and all his students, especially Marlys Barrera, Edwin Araúz and Carlos Jiménez in the ICBG AP3 laboratory at the University of Panama.

I thank Luis David Ureña, Michelle Ng, Alejandro Almanzo, Laura Maria Pineda, Liuris Herrera, Gina Della Togna and Roldolfo Contreras of the ICGB bioassay laboratory at INDICASAT.

I thank Alicia Hermosillo, Alicia Ibáñez, Beatriz Medina and Angela Capper, for their help in the field.

I thank Kerry McPhail for her cyanobacterial collection and cyanobacterial preservation advice.

I thank NAOS laboratories for use of their aquatic pavilion so that I could carry out an experiment. I thank the Liquid Jungle Lab for providing laboratory space during my fieldwork.

I thank Erika Garibaldo of ICBG for all her logistical support over the course of my thesis work.

I thank Carolyn Bowes the graduate program coordinator for the department of Plant Science at McGill University.

I thank Nilka Tejeira the academic coordinator of the NEO program in Panama.

I thank Cusiccoyllor Espinoza for editing my résumé.

I thank Andrew Hendry the current NEO program director and Catherine Potvin the former NEO program director.

I thank Bill Gerwick the project leader of the Panama ICBG and Biff Bermingham the acting director of STRI.

I thank Natalia Anaya, Jessica Ramírez, Sky Oestreicher, Cusiccoyllor Espinoza and Alicia Ibáñez for letting me stay on their sofas or air mattresses while I was writing and submitting my thesis.

I thank all of my friends and family for their support throughout my masters.

I thank my mother, Susan Clark, for her ongoing support and for editing my thesis.

LIST OF ABBREVIATIONS AND SYMBOLS

1 δH Chemical Shifts H-NMR 13 δC Chemical Shifts C-NMR 1H-NMR Proton Nuclear Magnetic Resonance 13C-NMR Carbon-13 Nuclear Magetic Resonance ACTs Artemisinin-based combination therapies AP Associate Program Ci Control Food Initial Weight Cf Control Food Final Weight d Doublet gwwt Grams of Wet Weight HPLC High Performance Liquid Chromatography

IC50 The concentration at which 50% of growth is inhibited ICBG International Cooperative Biodiversity Groups INDICASAT Institute of Advance Scientific Investigations and High Technology Services J Coupling Constant

LD50 The concentration at which 50% of death occurs in the test organism m Multiplet MCF-7 Human Estrogen Receptor-Positive Breast Cells m/z Mass/Charge MS Mass Spectrometry MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide n Number of replicates NMR Nuclear Magnetic Resonance P Probability PPP Public Private Partnerships s Singlet SIO Scripps Institute of Oceanography SMS Smithsonian Marine Station STRI Smithsonian Tropical Research Institute SPE Solid Phase Extraction Ti Treatment Food Initial Weight Tf Treatment Food Final Weight

vii

TABLE OF CONTENTS DEDICATION ...... ii ACKNOWLEDGEMENTS...... iii PERSONAL THANKS ...... v LIST OF ABBREVIATIONS AND SYMBOLS ...... vii TABLE OF CONTENTS ...... viii LIST OF TABLES ...... xi LIST OF FIGURES ...... xii RÉSUMÉ ...... xv Introduction...... 1 Literature Review...... 2 1.1 Marine Chemical Ecology ...... 2 1.1.1 Secondary Metabolite Sequestration...... 2 1.1.2 Metabolites in Predator Deterrence ...... 3 1.1.3 Ink Production...... 3 1.2 Cyanobacterial Secondary Metabolites...... 6 1.3 Dolabrifera dolabrifera ...... 6 1.4 Stylocheilus striatus ...... 9 1.5 Bioprospecting ...... 11 1.5.1 Dolastatin-10 ...... 11 1.5.2 Panama ICBG ...... 12 1.6 Neglected Diseases ...... 13 1.6.1 Malaria...... 14 1.6.2 Leishmanisis...... 14 Rationale and Objectives...... 16 Introduction to the Chapters ...... 17 Chapter 1: Isolation of α5,α8-Epidioxycholest-6-en-3-β-ol from the Digestive Gland of Opisthobranch Mollusc Dolabrifera dolabrifera...... 18 2.1 Background...... 18 2.1.1 Epidioxysterols ...... 18 2.1.2 Bioactivity of α5,α8-Epidioxycholest-6-en-3-β-ol...... 19 2.2 Materials and Methods...... 19 2.2.1 Study Sites ...... 19

viii

2.2.2 Study Organisms...... 19 2.2.3 Chemical Analyses ...... 21 2.2.3.1 Cyanobacteria...... 21 2.2.3.2 D. dolabrifera ...... 21 2.2.4 Bioassays ...... 23 2.3 Results...... 26 2.4 Discussion ...... 30 Linking statement ...... 31 Chapter 2: Feeding preferences of Dolabrifera dolabrifera...... 32 3.1 Background...... 32 3.2 Materials and Methods...... 32 3.2.1 Study Sites ...... 32 3.2.2 Study Organisms...... 33 3.2.3 Multiple Choice Assay ...... 35 3.2.4 No-Choice Assays ...... 35 3.3 Results...... 36 3.3.1 Multiple Choice Assay ...... 36 3.3.2 No-Choice Assay...... 36 3.4 Discussion ...... 39 Linking Statement...... 41 Chapter 3: The evaluation of a generic mechanism for secondary metabolite sequestration in sea hares Dolabrifera dolabrifera and Stylocheilus striatus...... 42 4.1 Background...... 42 4.1.1 Generic Mechanism in Sequestration...... 42 4.1.2 Stylocheilus striatus...... 46 4.1.2.1 Malyngamide A and B...... 46 4.1.2.2 Other Secondary Metabolites ...... 48 4.1.3 Cyanobacteria ...... 49 4.1.3.1 Toxicity and Defence ...... 50 4.1.3.2 Bioactive Sequestered Compounds...... 50 4.1.4 Bioassay-Guided Fractionation...... 51 4.2 Materials and Methods...... 51

4.2.1 Study Site ...... 51 4.2.2 Study Organisms...... 51 4.2.3 General Experimental Procedures...... 52 4.2.4 Chemical Analyses of L. majuscula ...... 52 4.2.4.1 Extraction and Analyses of Crudes...... 52 4.2.4.2 Fractionation of the EtOAc:MeOH extract (9401)...... 54 4.2.4.3 Isolation of compounds 9401D1 and 9401D2 ...... 54 4.2.4.4 Sequestration Experiment...... 58 4.2.5 Chemical Analyses of Sea Hares ...... 58 4.2.5.1 Dissection and Extraction ...... 58 4.2.5.2 Sequestration evaluation ...... 62 4.2.6 Bioassays ...... 62 4.3 Results...... 66 4.3.1 Sequestration Evaluation in S. striatus ...... 66 4.3.2 Sequestration Evaluation in D. dolabrifera ...... 66 4.3.3 Cyanobacterial Compounds 9401D1 and 9401D2 ...... 67 4.4 Discussion ...... 67 4.4.1 Generic Sequestration Mechanism in D. dolabrifera ...... 67 4.4.2 Sequestration of compounds by S. striatus ...... 69 General Discussion and Conclusions ...... 71 Suggestions for Future Research...... 74 Appendix A: NMR data of α5,α8-epidioxycholest-6-en-3-β-ol...... 75 Appendix B: MS data for the L. majuscula compounds ...... 76 List of References...... 77

LIST OF TABLES

Table 2-1: NMR Data for 5α,8α-epidioxycholest-6-en-3-β-ol (3) (CDCl3). Recorded at 400 MHz...... 28

Table 2-2: In vitro activity of 5α,8α-epidioxycholest-6-en-3-β-ol (3) in Panama

ICBG assays. Active results are in bold. Figures in brackets are IC50 values measured in micromolars (µM)...... 29

Table 4-1: Stylocheilus striatus feeding preference...... 47

Table 4-3: L. majuscula (9401) extract and SPE fraction bioassay results. Active screens and active IC50 values are in bold...... 56

Table 4-4: Bioassay values for L. majuscula fraction 9401D fractionated through HPLC, isolating compounds 9401D1 and 9401D2. Active samples are in bold.. 60

Table 4-5: Sea hare chemical analyses breakdown...... 64

Table 4-6: Animal tissue biological activity evaluation. Active samples are in bold...... 65

LIST OF FIGURES Figure 1-1: Chemical Structures of Dolabriferol (1) and Dolastatin-10 (2) ...... 4

Figure 1-2: Dolabrifera dolabrifera ...... 5

Figure 1-3: Phylogeny of Aplysiidae using bootstrap analysis of 37 morphological & histological characteristics. Used with consent. (Klussmann-Kolb, 2003) ...... 8

Figure 1-4: a) Stylocheilus striatus, b) with parapodia open, c) rearing ...... 10

Figure 2-1: The Coiba National Park, Panama, and the location of the Boca Grande Field site...... 20

Figure 2-2: Dolabrifera dolabrifera anatomy. Drawn by Kathryn E. Clark ...... 22

Figure 2-3: HPLC chromatogram at 254 nm (green) and 210 nm (red). Pure 5α,8α-epidioxycholest-6-en-3-β-ol eluted at 54 minutes...... 24

Figure 2-4: MS spectral data for a) D. dolabrifera crude digestive gland and b) pure 5α,8α-epidioxycholest-6-en-3-β-ol...... 24

Figure 2-5: Isolation of 5α,8α-epidioxycholest-6-en-3-β-ol...... 25

Figure 2-6: Structure of 5α,8α-epidioxycholest-6-en-3-β-ol (3) with carbon atoms numbered...... 27

Figure 3-1: Multiple choice assay food options a) mat-like L. majuscula, b) hair- like L. majuscula, c) Symploca sp., d) Spyridia sp. and e) Chaetomorpha sp.. No- choice assay foods a) L. majuscula and f) Cladophora sp. Scale bars indicate 1 cm of length...... 34

Figure 3-2: Multiple choice feeding assay using D. dolabrifera. The data shown are the mean of algal and cyanobacterial material consumed (blotted gwwt). Friedman’s ANOVA was used to analyse data (P > 0.05). Letters above bars refer to significant differences using Tukey’s post-hoc test. n = number of replicates. Mat-like L. majuscula is (1) and hair-like L. majuscula is (2)...... 37

xii

Figure 3-3: No choice feeding assay. Data shown are the mean proportion of food consumed by D. dolabrifera. Paired sample t-tests were used to analyse data (P < 0.05). n = number of replicates...... 38

Figure 4-1: Secondary metabolites (4, 5, 6, 7, 8, 17, 18, 21 & 22). Secondary L. majuscula metabolites sequestered by S. striatus (4, 5, 6, 7, 8, 17, 18, 21 & 22). Secondary metabolites that stimulate (16, 21* & 22*) and deter feeding (18, 19, 20, 21† & 22†) by S. striatus. S. striatus modified forms of L. majuscula secondary metabolites (15, 23 & 24). Measured at low* and high† natural concentrations..44

Figure 4-2: Secondary metabolites that S. striatus is capable of sequestering, from green algae (9 & 10), brown algae (11 & 12), and sponge (13 & 14) (Pennings and Paul, 1993)...... 45

Figure 4-3: Extraction, fractionation and compound isolation of hair type L. majuscula (2, TC-94). Compounds 9401D1 and 9401D2 were isolated. Bolded path indicates fractionation pathway...... 53

Figure 4-4: MS data from L. majuscula extracts (a) 9401 and (b) 9402...... 53

Figure 4-5: HPLC of L. majuscula fraction 9401D. Compounds 9401D1 eluted at

23 minutes and 9401D2 eluted at 31 minutes. Reverse phase C18 HPLC, 55%

MeCN, 45% H2O...... 57

Figure 4-6: Proton NMR data of (a) S. striatus digestive gland showing compound sequestration of L. majuscula compounds (b) 9401D2 and (c) 9401D1...... 59

Figure 4-7: HPLC chromatograms at 0.1 mg/mL. L. majuscula 9401D fraction (a), compound 9401D1 (b) and compound 9401D2 (c). S. striatus digestive gland (d) and skin (e). D. dolabrifera (fed L. majuscula) digestive gland (f) and skin (g). D. dolabrifera (non-feeding assay) digestive gland (h) and skin (i)...... 61

Figure 4-8: Anatomy of Stylocheilus striatus...... 63

xiii

ABSTRACT Dolabrifera dolabrifera, an anaspidean mollusc (sea hare) collected from Panama’s Coiba National Park, was subjected to its first marine chemical ecology study. Its digestive gland contained 5α,8α-epidioxycholest-6-en-3β-ol. This compound, documented for the first time in D. dolabrifera, demonstrated activity against the parasite responsible for leishmania (Leishmania donovani). An evaluation of its dietary repertoire revealed that D. dolabrifera significantly preferred the cyanobacterium Lyngbya majuscula over cyanobacterium Symploca sp., green alga Chaetomorpha sp., and red alga Spyridia sp.. A no- choice feeding assay using L. majuscula or green alga Cladophora sp. confirmed this preference. Through bioassay-guided fractionation, two novel cyanobacterial peptides were isolated. These compounds were determined active against Plasmodium falciparum, with one compound also active against L. donovani. These peptides were sequestered by sea hare Stylocheilus striatus, but not by D. dolabrifera. This work suggests that chemical ecological studies involving sea hares and cyanobacteria can guide researchers to compounds active against tropical parasites.

xiv

RÉSUMÉ Des opisthobranches (anaspidea) dont une espèce nommé Dolabrifera dolabrifera a été étudié au Parc National Coiba, une île au Panama. Cette étude de chimie écologique marine est la première pour cette espèce. Dans sa glande digestive, on a retrouvé 5α,8α-épidioxycholest-6-en-3β-ol. Ce composé possède des nouvelles activités contre le parasite de leishmanioses (Leishmania donovani). Lors d’une expérience d’alimentation D. dolabrifera a préféré la cyanobactérie Lyngbya majuscula, plutôt qu’une algue verte Chaetomorpha, une algue rouge Spyridia et une cyanobactérie Symploca. L’expérience suivante d’alimentation D. dolabrifera a mangé L. majuscula plutôt qu’une algue verte Cladophora. Deux peptides nouveaux étaient isolés de la L. majuscula, les deux actives en malaria (Plasmodium falciparum). Aussi un peptide actif en leishmanioses (L. donovani) et l’autre en malaria (P. falciparum). Ces composés étaient séquestrés dans un opisthobranche appelé Stylocheilus striatus. Ce travail suggère des études en chimie écologie chez les opisthobranches et les cyanobactéries qui peuvent amener à des composés ayant des activités contre les parasités tropicales.

Introduction As a part of the bioprospecting and drug discovery initiative and under the auspices of the Smithsonian Tropical Research Institute (STRI) and the Panama International Cooperative Biodiversity Groups (ICBG), the first marine chemical ecology study on the sea hare Dolabrifera dolabrifera (Opisthobranchia: Anaspidea) was carried out. Many species of sea hares are capable of sequestering dietary metabolites from algae and storing them almost exclusively in their digestive glands, rather than in external organs, ink or eggs (Pennings and Paul, 1993). This phenomenon has been identified as a generic mechanism for sequestration. Pennings and Paul (1993) presented the notion that sea hares have generic mechanisms for sequestering algal metabolites rather than mechanisms that are tightly linked to particular compounds, that these mechanisms do not differ dramatically between species, and that sequestered secondary metabolites are not located optimally for defense. I hypothesized that the sea hare D. dolabrifera also might utilize this generic mechanism. In order to test this hypothesis, I focused on a single site in the tidal waters off the shores of the Coiba Island located in the Eastern Pacific Ocean in Panama. Firstly, the digestive gland chemistry of D. dolabrifera was studied to evaluate the presence of dietary sequestered compounds. Secondly, as there was no documentation of its diet, its dietary repertoire was evaluated through feeding experiments. Thirdly, bioassay-guided fractionation was used to isolate compounds from the preferred food of two sea hare species D. dolabrifera and Stylocheilus striatus. S. striatus was used for comparative purposes, as it is known to sequester dietary metabolites (Pennings and Paul, 1993). Finally, the presence and the location of the sequestered algal metabolites were evaluated in the sea hare tissues.

Literature Review 1.1 Marine Chemical Ecology Marine chemical ecology considers the impacts of naturally occurring organic compounds and their role in the marine environment. This includes predator-prey interactions, competition, chemical communication, fouling, pathogen-host interactions, biosynthesis, reproduction, and the evolution of secondary metabolites (McClintock and Baker, 2001). The focus of the work described in this study involves predator-prey interactions, more specifically, herbivore-prey interactions between anaspidean opisthobranch molluscs (sea hares) and their prey which consists of algae and filamentous blue-green algae (cyanobacteria).

1.1.1 Secondary Metabolite Sequestration Like many marine mesograzers, sea hares sequester algal compounds from their diets (Paul and Pennings, 1991; Stallard and Faulkner, 1974); however, unlike nudibranchs (Opisthobranchia: Gastropoda) which store sequestered compounds in their mucus, skin, hermaphrodite gland and eggs (Becerro et al., 2006; Pitot et al., 2002; Pennings et al., 2001; Avila et al., 1990; Pawlik et al., 1988; Thompson et al., 1982), evidence suggests that sea hares concentrate the bulk of sequestered compounds in their digestive glands (Pennings et al., 1999; De Nys et al., 1996). Diet-derived compounds are found in lower concentrations in their skin, ink glands (De Nys et al., 1996) and secreted in ink, eggs and fecal matter (Capper et al., 2005). There may well be sequestered compounds that are concentrated in other organs besides the digestive gland that have not yet been discovered.

Since sea hares appear to concentrate sequestered compounds in their digestive glands rather than their exterior parts, the role for sequestered compounds in sea hares is heavily debated (Capper et al., 2005; Pennings and Paul, 1993). A distasteful nip of the skin would not kill the sea hare, but a bite to expose compounds concentrated in the digestive gland would leave the sea hare lethally wounded (Brower and Glazier, 1975). On the other hand, it has been proposed

that storage of sequestered compounds in the digestive gland aids in detoxifying a diet rich in toxins (Pennings et al., 1999; Pennings and Paul, 1993).

1.1.2 Metabolites in Predator Deterrence If and how dietary-derived and de novo metabolites aid in sea hare defence remains uncertain. For example, in a palatability study reef fish found S. striatus containing dietary-derived secondary metabolites (malyngamides A & B) and S. striatus not containing the metabolites equally palatable. The type of fish was a more significant factor in determining the sea hare’s vulnerability to predation, rather than the presence or absence of sequestered secondary metabolites (Pennings et al., 2001). In contrast, however, live Aplysia parvula containing dietary-derived secondary metabolites, (apakaochtodenes A & B) showed a significant distastefulness when fed to reef fishes in comparison to the A. parvula not containing the metabolites (Ginsburg and Paul, 2001).

Fish palatability studies have been conducted to test whether deterrence occurs between the skin and the digestive gland. To date, however, no significant difference has been observed between body parts’ palatability, regardless of the fact that secondary metabolites are concentrated in the digestive gland. This suggests that possibly important predator deterring compounds are concentrated in the skin and are de novo in origin (Ginsburg and Paul, 2001; Pennings, 1994). In two cases, compounds that could aid in sea hare defence have been reported from the skin of the animal. Dolabellanin B2 has been isolated from Dolabella auricularia (Iijima et al., 2003) and dolabriferol (1) (Fig. 1-1) from D. dolabrifera (Fig. 1-2) (Ciavatta et al., 1996). Compounds are numbered and bolded to indicate which compound is which in the figure.

1.1.3 Ink Production Ink is produced by many species of sea hares, most notably by Aplysia spp. (Johnson and Willows, 1999), S. striatus (Prince and Johnson, 2006) and Dolabella auricularia (Hermosillo et al., 2006).

Figure 1-1: Chemical Structures of Dolabriferol (1) and Dolastatin-10 (2)

Figure 1-2: Dolabrifera dolabrifera

Active and predator-deterring compounds in ink are not dependent on diet, which suggests that sea hares produce these compounds de novo (Kicklighter and Derby, 2006). The purple color in sea hare ink comes from chromophores a pigment derived from its algal food source (Johnson et al., 2006). Escapin, contains anti-bacterial activity, but it is not responsible for any adverse predator- deterrent effects (Kicklighter and Derby, 2006). It has been proposed that these colorants act as a chemical “smoke screen” (Johnson and Willows, 1999; Carefoot, 1987) or as a mimic device possibility imitating nudibranchs which have defensive compounds in their ink (Kicklighter et al., 2005).

1.2 Cyanobacterial Secondary Metabolites Cyanobacteria are well-recognized food sources of sea hares D. auricularia (Pennings et al., 1993), Bursatella leachii (Ramos et al., 1995) and S. striatus (Paul and Pennings, 1991). Cyanobacteria produce secondary metabolites that can stimulate or deter feeding (Nagle et al., 1998). Sea hares often prefer these chemically-defended foods and sequester their secondary metabolites (Rogers et al., 1995; Paul and Pennings, 1991). Sea hare generalists like D. auricularia (Pennings and Paul, 1993) prefer a varied diet of algae and cyanobacteria; whereas, sea hare specialists like S. striatus prefer and grow best on a single diet (Paul and Pennings, 1991). These species are known to consume cyanobacteria and sequester their secondary metabolites (Pennings and Paul, 1993). Compound sequestration can occur within the first day of exposure (Pennings and Paul, 1993) and compounds can be stored for up to three months after exposure (Stallard and Faulkner, 1974).

1.3 Dolabrifera dolabrifera The Sea hare Dolabrifera dolabrifera (Cuvier 1817; Dolabriferinae: Aplysiidae: Anaspidea: Opisthobranchia) has a long bulbous flat body, narrowing to its head. Its parapodia are fused along the animal’s body until the mantle. The gill is located in the mantle and is covered by the mantle flap. The mantle is located on its posterior right side and is protected by a small internal shell (Fig. 1-2). A foot

runs along its underside and the animal moves in a leach-like motion (Hermosillo et al., 2006). It is the most common opisthobranch in the Panamic region of the Tropical Eastern Pacific and is found under rocks in intertidal pools (Camacho- García et al., 2005). D. dolabrifera has been the subject of many studies due to the simplicity of its neurological system. D. dolabrifera is incapable of three types of non-associative learning: dishabituation, short term sensitization, and long term sensitization (Marinesco et al., 2003). Wright (1998) described this phenomenon as a phylogenetic lesion because all other sea hares tested were capable of these types of learning. This included Phyllaplysia taylori, in the Dolabriferinae sub-family, the same sub-family as D. dolabrifera (Fig. 1-3) (Marinesco et al., 2003; Erixon et al., 1999; Wright, 1998). Since D. dolabrifera lives on the underside of rocks and is nocturnal it may also be relatively free from predators (Kay, 1979), Erixon et al. (1999) suggested that ecological studies should be carried out to determine whether restricted habitat, among other ecological factors, plays a role in the behavioural plasticity of D. dolabrifera.

D. dolabrifera has several documented forms of physical, behavioural and chemical defences. Physically, it has some protection from a reduced shell. Behaviourally, it is nocturnal and cryptic (Hermosillo, Personal Communication). Chemically, a water-soluble extract taken from the mid-gut gland has shown lethality when injected into mice at 200 mg/kg (Waston, 1973) and its egg masses have been active in an anti-bacterial zone of inhibition assay (Benkendorff et al., 2001). It is unable to produce ink (Prince and Johnson, 2006), but produces a white milky secretion from its mantle when threatened (Ghazali, 2006).

Dolabriferol (1) (Fig. 1-1), a polypropionate, was isolated from the skin of D. dolabrifera (Ciavatta et al., 1996). No cytotoxicity or bioactive studies have been carried out on this compound. It is probable that Dolabriferol (1) (Fig. 1-1) aids in the chemical defence of this animal.

Figure 1-3: Phylogeny of Aplysiidae using bootstrap analysis of 37 morphological & histological characteristics. Used with consent. (Klussmann-Kolb, 2003)

1.4 Stylocheilus striatus The sea hare Stylocheilus striatus (Quoy & Gaimard, 1832; Opisthobranchia: Anaspidea) has an elongated body covered with the compound papillae. Most animals are translucent grey or brown in colour with blue or pink eye spots and dark longitudinal striations along their body (Fig. 1-4 a) (Camacho-García et al., 2005). S. striatus has lost its shell and its parapodia are not fused, thus exposing its mantle cavity (Fig. 1-4 b). The mantle cavity contains two major ink glands, namely, a purple gland on the roof of the mantle which produces a purple ink and an opaline gland on the floor of the mantle cavity which secretes a thick white fluid (Rudman, 2002a). A foot runs along the entire underside of the animal and moves by attaching the anterior foot to the substrate, contracting its posterior foot and attaching it to the substrate and then, elongating its body. It has a circum- tropical distribution (Camacho-García et al., 2005) and is commonly found in the Indio-West Pacific and Atlantic Oceans (Rudman, 1999).

S. striatus demonstrates several documented forms of behavioural and chemical defences. Behaviourally, it grazes and lives on the chemically defended cyanobacterium L. majuscula, which provides a safe haven (Paul et al., 2001) by lowering encounter rates with reef predators (Paul and Pennings, 1991). When threatened, it responds by a rearing behaviour consisting of raising its anterior region and mantle and extending its oral tentacles (Fig. 1-4 c) (Ghazali, 2006). In opisthobranchs, rearing is associated with repulsion or with escape (Behrens, 2005). In addition, it produces a purple ink when threatened or distressed (Ghazali, 2006). Chemically, an ether-soluble extract from its mid-gut gland showed lethality when injected into mice at 25 mg/kg (Waston, 1973) and its egg masses have demonstrated activity against four marine pathogenic bacteria with inhibition between 5-10 mg/mL (Benkendorff et al., 2001).

Figure 1-4: a) Stylocheilus striatus, b) with parapodia open, c) rearing

Although potential chemical defences derived from its diet have been studied, no definitive links between dietary compounds and their purpose as a defence mechanism have been made.

1.5 Bioprospecting 1.5.1 Dolastatin-10 The search for novel medicines from natural sources such as plants and marine organisms is often referred to as bioprospecting (Reid et al., 1993). The best known example of a compound isolated from a sea hare is the potent antiproliferative agent, dolastatin 10 (2) (Fig. 1-1) which was originally isolated from D. auricularia extracted whole, at a very low yield of ~1 mg/100 kg. The -5 dose at which 50% of murine PS leukemia cells were killed (ED50) was 4.6 x 10 µg/mL (0.046 ng/mL). Because there was a very low compound yield of ~1 mg/100 kg, this led Pettit et al. (1987) to believe that only an extremely low concentration of dolastatin-10 was needed for the animal’s defence. Later it was revealed that dolastatin 10 (2) (Fig. 1-1) was dietary in origin from cyanobacterium Symploca VP642 species (Luesch et al., 2001).

Dolastatin 10 (2) (Fig. 1-1) which is a powerful microtubule inhibitor (Simmons et al., 2005) has shown antimalarial activity towards Plasmodium falciparum (Fennell et al., 2003). As a cancer drug, it has advanced to phase II clinical trials and has been tested against platinum-sensitive ovarian carcinoma (Hoffman et al., 2003), metastatic colorectal carcinoma (Aguayo et al., 2000), metastatic melanoma (Margolin et al., 2001), non-small cell lung carcinoma (Krug et al., 2000), hormone-refractory metastatic prostate adenocarcinoma (Vaishampayan et al., 2000), advanced breast cancer (Perez et al., 2005) and advanced pancreaticobiliary cancer (Kindler et al., 2005) and advanced renal cell carcinoma (Pitot et al., 2002). The reports conclude however that the compound lacks clinical activity (Vaishampayan et al., 2000) or that the compound contains minimal clinical activity using the experimental dose and schedule followed

(Hoffman et al., 2003). Although dolastatin-10 may never become a cancer drug, several natural and synthetic analogs have the potential (Simmons et al., 2005).

1.5.2 Panama ICBG The Panama International Cooperative Biodiversity Group (ICBG) is dedicated to the isolation of bioactive compounds from cyanobacteria, endophytic fungi, and plants collected from the national parks and the protected areas in the Republic of Panama. The objectives of the Panama ICBG are to isolate compounds with potential applications against cancer and tropical disease, to promote the conservation of Panamanian biodiversity, to promote the development of the country’s scientific infrastructure and to train scientists (Capson, 2007; Kursar et al., 2006).

All bioassays in this study were performed in the Panama ICBG laboratories at the Institute of Scientific Research and High Technology Services (INDICASAT) in Panama City, Panama. Six in vitro bioassays were employed which detect compounds with activity against Plasmodium falciparum (malaria) (Corbett et al., 2004), Trypanasoma cruzi (Chagas’ disease) (Buckner et al., 1996), Leishmania donovani (Leishmania) (Calderón et al., 2006), dengue fever (Ureña, Personal Communication) and MCF-7 (breast cancer cell line) (Monks et al., 1991). Cytotoxicity was measured in Vero mammalian cells (van de Loosdrecht et al., 1994).

Screening for bioactivity which is the first step in the bioassay process consists of testing the samples at 10 µg/mL. In the Leishmania bioassay, a sample is considered active when parasitic growth is less than 50%. In the cancer bioassay, a sample is considered active when cancer cell growth is less than 50%. In the malaria and Chagas’ disease bioassay, a sample is considered active when parasitic inhibition is more than 50% when tested at a single concentration of 10 µg/mL. In the Dengue bioassay, a sample is considered to have good activity

when cell protection is 50% or greater. When a sample provides less than 50% cell protection and is not cytotoxic it is considered moderately active.

Once a sample is determined to be active, an IC50 is determined. This process consists of sampling bioactivity at several different concentrations (10, 2, 0.4 and 0.08 µg/mL) to determine the concentration at which 50% of the parasite, virus, cancer cell, or normal cell is inhibited. When the mass is known for a compound

IC50 values are calculated in micromolars (µM). The therapeutic window is the range at which a substance, compound or drug is effective in killing the disease or parasite while not harming normal human cells. It is calculated by dividing its

IC50 value for cytotoxicity by its IC50 value towards the disease.

1.6 Neglected Diseases Neglected diseases such as malaria, leishmanisis, Chagas and Human African trypanosomiasis (sleeping sickness), ten years ago accounted for 90% of the world’s health problems (Drugs for Neglected Diseases, 2003), yet only 10% of funding was allocated for research of these diseases. This phenomenon is known as the “10/90” gap (Drugs for Neglected Diseases, 2004).

Recently, however, there has been more focused attention on developing treatments for malaria and other neglected diseases, mainly through the funding of Public Private Partnerships (PPPs). PPPs are not-for-profit organizations that work in collaboration with industry to fund drug development projects for neglected diseases (Moran, 2005). Drugs for Neglected Disease initiative (DNDi), Medicines for Malaria Venture (MMV), Institute for OneWorld Health (iOWH) Training in Tropical Diseases (TDR) and the Global Alliance for Tuberculosis Drug Development (TB alliance) are the most important PPPs in neglected disease research and development (Moran, 2005).

1.6.1 Malaria Every 30 seconds a child dies from malaria and each year more than 500 million people are made seriously ill from the disease. One million people die each year (World Health Organization, 2007c), and 80% of the deaths occur in sub-Saharan Africa (World Health Organization, 2006). Forty per cent of the world’s population is at risk, most of whom live in the world’s poorest countries (World Health Organization, 2007c). Malaria is transmitted through the bites of infected mosquitoes. The parasite multiples in the liver and then infects red blood cells (World Health Organization, 2007c). With early diagnosis and treatment, malaria is usually not life threatening (World Health Organization, 2007c; Bjorkman and Bhattarai, 2005). There are four species of malaria that infect humans: P. falciparum, P. vivax, P. malariae and P. ovale (Bjorkman and Bhattarai, 2005). P. falciparum is one of the most common and the most deadly of the malaria parasites (World Health Organization, 2007c). P. falciparum and P. vivax have developed resistance to the inexpensive drugs chloroquine, sulfadoxine and pyrimethamine (World Health Organization, 2006). Currently artemisinin-based combination therapies (ACTs) are drug treatments suggested by the World Health Organization to treat people infected with P. falciparum (World Health Organization, 2007c; Bjorkman and Bhattarai, 2005). Cure rates using ACTs are from three to seven days (World Health Organization, 2006). Signs of resistance of P. falciparum toward artemisinin have been noted in recent years (Jambou et al., 2005). The development of P. falciparum resistance to antimalarial drugs has a large impact on public health (Bjorkman and Bhattarai, 2005).

1.6.2 Leishmanisis Leishmaniasis is prevalent in the tropics and sub-tropics, especially in Asia, parts of Europe, Africa and the Americas (Carmargo and Langoni, 2006). It is estimated that 12 million people are infected world wide, with two million new cases each year (World Health Organization, 2007b). Over 350 million people are at risk and it is prevalent in 88 countries (World Health Organization, 2007d). Leishmania is a zoonotic protozoan parasite, from the genus Leishmania which

comprises 20 species (Carmargo and Langoni, 2006). The parasite is passed when the vector, the female phlebotomine sandfly bites humans or animals (Carmargo and Langoni, 2006). The disease is prevalent in both urban and wild environments and can cause epidemic outbreaks (Carmargo and Langoni, 2006). In humans there is cutaneous, diffused cutaneous, mucocutaneous and visceral forms of the disease (World Health Organization, 2007a). L. donovani falls in the visceral disease group (World Health Organization, 2007d), affecting internal organs such as the spleen, liver, lymph nodes and bone marrow (Carmargo and Langoni, 2006). It is estimated that 500,000 suffer from this form of the disease (World Health Organization, 2007b). Current leishmania drugs have some serious possible side-effects which include death (World Health Organization, 2007d). Treatment drugs can be highly priced and must be administered intravenously to a patient for 15 to 30 days under close doctor supervision (World Health Organization, 2007b). Liposomal amphotericin, miltefosine and paromonycin are three new antimonial drug treatments (Bryceson et al., 2007).

Rationale and Objectives The sea hare, D. dolabrifera stood out as an interesting study candidate as it had never been studied from a marine chemical ecology perspective. As well, it was accessible on Coiba Island, Panama. As the feeding preferences of this generalist herbivore were unknown, feeding studies were implemented, with the intention of laying the ground work for future marine chemical ecology research of this species. As a further exploration of the Pennings and Paul (1993) study, in which they suggested that sea hares contain a generic mechanism for processing dietary secondary metabolites, I hypothesised that sea hare D. dolabrifera was likewise capable of sequestering dietary derived compounds and concentrating them in its digestive gland.

The objectives of this study were to determine: 1) the feeding preferences of D. dolabrifera, 2) if D. dolabrifera is capable of sequestering secondary metabolites from its diet, 3) where D. dolabrifera stores the sequestered compounds tested and 4) the bioactivity of all samples and compounds from this study. Before determining the feeding preferences of generalist herbivore D. dolabrifera, an evaluation its digestive gland chemistry was carried out. Through this work, α5,α8-epidioxycholest-6-en-3-β-ol a major digestive compound was isolated. It was determined to be neither algal nor cyanobacterial in origin. Thus, it was apparent that a different strategy was necessary to determine whether or not D. dolabrifera sequesters dietary compounds. Thus, after determining what foods D. dolabrifera preferred, secondary compounds were isolated directly from its preferred food source. Subsequent to this, an evaluation of the compound sequestration was carried out in the sea hare’s tissues. Another sea hare, S. striatus, which is known to sequester secondary metabolites (Paul and Pennings, 1991) was used as an to evaluate whether or not sequestration of the cyanobacterial compounds was indeed possible. Finally, as part of the Panama ICBG drug discovery initiative, bioassay-guided fractionation was used to isolate secondary metabolites from the preferred food source L. majuscula and to test them for activity towards several tropical parasites, dengue fever and cancer.

Introduction to the Chapters Three chapters form the core of the thesis. Each chapter has its own introduction, materials and methods, results and discussion. There are linking statements joining the chapters, explaining why the work in the preceding chapter led to the work in the subsequent chapter.

In chapter one I collaborated with many researchers. I extracted the samples in Dr. Valerie Paul’s lab at SMS in Fort Pierce. Dr. Paul’s post-doc Dr. Angela Capper helped me formulate the extraction methodologies. Then I isolated the epidioxysterol under advice of Dr. Roger Linington from the Panama ICBG, in the laboratories of Dr. Luis Cubilla at the University of Panama. Dr. Linington ran MS and NMR analyses on my samples.

For chapter two I collaborated with Dr. Valerie Paul’s post-doc Dr. Angela Capper. Under her advice I created the feeding assay methodologies. We conducted the statistical analyses together.

In chapter three I collaborated with Dr. Marcy Balunas and Dr. Valerie Paul. I extracted the samples in Dr. Paul’s lab under the supervision of Dr. Angela Capper. Following this I isolated two novel cyanobacterial peptides in Dr. Marcy Balunas’ Panama ICBG laboratory in INDICASAT in Panama. I used HPLC and SPE procedures under her instruction to isolate these compounds. I carried out the HPLC sequestration experiment with advice from Dr. Balunas. She ran all NMR and MS analyses and will carry out the structural elucidation of the compounds. We conducted the NMR sequestration experiment together.

Cyanobacteria identification was carried out by Cameron Coates from the University of California in San Diego. Algal identification was carried out by Dr. Carlos Frederico Gurgel at SMS. Bioassays were run by the Panama ICBG bioassay technicians in INDICASAT in Panama.

Chapter 1: Isolation of α5,α8-Epidioxycholest-6-en-3-β-ol from the Digestive Gland of Opisthobranch Mollusc Dolabrifera dolabrifera

2.1 Background 2.1.1 Epidioxysterols Epidioxysterols, compounds which contain a sterol nucleus and an endoperoxide moiety, have been reported from a wide variety of marine invertebrates including species of sea hare, Cnidaria, tunicates, pillar coral, cone snail, sea urchin and sponge (Min-Hsiung et al., 2007; Mansoor et al., 2006; Minh et al., 2004; Abourriche et al., 2000; Gauvin et al., 2000; Sheu et al., 2000; Sera et al., 1999; Aknin et al., 1998; Jiménez et al., 1989; Kahlos et al., 1989; Jiménez et al., 1986; Gunatilaka et al., 1981).

Many researchers believe that epidioxysterols are of dietary origin (Gunatilaka et al., 1981) as invertebrates do not always contain similarly structured precursors and epidioxysterols (Jiménez et al., 1989). In an in vivo 14C incorporation experiment, Jiménez et al. (1989) proved that cnidarians are capable of oxygenating precursors forming epidioxysterols. Additionally, when comparing cnidarians collected from different sites, Jiménez et al. (1989) found that the animals contained different epidioxysterols depending on where they were collected. Thus they suggested that there was an external source for these compounds, most probably from their mussel prey. In sea hares Aplysia spp. it has been suggested that epidioxysterol precursors are dietary in origin. Jiménez et al. (1986) found a sterol from the epidioxysterol family in A. punctata which they did not find in A. depilans. They suggested that this difference could be due to the difference in the diet of the sea hares, as green and red algae were found in the stomach contents of A. punctata, but only a green alga was found in the stomach contents of A. depilans. A more recent hypothesis suggests that since epidioxysterols are so wide spread among different marine invertebrates that perhaps the source of epidioxysterol precursors may be derived from common symbiotic micro-organisms (Gauvin et al., 2000).

Epidioxysterols have shown cytotoxicity against breast, lung, stomach, kidney, colon, ovary, central nervous system, prostate, and melanoma tumour cell lines.

The growth inhibition at 50% (IC50) varied between cell lines, with activities between 0.6 to 2.7 µg/mL (Iwashima et al., 2002; Sheu et al., 2000). In addition, epidioxysterols have shown cytotoxicity against lymphotrophic T-cell leukemia cells with an IC50 of 0.3 mg/mL (Gauvin et al., 2000).

2.1.2 Bioactivity of α5,α8-Epidioxycholest-6-en-3-β-ol α5,α8-Epidioxycholest-6-en-3-β-ol was shown by Minh et al. (2004) to be significantly cytotoxic against KB (human epidermoid carcinoma) with an IC50 of

2.0 µg/mL, FL (fibrillary sarcoma of the uterus) with an IC50 of 3.93 µg/mL, and

Hep-2 (Human hepatocellular carcinoma) with an IC50 of 2.4 µg/mL. The compound was also shown to have an anti-bacterial zone of inhibition of 6 to 18 mm, an anti-fungal zone of inhibition of 7 to 15 mm, and a lethal dose of 50% of the testing population (LD50), in this case to brine shrimp at 71.49 µg/mL (Abourriche et al., 2000).

2.2 Materials and Methods 2.2.1 Study Sites Collections were made mid March 2006 from Boca Grande, a rocky and sandy beach from the Coiba Island National Park, Panama (Fig. 2-1) (07°23´ 41.7" north, 81° 39´ 03.7" west).

2.2.2 Study Organisms Three species of cyanobacteria were collected. Lyngbya majuscula grew as long hair-like filamentous strands, Symploca sp. grew as small, firm, leaf-like tuffs; and Oscillatoria sp. grew as a green mass and contained air bubbles which gave it buoyancy. Samples used for chemical analyses as well as voucher samples were preserved in 75% ethanol (EtOH): 25% sea water at -20°C.

Figure 2-1: The Coiba National Park, Panama, and the location of the Boca Grande Field site.

Thirty-five D. dolabrifera were collected from the underside of uneven boulders in intertidal pools. They were maintained in aquaria, fasted for 24 hours to evacuate their digestive system, euthanized in freezing seawater, and stored at -20°C.

2.2.3 Chemical Analyses 2.2.3.1 Cyanobacteria Samples were blended, extracted three times for 12 to 24 hours in 1:1 ethanol (EtOH):ethyl acetate (EtOAc) and filtered using Whatman 1 qualitative circles (185 mm, Whatman International Ltd., Maidstone England). Extracts were dried down and resolubilized in a mixture of 1:1 EtOAc:water (H2O) and partitioned using a separation funnel. Three parts of 1-Butanol were added to the water layer and partitioned using a separation funnel, and the water was discarded. The EtOAc and 1-butanol extracts were analysed by mass spectrometry (MS) on a JEOL JMS LC-mate mass spectrometer (Tokyo, Japan).

2.2.3.2 D. dolabrifera D. dolabrifera were divided into seven replicates and dissected into skin (parapodia, foot and head), digestive gland and body parts (all other internal organs) (Fig. 2-2). Replicates were freeze-dried, weighed and pulverized. 1:1 EtOAc: methanol (MeOH) was added, the sample was sonicated for 20 minutes and left for 24 hours to extract. The liquid was then filtered under vacuum, using a Buchner funnel and Whatman 4 qualitative circles (90 mm, Whatman International Ltd., Maidstone England). Using the above solvent mixture, samples were then extracted for 5 minutes with sonication, the liquid was filtered under vacuum, the sample was rinsed in the solvent mixture, and filtered under vacuum.

Initial separation of the crude digestive gland was carried out using normal-phase high performance liquid chromatography (HPLC). The extract was solubilized in 200 µl of hexanes and filtered at 0.45 µm (Altech 17mm PTFE syringe filters).

Figure 2-2: Dolabrifera dolabrifera anatomy. Drawn by Kathryn E. Clark

An Econosphere Silica 10 µm column (10 x 250 mm, Altech Associates, Inc. Deerfield IL) and an Econosphere silica 5 µm guard column (7.5 x 4.6 mm, Altech Associates, Inc.) were used in a gradient system of 100% hexanes to 100% EtOAc over 60 minutes, monitoring at 260 nm, with a flow rate of 2 mL/min. Proton nuclear magnetic resonance (1H-NMR; JEOL 600 MHz) was carried out in order to determine fractions of interest. Purification of one fraction which eluted from 37 to 38 minutes was accomplished by high performance liquid chromatography (HPLC) under reverse phase using an XTerra C18 analytical column (5 µm, 4.6 x 150 mm, Waters Inc., Ireland) and a Spherisorb/ODS 2 guard column (5 µm, 4.6 x 30 mm). HPLC was conducted using a Waters HPLC 600 controller, containing dual pumps (600E) and a photodiode array detector (waters 2996) and a dual wavelength detector (Waters 2487), monitoring at 210 nm and 254 nm. An isocratic system of 7:3 acetonitrile (MeCN):H2O over 60 minutes, with a flow rate of 1 mL/min. The compound of interest eluted from 52 to 56 minutes (Fig. 2-3). Proton nuclear magnetic resonance (1H-NMR) and carbon- 13 nuclear magnetic resonance (13C-NMR) were recorded in chloroform-d

(CDCl3, 99.8 atom %, Sigma-Aldrich, St. Louis, MO), using a JEOL Eclipse 400 MHz spectrometer (United Kingdom). Proton NMR for the crude EtOAc:MeOH extracts from the D. dolabrifera skin, body parts, mucus and excrement, and S. striatus skin, digestive gland and excrement were evaluated as well using the above protocol (Chapter 3). The compound and the crude digestive gland were analysed on a JEOL JMS LC-mate mass spectrometer (Fig. 2-4 a, b & Fig. 2-5).

2.2.4 Bioassays The P. falciparum assay is a DNA-based fluorimetric assay using the W2 strain of the parasite which is chloroquine resistant (Corbett et al., 2004). The T. cruzi assay uses a colorimetric method based on amastigotes of a T. cruzi strain expressing β-galactosidase (Buckner et al., 1996). The Leishmania donovani assay uses a DNA-based fluorimetric bioassay for the amastigote form of the parasite (Calderón et al., 2006). The dengue fever assay uses a colorimetric method, in which extract-treated cells are exposed to the virus and cell protection

Figure 2-3: HPLC chromatogram at 254 nm (green) and 210 nm (red). Pure 5α,8α-epidioxycholest-6-en-3-β-ol eluted at 54 minutes.

Figure 2-4: MS spectral data for a) D. dolabrifera crude digestive gland and b) pure 5α,8α-epidioxycholest-6-en-3-β-ol.

Figure 2-5: Isolation of 5α,8α-epidioxycholest-6-en-3-β-ol

is measured (Ureña, Personal Communication). The cancer assay uses MCF-7 breast cancer cell line in a colorimetric bioassay (Monks et al., 1991). The general cytotoxicity uses green monkey Vero kidney cells in MTT cell proliferation colorimetric assay (van de Loosdrecht et al., 1994).

2.3 Results A white crystalline solid was isolated. Its 13C-NMR spectra was compared to the literature and matched the known compound, 5α,8α-epidioxycholest-6-en-3-β-ol (3) (Fig. 2-6 & Table 2-1) (Abourriche et al., 2000; Gauvin et al., 2000; Aknin et al., 1998; Miyamoto et al., 1988). The proton NMR spectra was compared with the literature and matched the known compound 5α,8α-epidioxycholest-6-en-3-β- ol (Table 2-1) (Abourriche et al., 2000; Gauvin et al., 2000; Aknin et al., 1998).

MS data yielded m/z 416.4, 398.2, 382.4 and 364.3, consistent with 5α,8α- epidioxycholest-6-en-3-β-ol and its compound fragments (Gauvin et al., 2000; Sera et al., 1999; Miyamoto et al., 1988; Gunatilaka et al., 1981).

Activity was shown in two in vitro Panama ICBG bioassays. There was strong activity of 5α,8α-epidioxycholest-6-en-3-β-ol towards L. donovani at IC50 of 4.9 µm (Table 2-2). The compound was also tested for activity in Plasmodium falciparum (the causative agent of malaria), Trypanosoma cruzi (the causative agent of Chagas’ disease), and in MCF-7 (human estrogen receptor-positive breast cells) tumour cell line, but it showed no detectable activity (IC50 > 10

µg/mL). The cytotoxicity towards mammalian Vero cells has an IC50 of 281 µM. The therapeutic window of 5α,8α-epidioxycholest-6-en-3-β-ol towards L. donovani is 57.3.

Figure 2-6: Structure of 5α,8α-epidioxycholest-6-en-3-β-ol (3) with carbon atoms numbered.

Table 2-1: NMR Data for 5α,8α-epidioxycholest-6-en-3-β-ol (3) (CDCl3). Recorded at 400 MHz.

Atom Number δH [multiplicities, J(Hertz)] δC (multiplicities)

1 34.667 (CH2)

2 30.102 (CH2) 3 3.9660 (m) 66.470 (CH)

4 36.923 (CH2) 5 82.139 (C) 6 6.2332 (d, 8.4) 135.369 (CH) 7 6.4983 (d, 8.8) 130.765 (CH) 8 82.139 (C) 9 51.039 (CH) 10 36.923 (C)

11 23.395 (CH2)

12 39.408 (CH2) 13 44.715 (C) 14 51.559 (CH)

15 20.604 (CH2)

16 28.228 (CH2) 17 56.400 (CH)

18 0.7880 (s) 12.606 (CH3)

19 0.8714 (s) 18.158 (CH3) 20 35.210 (CH)

21 0.8888 (d, 6.6) 18.556 (CH3)

22 35.929 (CH2)

23 23.778 (CH2)

24 39.408 (CH2) 25 27.969 (CH)

26 0.8504 (d, 6.6) 22.791 (CH3)

27 0.8549 (d, 6.6) 22.524 (CH3)

Table 2-2: In vitro activity of 5α,8α-epidioxycholest-6-en-3-β-ol (3) in Panama

ICBG assays. Active results are in bold. Figures in brackets are IC50 values measured in micromolars (µM).

§ IC50 µg/mL

(IC50 µM)* Leishmania Malaria Chagas’ disease Cancer Cytotoxicity

L. donovani P. falciparum T. cruzi MCF-7 Vero cells

2.05 > 10 > 10 > 10 117 (4.9) (281)

tested at 10, 2, 0.4, 0.08 and 0.016 µg/mL § activity < 10 µg/mL * activity < 10 µM

2.4 Discussion 5α,8α-Epidioxycholest-6-en-3-β-ol (3) (Fig. 2-6) was the sole detectable component in the D. dolabrifera digestive gland (Fig. 2-4 a, b). Proton NMR revealed no sign of the epidioxysterol in the skin, body parts, albumen gland, mucous or excrement of the D. dolabrifera. In D. dolabrifera the compound was concentrated in its digestive gland rather than any other tissue in its body. This is unlike the findings of Jiménez et al. (1986) who examined its prostate glands, digestive glands and other body parts of two Aplysia spp. and found that the epidioxysterol composition was distributed evenly throughout the bodies. Additionally, no sign of this compound was found in the digestive gland, skin or excrement of S. striatus (Chapter 3). This suggests that the presence and location of epidioxysterols differs between sea hare species. The reason why epidioxysterols are evenly distributed throughout certain sea hares (Jiménez et al., 1986), are concentrated in its digestive gland, or are lacking entirely from its body, has yet to be studied.

Through a comparative analysis of the MS data of the three species of cyanobacteria collected from the same tidal pools as D. dolabrifera it was determined that neither the epidioxysterol nor its precursors appear to be of cyanobacterial origin. These samples did not contain m/z 416 or its fragments m/z 384, 369, 366, 351, 271, 253 or 251. It has been suggested that D. dolabrifera feeds on the microalgae and microbial film on the underside of rocks (Paul, Personal Communication; Rudman, 2002b).

Linking statement As noted in Chapter 1, through direct evaluation of the D. dolabrifera digestive gland, it was not possible to identify diet-derived compounds. The hypothesis of this thesis states that because D. dolabrifera is a sea hare it too should contain a generic mechanism for sequestration of diet-derived compounds, as described by Pennings and Paul (1993). Following the chemical analysis of the D. dolabrifera digestive gland in Chapter 1, in order to evaluate whether the sea hare is capable of sequestering diet-derived compounds, compounds needed to be isolated from the preferred food of D. dolabrifera, then their presence evaluated in the sea hare’s tissues. As such, as a first step, in Chapter 2, I evaluated the preferences of D. dolabrifera using several species of cyanobacteria and algae found in its habitat.

Chapter 2: Feeding preferences of Dolabrifera dolabrifera 3.1 Background D. dolabrifera is a generalist herbivore (Paul, Personal Communication) that feeds on algae (Prince and Johnson, 2006; Camacho-García et al., 2005) and on bacterial and algal film found on the underside of rocks (Hermosillo, Personal Communication; Paul, Personal Communication; Rudman, 2002b). In a recently published study on sequestered anti-predator proteins in ink, Prince and Johnson (2006) fed D. dolabrifera either the cyanobacterium Lyngbya majuscula or the cyanobacterium Hormothamnion enteromorphoides or the green alga Entermorpha clathrata. Through monitoring fecal matter production and the disappearance of food, they confirmed that D. dolabrifera fed upon L. majuscula and E. clathrata. D. dolabrifera which were fed the cyanobacterium H. enterophoides died during the feeding experiment (Prince and Johnson, 2006).

It has been documented that cyanobacteria constitute part of the diets of the sea hares Stylocheilus striatus (Paul and Pennings, 1991), D. auricularia (Pennings et al., 1993) and Bursatella leachii (Capper et al., 2005). As the article by Prince and Johnson (2006), had not been published at the time of our feeding studies, it was unknown whether or not the generalist grazer, D. dolabrifera, fed on cyanobacteria. Even still, the question remained whether D. dolabrifera preferred certain types of algae and cyanobacteria over others.

3.2 Materials and Methods 3.2.1 Study Sites Collections were made at the end of May 2006 from Boca Grande, a rocky and sandy beach from the Coiba Island National Park, Panama (Fig. 2-1) (07°23´ 41.7" north, 81° 39´ 03.7" west). Boca Grande is composed of a giant sedimentary rock cliff, with its peninsula eroded to sea level at low tide. During low tide, this site covers approximately 60 x 30 m2 of beach, which is covered during high tide.

3.2.2 Study Organisms In small, hot, intertidal puddles during the lowest part of the tidal cycle three cyanobacterial samples were collected, including Lyngbya majuscula, which grew as long hair-like filamentous 10 cm long strands, in small clumps, on rock and sand (Fig. 3-1 a), Lyngbya majuscula, which grew as a mat (~ 5 m2) on sand (Fig. 3-1 b); and Symploca sp., which grew as small, firm, leaf-like tuffs, 3 cm tall, attached to sand (Fig. 3-1 c).

Algal samples were also collected, including Chaetomorpha sp. (Fig. 3-1 d), a green alga, which grew as long woolly masses attached to red alga Spyridia sp., Cladophora sp., a green alga, which grew as feathery masses attached to sand (Fig. 3-1 e); and Spyridia sp., a red alga, which grew in abundance in a bush like mass attached to sand (Fig. 3-1 f).

Collections of the cyanobacteria and algae were maintained in aerated aquaria in fresh seawater until they were needed for each experiment. A large portion of each algal and cyanobacterial collections were frozen at -20 ºC in a minimal amount of seawater for chemical analyses. Voucher samples were preserved in

75% EtOH to 25% H2O for identification.

Eighteen D. dolabrifera were collected from the underside of uneven boulders in intertidal pools, maintained in a seawater aquarium, and fasted for 24 hours. Five non-feeding assay animals were euthanized in freezing seawater and stored at - 20 ºC. The remaining 13 animals were maintained in aquaria, in fresh seawater and were used in both the multiple choice and no-choice feeding assays. After the feeding preference assays, these animals were fasted for 24 hours to evacuate their digestive system, euthanized in freezing seawater, and stored at - 20°C.

3.2.3 Multiple Choice Assay Animals (n = 13) were placed individually in separate 2 L aluminium aquaria in fresh seawater. Each sea hare was offered a choice between five food types. The three species of cyanobacteria were mat-like Lyngbya majuscula, hair-like Lyngbya majuscula and Symploca sp.; and two species of algae, the red alga, Spyridia sp., and the green alga, Chaetomorpha sp.. Food pieces were blotted dry, weighed (to the nearest 0.01 g), and divided into treatment and no-herbivore control groups. A no-herbivore control group was used to measure natural changes in the algal mass, caused by any change in the absence of the herbivore. For example, weight changes could be caused by algae kept in aquaria under conditions outside of their natural environment. After 2.5 days, the blotted wet weight was taken for the food pieces. The control adjusted mean for each replicate was calculated for the five food types using the equation [(T(i-f)- C(i-f)], where Ti and Tf are the initial and final weights of the treatments, and Ci and Cf are the initial and final weights of the controls. Results from the assay were analysed using Friedman’s repeated measures ANOVA, followed by Tukey’s post-hoc non-parametric test.

3.2.4 No-Choice Assays The no-choice assays consisted of the consumption by D. dolabrifera on two separate diets consisting of the hair-like cyanobacterium L. majuscula or the green alga Cladophora sp.. The food pieces were blotted dry, weighed (to the nearest 0.1 mg), and divided into treatment and no-herbivore control groups. Cyanobacterium or alga were provided to sea hares (n = 7 fed L. majuscula, and n = 6 fed Cladophora sp.), kept in separate 2 L aluminium aquaria in fresh seawater. No-herbivore controls again were used to control for changes in algal mass throughout the experiment in the absence of herbivores. After 2.5 days, the food pieces were blotted and wet weights taken. The control adjusted consumption was measured as a proportion using the formula [(Ti-Tf)/Ti - (Ci- Cf)/Ci.]. The relative proportion was calculated to compensate for variation between replicates which was caused by failing to standardize initial food weight

(0.5 g). A T-test for two-samples assuming unequal variances was used to look for significance between the algal and the cyanobacterial fed groups.

3.3 Results 3.3.1 Multiple Choice Assay Thirteen D. dolabrifera were used at the beginning of the experiment, but only 10 were used in the statistical analyses, excluding, one sea hare that died and two sea hares that consumed less than 5% of food offered to them, as animals who consumed less-than 5% or more-than 95% of food were considered outliers. Friedman’s repeated measures ANOVA was used to detect differences in consumption of the five food types by D. dolabrifera. The control adjusted mean, standard deviation, and standard error of each food type was used in the analysis. A significance of P=0.002 was detected. In order to detect where the significance was coming from, a Tukey’s Post hoc test was used to examine all possible pair combinations for significant differences between groups. This procedure revealed a difference between the L. majuscula strains and the red alga Spyridia sp., but no difference between the L. majuscula strains and the Symploca sp. or to the green alga Chaetomorpha sp. (Fig. 3-2).

3.3.2 No-Choice Assay Six animals were in the group that was fed the alga and seven were in the group that was fed the cyanobacterium. One animal was removed from the cyanobacterial group’s analysis in order to maintain an equal number of replicates in both treatment groups. The animal excluded had a weight three to seven times greater than any other replicate.

D. dolabrifera statistically preferred cyanobacterium L. majuscula over green alga Cladophora sp., P=0.0062. These results showed that Cladophora sp. was not readily consumed, at -0.5%, whereas the L. majuscula was readily consumed at 15% (Fig. 3-3).

Figure 3-3: No choice feeding assay. Data shown are the mean proportion of food consumed by D. dolabrifera. Paired sample t-tests were used to analyse data (P < 0.05). n = number of replicates.

3.4 Discussion It would appear that a broad generalist diet consisting of algae (Camacho-García et al., 2005), algal and bacterial film found on the underside of rocks (Hermosillo, Personal Communication; Paul, Personal Communication; Rudman, 2002b), and strains of cyanobacterium Lyngbya majuscula, would help D. dolabrifera survive a variety of different environmental and ecological conditions as they would not be dependent on only a few types of food for their survival. For example, it was noted that during the March collecting trip, there were many different species of algae and cyanobacteria, and 35 D. dolabrifera were collected. During the collecting trip the weather was sunny and the beach was undisturbed. However, 2.5 months later at the end of May during the feeding assay collection trip, there were only five collectable species of algae and cyanobacteria, lightly dispersed about the beach. Moreover, there were no algae or cyanobacteria in the same tidal pools where the 20 D. dolabrifera were collected. It was stormy the entire collecting trip and subsequently, the beach was very disturbed which may account for the differences in the abundance of cyanobacteria and algae. Thus, it would be interesting to study whether the local population of D. dolabrifera is lower when the beach is disturbed and when macroalgae and cyanobacteria are lower in abundance.

D. dolabrifera is the only sea hare from the sub-family Dolabriferinae known to feed on cyanobacteria (Fig. 1-3). Phyllaplysia padinae feeds on the brown alga Padinae and on the eel grass Zostera; Phyllaplysia taylori feeds on the eel grass Zostera (Behrens and Hermosillo, 2005; Camacho-García et al., 2005), and Petalifera petalifera feeds on sea grass leaves and algae (Rudman, 2005).

From the sub-family Aplysiinae, Aplysia spp. feed on red, brown and green algae (Behrens and Hermosillo, 2005; Camacho-García et al., 2005) and Syphonota geographica feeds on sea grass (Gavagnin et al., 2005). Sea hares from the sub- family Dolabellinae, such as D. auricularia, feeds on cyanobacteria (Luesch et al., 2001) and on green, red and brown algae (Pennings et al., 1993). From the sub-

family Notarchinae, Bursatella leachii, feeds on cyanobacteria (Ramos et al., 1995) and, as previously stated, Stylocheilus striatus specializes on the cyanobacterium Lyngbya majuscula (Paul and Pennings, 1991). Beside D. dolabrifera, all the known cyanobacteria grazers are from the Notarchinae and Dolabellinae sub-families (Fig. 1-3).

Linking Statement Having determined that D. dolabrifera feed on and prefer cyanobacteria through two types of feeding assays, I then sought to determine whether D. dolabrifera is capable of sequestering cyanobacterial metabolites and, if so, where these cyanobacterial metabolites are stored in the organism.

Chapter 3: The evaluation of a generic mechanism for secondary metabolite sequestration in sea hares Dolabrifera dolabrifera and

Stylocheilus striatus

4.1 Background 4.1.1 Generic Mechanism in Sequestration Marine chemical studies have been conducted in order to analyze the food ingested by marine life to determine if the organisms sequester dietary metabolites. These sequestered compounds have shown promise in the area of drug discovery in some cases, the best known example of which is dolastatin-10 (Pettit et al., 1987). Of particular interest to marine chemical ecologists is the evaluation of the ecological roles and locations of sequestered cyanobacterial secondary metabolites in marine organisms (Cruz-Rivera and Paul, 2007). In sea hares, compounds sequestered from dietary sources have been found in the organism’s digestive gland. From their natural diet of the cyanobacterium L. majuscula, S. striatus have been known to sequester malyngamide A (4) and B (5) (Paul and Pennings, 1991), lyngbyatoxin A (6) (Capper et al., 2005), aplysiatoxin (7), and debromoaplysiatoxin (8) (Fig. 4-1) (Kato and Scheuer, 1974). While these compounds are stored principally in their digestive gland, trace amounts have also been detected in their skin, eggs, and ink glands (Capper et al., 2005; Paul and Pennings, 1991).

Three species of sea hares, Aplysia californica, D. auricularia and S. striatus, are thought to have generic mechanisms for the sequestration of secondary metabolites (Pennings and Paul, 1993). This mechanism allows them to sequester compounds from a variety of sources and store them almost exclusively in their digestive glands rather than in their skin or external parts.

Figure 4-2: Secondary metabolites that S. striatus is capable of sequestering, from green algae (9 & 10), brown algae (11 & 12), and sponge (13 & 14) (Pennings and Paul, 1993).

For example, through a no-choice feeding assay in which pure compounds were individually incorporated into an artificial food, S. striatus sequestered and stored the following metabolites in its digestive gland: the green algae metabolites caulerpenyene (9) and halimedatetraacetate (10), the brown algae metabolites pachydictyol A (11) and ochtodene (12), the sponge metabolites luffariellolide (13) and brominated diphenyl ether (14), and L. majuscula metabolites malyngamide A (4) and B (5) (Fig. 4-1 & Fig. 4-2) (Pennings and Paul, 1993).

The three focal questions of this Chapter are 1) Does the sea hare D. dolabrifera have a generic mechanism for sequestering as described by Pennings & Paul (1993) and storage of dietary derived compounds? 2) Does the sea hare S. striatus contain a generic mechanism? 3) Can chemical ecological studies of D. dolabrifera and S. striatus lead to the discovery of novel compounds with the potential to serve as medicines?

4.1.2 Stylocheilus striatus S. striatus is a specialist feeder of the cyanobacterium, L. majuscula (Pennings and Paul, 1993) which it significantly prefers over the cyanobacteria Schizothrix and Tolyphthrix, the seagrasses Zostera and Halophila, the red alga Acanthophora, the green algae Bryopsis, Caulerpa and Enteromorpha, and the brown algae Padina, Sargassum, Dictyota and Lobophora (Table 4-1) (Capper et al., 2006b; Cruz-Rivera and Paul, 2006; Paul and Pennings, 1991).

4.1.2.1 Malyngamide A and B When fed to S. striatus, malyngamide A (4) and B (5) were shown as feeding stimulants at their lower natural algal concentrations and feeding deterrents at their higher natural algal concentrations (Fig. 4-1) (Nagle et al., 1998). This suggests S. striatus are sensitive to chemical cues from their cyanobacterial food source L. majuscula and may choose not to graze on their preferred food source if secondary metabolite concentrations are too high (Nagle et al., 1998).

Table 4-1: Stylocheilus striatus feeding preference

Algal Food Algal Food General Name Type of Result Source Option Option Preference (by genus) (by species) Assay Lyngbya majuscula cyanobacterium Multiple (P < 0.001) (Cruz- Acanthophora spicifera red alga Choice preferred Rivera and Bryopsis pennata green alga Padina tenuis brown alga Lyngbya Paul, Sargassum cristaefolium brown alga majuscula 2006) Tolypothrix sp. cyanobacterium

Lyngbya majuscula cyanobacterium Multiple (P < 0.0001) (Capper et Zostera capricorni seagrass Choice preferred al., 2006b) Halophila ovalis seagrass Acanthophora specifera red alga Lyngbya Caulerpa taxifolia green alga majuscula Dictyota dichotoma brown alga Sargassum flavicans brown alga Lobophora variegata brown alga

Lyngbya majuscula cyanobacterium Paired (0.031 ≤ P (Paul and vs. Choice ≥ 0.008) Pennings, Enteromorpha clathrata green alga Dictyota cervicornis brown alga Test preferred 1991) Padina tenuis brown alga Lyngbya Schizothrix calcicola cyanobacterium Schizothrix mexicana cyanobacterium majuscula (tufted) Schizothrix mexicana cyanobacterium (prostrate)

In live feeding assays, fish predation was not significantly different upon S. striatus previously fed a diet containing malyngamides A (4) and B (5) as opposed to S. striatus fed a diet which did contain these metabolites (Fig. 4-1). Fish identity was a more significant factor on S. striatus palatability, than the presence or absence of the secondary metabolites it contained (Pennings et al., 2001). Malyngamide A and B are deterrents towards the omnivorous pufferfish Canthigaster solandri, the crab Leptodius spp. (Pennings et al., 1996), the juvenile rabbitfish Siganus spinus, and the juvenile parrotfish Scarus schlegeti (Thacker et al., 1997). Malyngamide B acetate (15) (Fig. 4-1), a less toxic form derived by S. striatus from malyngamide B (Paul and Pennings, 1991), did not deter feeding (Pennings et al., 1996). Malyngamide A and B deter feeding in a wide variety of mesograzers. This suggests that S. striatus that live on this chemically protected habitat could benefit from the lack of predation, from crabs and fish, as predators avoid L. majuscula, containing secondary metabolites malyngamide A and B (Paul et al., 2001).

4.1.2.2 Other Secondary Metabolites Other L. majuscula secondary metabolites also influence S. striatus feeding behaviour and have been well studied. At natural concentrations, barbamide (16) (Nagle et al., 1998) and pitipeptolide A (17) (Cruz-Rivera and Paul, 2007) stimulate feeding (Fig. 4-1). By contrast, ypaoamide (18) , malyngolide (19) , and microcolin B (20) deter feeding at natural concentrations (Fig. 4-1) (Nagle et al., 1998). However, at lower concentrations, S. striatus are indifferent to ypaoamide (18) and malyngolide (19) (Fig. 4-1) (Nagle et al., 1998). Some secondary metabolites act as feeding deterrents at high natural concentrations and feeding stimulants at low natural concentrations. Majusculamides A (21) and B (22) (Fig. 4-1) (Nagle et al., 1998) are examples of this. This then suggests that chemical cues produced by L. majuscula may influence S. striatus grazing behaviour (Fig. 4-1) (Nagle et al., 1998).

The effects of other mesograzer feeding on L. majuscula have shown that crude extracts deter feeding towards amphipods Parhyale hawaiensis and Cymadusa imbroglio, majid crab Menaethius monoceros, urchin Echinometra mathaei, cephalaspidean Diniatys dentifer, wild caught rabbitfish Siganus fuscescens (Capper et al., 2006a), adult rabbitfish Siganus arenteus (Paul et al., 1990), surgeonfish Zebrasoma flavescens (Wylie and Paul, 1988) and herbivorous and carnivorous reef fish (Paul and Pennings, 1991).

Although the secondary metabolites of L. majuscula vary according to geographic location, in general, L. majuscula crude extract and its defensive secondary metabolites have been shown to deter most generalist herbivores at natural concentrations. At the same time, many of these secondary metabolites act as feeding stimulants or deterrents to S. striatus depending on their concentration (Cruz-Rivera and Paul, 2007; Nagle et al., 1998; Thacker et al., 1997).

4.1.3 Cyanobacteria Cyanobacteria, also known as blue-green algae, are among the world’s oldest organisms, originating 2.7 billion years ago (Brocks et al., 1999). They are ubiquitous in oceans, fresh water, and terrestrial environments. Cyanobacteria carry out photosynthesis and were responsible for forming the earth’s oxygenated atmosphere (Garcia-Pichel, 1998). Primary producers, also known as autotrophs, produce organic molecules from inorganic nutrients mainly through photosynthesis. Cyanobacteria are the dominant primary producers in the ocean’s intertidal zone (Whitton and Potts, 1982) and make an important contribution to the ocean’s primary productivity as a whole (Waterbury et al., 1979).

Cyanobacteria are prolific producers of secondary metabolites (Simmons et al., 2005). Oscillatoriales are filamentous cyanobacteria, are responsible for 49% of the 798 natural products reported from cyanobacteria. Lyngbya majuscula is responsible for 83% (236/389) of the natural products from Oscillatoriales

(Gerwick, 2007). The majority of the natural products isolated from L. majuscula are amino acids and are polyketide in origin (Garson, 2001).

4.1.3.1 Toxicity and Defence Cyanobacteria are capable of dramatically increasing their growth rate and forming mats which can provide both a habitat and a food source for reef mesograzers (Garson, 2001). In order to restrict predation (Nagle and Paul, 1998) and to compete for nutrients (Burja et al., 2001), L. majuscula produce secondary metabolites to kill predators or to deter feeding of these predators. Secondary metabolites that a cyanobacterium produces affect its survivorship in areas of strong herbivory (Garson, 2001). In addition, it limits the growth of other cyanobacteria and algae (Burja et al., 2001). Cyanobacteria can grow into bloom formations which produce toxic compounds such as ypaoamide (18) (Nagle and Paul, 1998), lyngbyatoxin-A (6), and debromoaplysiatoxin (8) (Fig. 4-1) (Capper et al., 2006a). Bloom formations kill fish on a large scale (Nagle and Paul, 1998) and cause dermatitis or “swimmers’ itch” in humans (Cardellina et al., 1979). As one of the most abundant benthic cyanobacteria and as one of the most toxic cyanobacterial genera, L. majuscula is a popular and most promising candidate for scientists in the fields of marine chemical ecology, natural products chemistry, and drug discovery (Burja et al., 2001).

4.1.3.2 Bioactive Sequestered Compounds Many interesting compounds such as Dolastatins from D. auricularia were first isolated from the sea hare and later discovered as sequestered compounds from their algal food sources (Luesch et al., 2002). There are several examples of potential anti-cancer agents that S. striatus sequesters from L. majuscula (Luesch et al., 2002). These compounds are maintained or transformed to a less toxic acetate in the animal’s digestive gland (Pennings and Paul, 1993). S. striatus sequesters malyngamide A (4) (Gerwick et al., 2001) and modifies it to malyngamide O (23) (Fig. 4-1) (Winklet et al., 2000) or stores it unmodified (Paul and Pennings, 1991). It sequesters lyngbyatoxin A (6) (Cardellina et al., 1979)

and then modifies it to lyngbyatoxin A acetate (24) (Gallimore et al., 2000) or stores it as the original natural product (6) (Fig. 4-1)(Capper et al., 2005). S. striatus also sequesters aplysiatoxin (7) (Fig. 4-1) (Fujiki et al., 1985) and stores it in its unmodified form (Kato and Scheuer, 1974).

4.1.4 Bioassay-Guided Fractionation Bioassay-guided fractionation is used to isolate compounds based upon their biological activity. For example, a crude sample is fractionated into approximately 6 to 8 parts and the crude and each fraction are tested for bioactivity. If activity is present in a given fraction, chemical techniques such as liquid chromatography are used to isolate the compounds from the fraction and, when warranted, the chemical structures are determined with a combination of spectroscopic techniques, principally nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS). In marine chemical ecological studies such as this one, bioassay guided-fractionation can also facilitate the study of sequestration of biologically active compounds by the animals in question.

4.2 Materials and Methods 4.2.1 Study Site As in section 3.2.1 on page 32.

4.2.2 Study Organisms Refer to Chapter 2 (section 3.2.2 page 33) for collection details of D. dolabrifera and L. majuscula (hair type). Nine S. striatus were found grazing on L. majuscula (hair type) that was collected for use in the feeding experiments. They were maintained in an outdoor flow-through seawater aquarium with a 12:12 hour light:dark cycle. They were allowed to graze for five days, fasted for 24 hours, euthanized in freezing seawater and then stored at -20ºC.

4.2.3 General Experimental Procedures Low-resolution mass spectra (MS) were measured in MeOH and analysed by direct-injection on a JEOL LC mate mass spectrometer (Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra data was collected using a JEOL Eclipse 400 MHz spectrometer (United Kingdom). Fractionation was accomplished using a reverse-phase solid phase extraction (SPE) cartridge (Supelco Discovery DSC- 18, 60 mL Tubes, 10 g, Bellefonte, PA) under manually applied pressure. High performance liquid chromatography (HPLC) was carried out in reverse phase using a Prontosil-120 C18 analytical column (4.6 x 250 mm, Bischoff Chromatography, Leonberg, Germany) and a Merck Hitachi HPLC (Tokyo, Japan) containing dual pumps (L-7100) and a diode array detector (L-7455) monitoring at 210 nm. All of the experimental procedures were carried out at the INDICASAT laboratories.

4.2.4 Chemical Analyses of L. majuscula 4.2.4.1 Extraction and Analyses of Crudes L. majuscula (hair type,1, TC-94) was freeze dried, blended, and extracted three times for 24-36 hour periods in 1:1, EtOAc:MeOH. After each extraction, the solvent was filtered and concentrated (9401). The same L. majuscula sample was then extracted twice for 24-36 hour periods in 1:1, EtOH:H2O, and the solvent was filtered and concentrated (9402) (Fig. 4-3). The EtOAc:MeOH extract

(9401) and the EtOH:H2O extract (9402) were analysed by MS. The EtOAc:MeOH extract (9401) revealed the following mass peaks: m/z = 887.1, 717.5, 703.5, 537.4, 313.3, 279.2, 245.2, 227.2 and 213.2 (Fig. 4-4 a); whereas, the EtOH:H2O extract (9402) had only two non-solvent peaks m/z = 717.5 and 285.2 (Fig. 4-4 b). Since the EtOAc:MeOH extract (9401) contained more potential for compound isolation as determined by MS data, it became the focus of further fractionation.

Figure 4-3: Extraction, fractionation and compound isolation of hair type L. majuscula (2, TC-94). Compounds 9401D1 and 9401D2 were isolated. Bolded path indicates fractionation pathway.

Figure 4-4: MS data from L. majuscula extracts (a) 9401 and (b) 9402

4.2.4.2 Fractionation of the EtOAc:MeOH extract (9401) The EtOAc:MeOH extract (9401) was fractionated using a reverse-phase SPE cartridge in a stepwise fashion, starting with a more polar solvent system moving towards a less polar solvent system (Table 4-2, Fig. 4-3). The SPE column was equilibrated in 50:50 MeOH:H2O, and 500 mg of the EtOAc:MeOH (9401) extract was loaded onto the cartridge. The sample was sequentially eluted with 150 mL each of MeOH:H2O in the ratios of 50:50, 60:40, 70:30, 80:20 and 90:10 followed by 150 mL each of MeOH, EtOAc and acetone (Table 4-2).

The eight SPE fractions (9401A through 9401H) were screened in bioassays for activity in L. donovani, MCF-7 breast cancer cell lines, T. cruzi, P. falciparum and dengue fever infected Vero cells. IC50 values were determined for active fractions in each assay (Table 4-3). The fraction 9401D showed activities in the cancer bioassay with an IC50 value of 6.0 µg/mL, and in the malaria bioassay with an 1 IC50 of 1.0 µg/mL. Following this, fraction 9401D was then analyzed by H-NMR in chloroform-d (CDCl3, 99.8 atom %, Sigma-Aldrich, St. Louis, MO). The proton spectral data revealed several peptide-like signals. Due to strong activity in malaria and the promising NMR results, fraction 9401D was subjected to bioassay-guided fractionation.

4.2.4.3 Isolation of compounds 9401D1 and 9401D2 Compounds were isolated from fraction 9401D using reverse-phase HPLC (Fig. 4-3). Fraction 9401D was dissolved in MeOH and filtered at 0.45 µm (Altech 17 mm PTFE syringe filters). Compound separation was accomplished using an isocratic system of 55% acetonitrile (MeCN) and 45% H2O, with a flow rate of 1 mL/min. Pure compounds 9401D1 and 9401D2 eluted at 23 and 31 minutes respectively. All eluent other than peaks one and two were collected together (9401DAE) (Fig. 4-5). The column was washed in 100% MeOH and collected (9401DW). The compounds dried as white solids and had ultraviolet (UV) absorbance at 200 nm.

Table 4-2: L. majuscula EtOAc:MeOH extract (9401) fractionation using reverse phase solid phase extraction (SPE).

Fraction Solvent mixture Volume Mass (mL) (mg)

9401A 50% MeOH, 50% H2O 150 156.9

9401B 60% MeOH, 40% H2O 150 16.2

9401C 70% MeOH, 30% H2O 150 29.2

9401D 80% MeOH, 20% H2O 150 24.6

9401E 90% MeOH, 10% H2O 150 172.0 9401F 100% MeOH 150 85.4 9401G 100% EtOAc 150 54.2 9401H 100% Acetone 150 0.8

Table 4-3: L. majuscula (9401) extract and SPE fraction bioassay results. Active screens and active IC50 values are in bold.

Category Anti- Anti-cancer Anti-malaria Anti-T.cruzi Cytotoxicity leishmania Bioassay L. donovani MCF-7 P. falciparum Trypanomas Vero 225 amastigote (W2) Intracel cell † † ‡ ‡ § Measure- % growth % growth % inhibition % inhibition IC50 µg/mL § § ment (IC50 µg/mL) (IC50 µg/mL)

9401 81.85 131 8.8 9401A 124.01 110 -8.3 10.53 74 9401B 112.08 66 12.3 11.02 33 9401C 83.63 -76 66.8 58.67 22 (6) (> 10) 9401D 81.67 -70 73.5 9.69 27 (6.3) (1) 9401E 102.71 111 7.9 13.47 77 9401F 101.89 111 5.2 7.94 82 9401G 95.92 86 -4.3 5.10 84 9401H 97.31 102 0.4 8.32 27 † activity < 50% growth ‡ activity > 50% inhibition § IC50 activity < 10 µg/mL No cell protection against dengue fever infected vero cells was observed in any of the samples

Figure 4-5: HPLC of L. majuscula fraction 9401D. Compounds 9401D1 eluted at

23 minutes and 9401D2 eluted at 31 minutes. Reverse phase C18 HPLC, 55%

MeCN, 45% H2O.

The pure compounds eluted as 2-3 peaks, possibly due to conformational changes within each compound. For compounds 9401D1 and 9401D2, 1H-NMR spectra were recorded in CDCl3, and 1D and 2D NMR spectra were recorded in methylene chloride-d2 (CD2Cl2 “100%”, Cambridge Isotope Laboratories, Inc., Andover, MA, USA) (Fig. 4-6 b, c).

Compounds 9401D1 and 9401D2 were analysed by MS. The molecular weight of 9401D1 is m/z = 704 [M+1] and for 9401D2 is m/z = 718 [M+1] (Appendix B). Compounds 9401D1 and 9401D2, and fractions 9401DAE and 9401DW were screened for bioactivity in L. donovani, MCF-7 cancer cell lines, P. falciparum, T. cruzi and dengue fever. IC50 values were determined for active fractions or compounds. The compounds’ cytotoxicity to mammalian Vero cells were measured as IC50 values. The IC50 values for the compounds were measured in micromolars (µM) (Table 4-4).

4.2.4.4 Sequestration Experiment In order to evaluate compound sequestration by the sea hares, fraction 9401D and pure 9401D1 and 9401D2 were re-injected in the HPLC at 0.1 µg/mL in an isocratic system of 68% MeCN and 32% H2O at 1 mL/min (Fig. 4-7 a, b, c).

4.2.5 Chemical Analyses of Sea Hares 4.2.5.1 Dissection and Extraction Three D. dolabrifera that were not exposed to the L. majuscula and were not used in the feeding experiments, were divided into three replicates and dissected into skin (parapodia, foot and head), digestive gland, albumen gland and remaining internal organs (body parts) (Fig. 2-2). Six D. dolabrifera that were exposed to the L. majuscula in the feeding experiments were divided into three replicates and dissected into skin (parapodia, foot and head), digestive gland, albumen gland and remaining internal organs (body parts) (Fig. 2-2).

Figure 4-6: Proton NMR data of (a) S. striatus digestive gland showing compound sequestration of L. majuscula compounds (b) 9401D2 and (c) 9401D1.

Table 4-4: Bioassay values for L. majuscula fraction 9401D fractionated through HPLC, isolating compounds 9401D1 and 9401D2. Active samples are in bold.

Category Anti- Anti-cancer Anti-malaria Anti-T.cruzi Cytotoxicity leishmania Bioassay L. donovani MCF-7 P. falciparum Trypanomas Vero 225 amastigote (W2) Intracel cell § § § § § Measure- IC50 µg/mL IC50 µg/mL IC50 µg/mL IC50 µg/mL IC50 µg/mL

ment (IC50 µM)* (IC50 µM)* (IC50 µM)*

9401D > 10 > 10 1 > 10 27 9401D1 4.83 > 10 3.0 > 10 24 (6.87) (4.27) (34.14) 9401D2 > 10 > 10 3.0 > 10 21 (> 10) (4.18) (29.29) 9401DAE 1 1.23 5.5 3.0 > 10 1.2 9401DW > 10 > 10 >10 > 10 100 § activity < 10 µg/mL * activity < 10 µM No cell protection against dengue fever infected vero cells was observed in any of the samples

1 Proton NMR spectral analysis of this fraction, revealed cyclopropyl signals, which had chemical shifts between 0.150 - 0.125 and 0.138 - 0.148. Further investigations of these signals revealed that the signals were that of phthalate esters, a common contaminant from plastics encountered in natural products research and most probably the source of this fraction’s activities.

Nine S. striatus collected grazing on the L. majuscula (L. majuscula exposed S. striatus) were divided into three replicates and dissected into skin (parapodia, foot and head), digestive gland and remaining internal organs (body parts) (Fig. 4-8). These animal tissues along with excrement from the D. dolabrifera and S. striatus fed L. majuscula during their 24 hour post feeding fast, an egg mass from a S. striatus and mucous from D. dolabrifera were dried and extracted as described in section 2.2.3.2 on page 21.

4.2.5.2 Sequestration evaluation The animal tissue extracts evaluated by HPLC consisted of the following: (i) S. striatus digestive gland replicates 1-3 and skin replicates 1-3, (ii) L. majuscula fed D. dolabrifera digestive gland and skin replicates 1-3 and (iii) non-L. majuscula fed D. dolabrifera digestive gland and skin replicates 1-3 (Table 4-5). D. dolabrifera digestive gland for both the feeding assay and non-feeding assay groups were combined for chemical analysis. The tissue samples were evaluated for presence of compounds 9401D1 and 9401D2 by injecting 0.1 mg/mL per sample using the same method that was described in section 4.2.4.4 on page 58 (Fig. 4-7 c-i).

In addition, 1H-NMR of extracts of animal tissues, excrement, mucous and egg mass were recorded in CDCl3, to compare the animal tissues with the compounds. The digestive gland and skin from each animal group was screened at 10 µg/mL for activity against L. donovani, T. cruzi, P. falciparum and MCF-7 cancer cell lines and for cytotoxicity to mammalian Vero cells. IC50 values were determined for samples that showed activity in the initial screening assay (Table 4-6).

4.2.6 Bioassays As in section 2.2.4 on page 23.

Figure 4-8: Anatomy of Stylocheilus striatus

Table 4-5: Sea hare chemical analyses breakdown

Animal Part No. of Body parts and No. of weight range weight range replicates b a a animals secretions (gwwt) (gwwt) L. majuscula 0.29 (±0.05) 0.18 (±0.04) 9 Digestive gland 3 (3) exposed 0.30 (±0.06) Skin 3 (3) S. striatus L. majuscula 1.12 (±0.11) 0.09 (±0.01) 6 Digestive gland 6 (1) exposed 0.70 (±0.10) Skin 2 (3) D. dolabrifera Non-L. 1.02 (±0.50) 0.04 (±0.01) 3 Digestive gland 3 (1) majuscula 0.29 (±0.05) Skin 1 (3) exposed D. dolabrifera a blotted wet weight in grams & number in parenthesis are standard errors b Number outside parenthesis is the amount of animals per replicate and number inside parenthesis is the number of replicates.

Table 4-6: Animal tissue biological activity evaluation. Active samples are in bold.

Category Anti- Anti-cancer Anti-malaria Anti-T.cruzi Cytotoxicity leishmania Bioassay L.donovani MCF-7 P. falciparum Trypanomas VERO 225 amastigote (W2) Intracel cell † † ‡ ‡ § Measurement % growth % growth % inhibition % inhibition IC50 µg/mL § (IC50 µg/mL)

S. striatus 81.27 104.1 92.0 22.32 10 DG a (3.0) S. striatus 106.69 107.4 5.5 9.60 Skin a D. dolabrifera 114.00 88.7 41.9 18.15 46 DG a D. dolabrifera 109.96 109.7 -4.0 9.90 >100 Skin a D. dolabrifera 113.92 98.2 49.3 10.81 99 DG b D. dolabrifera 93.91 108.7 -6.7 9.26 >100 Skin b Digestive gland (DG) a L. majuscula exposed b Non-L. majuscula exposed † activity < 50% growth ‡ activity > 50% inhibition § IC50 activity < 10 µg/mL

4.3 Results 4.3.1 Sequestration Evaluation in S. striatus Sequestration evaluation using HPLC revealed that S. striatus sequestered compounds 9401D1 and 9401D2 and stored them in its digestive gland (Fig. 4-7 d). Sequestration was confirmed in all three replicates. The 1H-NMR spectra data was used to compare the chemical shifts of the compounds isolated from L. majuscula to the chemical shift data in the S. striatus digestive gland extract revealing that sequestration had occurred. Most notably the compounds had the following chemical shifts: δ = 1.10 - 1.05, 2.90 - 3.00, 3.15, 3.45 - 3.65 and 3.85 - 4.20 (Fig. 4-6 a, b, c). Bioassay results indicated that the S. striatus digestive gland showed an antimalarial activity of 92.0% parasite inhibition when tested at a single concentration of 10 µg/mL (Table 4-5).

Compounds 9401D1 and 9401D2 were not stored in the skin of S. striatus; however, a small peak observed at 20.3 minutes seemed to indicate the presence of minute quantities of the compounds (Fig. 4-7 e). Proton NMR spectra data of the S. striatus skin, however, showed no evidence for either compound.

Additionally, proton NMR spectral data of the other internal organs from S. striatus (ink glands, buccal mass, crop, gizzard/stomach, copulatory bursa, hermaphrodite gonad, albumen gland, penis and gill) (Fig. 4-8) showed evidence for the sequestration of the compounds, as no signals from the compounds 9401D1 or 9401D2 were seen.

4.3.2 Sequestration Evaluation in D. dolabrifera In HPLC and 1H-NMR comparison experiments, no evidence of 9401D1 and 9401D2 was found in the D. dolabrifera tissues. Although, the D. dolabrifera fed significantly on L. majuscula, over the course of the feeding assays (see section 3.3 page 36), there was no HPLC evidence of the presence of the compounds 9401D1 and 9401D2 in either its digestive gland (Fig. 4-7 f) or its skin (Fig. 4-7 g). This was confirmed by proton NMR. Furthermore, proton NMR showed no

sequestration of the compounds in its albumen gland, other internal organs, mucous or excrement.

Through HPLC and 1H-NMR experiments it was revealed that the D. dolabrifera not used in the feeding assays, and which, to our knowledge, had not been previously exposed to L. majuscula, showed no evidence for sequestration of compounds 9401D1 or 9401D2 in either its digestive gland (Fig. 4-7 h) or its skin (Fig. 4-7 i).

4.3.3 Cyanobacterial Compounds 9401D1 and 9401D2 Structural elucidations of the L. majuscula peptides are to be carried out by Dr. Marcy Balunas of the Panama ICBG. The peptides are composed of a series of modified amino acids. Peptide 9401D1 showed strong in vitro activity to L. donovani with an IC50 of 6.9 µM and P. falciparum with an IC50 of 4.3 µM. Compound 9401D1 showed a moderate cytotoxicity to mammalian Vero cells with an IC50 of 34.1 µM. Compound 9401D1 has a therapeutic widow of five towards L. donovani and eight towards P. falciparum. Peptide 9401D2 showed a strong in vitro activity to P. falciparum with an IC50 of 4.2 µM and showed a moderate cytotoxicity to mammalian Vero cells with an IC50 of 29.3 µM. Compound 9401D2 has a therapeutic window of seven towards P. falciparum (Table 4-4).

4.4 Discussion 4.4.1 Generic Sequestration Mechanism in D. dolabrifera Generic sequestration mechanisms as suggested by Pennings & Paul (1993) states that sea hares as a rule sequester dietary compounds and store them almost exclusively in their digestive gland rather than in their external parts. Prior to this study, however, it was uncertain whether the particular sea hare D. dolabrifera was, capable of sequestering dietary derived metabolites and subsequently possessed this type of generic mechanism.

While I could not prove that D. dolabrifera is capable of sequestering 9401D1 and 9401D2 in this study, it is possible that D. dolabrifera may sequester other dietary derived compounds which were not detected. It is also possible that D. dolabrifera may sequester the L. majuscula compounds 9401D1 and 9401D2 under different experimental conditions. In this study the cyanobacterial exposure was comparatively short as D. dolabrifera were exposed for five days. In other compound sequestration evaluation feeding experiments, S. striatus was fed cyanobacteria for up to 20 days (Capper et al., 2005; Pennings and Paul, 1993). Sequestration rates have been shown to be different between sea hare species (Capper et al., 2005).

Differences in L. majuscula consumption between sea hare species exist as well (Capper et al., 2006b). While the consumption of L. majuscula by D. dolabrifera was 0-0.012 g/day in the no-choice feeding assay (Chapter 2), other studies showed that S. striatus consumed 0-5 g/day depending on the concentration of the secondary metabolites (Capper et al., 2006b). Even though L. majuscula consumed significantly more than any other food choice offered, perhaps the quantity of food ingested was much lower than required to properly quantify the sequestration of secondary metabolites.

Another possible explanation is that D. dolabrifera might sequester these compounds but they could be broken down into other forms that were not detected. While S. striatus is able to store some compounds like 9401D1 and 9401D2 with no obvious detrimental impacts, D. dolabrifera may not be able to tolerate L. majuscula compounds and may have to metabolize metabolites from the cyanobacterium.

The fate of L. majuscula secondary compounds varies among sea hares. For example when sequestration of lyngbyatoxin A (6) was evaluated in two species of sea hare, S. striatus and B. leachii, researchers found that S. striatus sequestered the metabolite transforming it to lyngbyatoxin A acetate (24) and

stored it in its digestive gland (Fig. 4-1) (Pennings et al., 1996). B. leachii, on the other hand, sequestered the compound, stored it in its original form in its digestive gland, and excreted it in its ink and fecal matter (Capper et al., 2005). This study is another example of how the dynamics of compound sequestration can vary among sea hare species. Again, this suggests that the generic mechanism is not contained in all sea hare species, including D. dolabrifera and B. leachii.

4.4.2 Sequestration of compounds by S. striatus This research shows that S. striatus sequesters two novel compounds 9401D1 and 9401D2 from L. majuscula. S. striatus has also been shown to sequester other L. majuscula metabolites including, malyngamide A (4) and B (5) (Paul and Pennings, 1991), lyngbyatoxin A (6) (Capper et al., 2005), aplysiatoxin (7), and debromoaplysiatoxin (8) (Kato and Scheuer, 1974), pitipeptolide (17) (Cruz- Rivera and Paul, 2007), ypaoamide (18) (Nagle et al., 1996), and majusculamides A (21) and B (22) (Nagle et al., 1998). This is the second example of a peptide sequestered by S. striatus, after pitipeptolide (17) (Fig. 4-1) (Cruz-Rivera and Paul, 2007).

S. striatus provided me with an excellent opportunity to compare sequestration in an easily maintained test organism with an animal whose capability of sequestering was uncertain, in this case D. dolabrifera. While it is possible that S. striatus did not sequester the L. majuscula compounds 9401D1 and 9401D2, and instead either produced them de novo or sequestered them from another source. Given that S. striatus has been shown in this study and others to assimilate compounds from dietary sources, I suggest that S. striatus is a good species for comparative studies in other sea hares to evaluate secondary metabolite sequestration.

S. striatus concentrated sequestered dietary secondary metabolites in its digestive gland and other internal organs, rather than the skin. This is consistent

with other sea hare sequestration studies in which dietary metabolites have been found in significantly high concentrations in the digestive gland of S. striatus (Pennings and Paul, 1993), D. auricularia (Pennings et al., 1999) and B. leachii (Capper et al., 2005) rather than in the skin.

The function of sequestered dietary metabolites in sea hares is heavily debated. Compounds concentrated in the internal organs rather than its skin are not optimally placed for defensive purposes (Pennings et al., 1999). It has been suggested that one purpose of concentrating sequestered compounds in the digestive gland might be to aid in detoxifying a diet rich in secondary metabolites (Pennings et al., 1999) or that storing them could be less metabolically expensive than detoxifying them (Pennings and Paul, 1993). No study exists at this point which identifies the ecological roles of compounds found in D. dolabrifera.

S. striatus stored the sequestered L. majuscula compounds 9401D1 and 9401D2 in its digestive gland. These compounds are active towards P. falciparum (Table 4-4). Interestingly, an extract from the S. striatus digestive gland was also active against P. falciparum (Table 4-6). Although the sequestered peptide was not isolated from the S. striatus digestive gland, it is probable that the sequestered compound is responsible for much of the activity seen in this sample.

General Discussion and Conclusions Given that many species of sea hares sequester dietary derived metabolites and store them in their digestive glands (Pennings and Paul, 1993), I hypothesized that the sea hare D. dolabrifera may also sequester dietary metabolites and concentrate them in its digestive gland. In the three preceding Chapters, I assessed whether: 1) the sea hare D. dolabrifera contained diet-derived cyanobacterial secondary metabolites by conducting a general study of the chemical compounds present in its digestive gland, 2) D. dolabrifera showed dietary preferences for one or more cyanobacteria, 3) sea hares D. dolabrifera and S. striatus were capable of sequestering the same dietary compounds and where those compounds were stored, and 4) the preferred algal food of D. dolabrifera and S. striatus contained any novel and/or bioactive compounds.

Firstly, through an examination of the digestive gland chemistry of D. dolabrifera in search of diet-derived metabolites, I isolated one of the gland’s major compounds 5α,8α-epidioxycholest-6-en-3-β-ol (3) (Fig. 2-6). Although it is not a cyanobacterial compound, it is the first time an epidioxysterol has been isolated from this sub-family. Further studies are needed to understand fully the dynamics of epidioxysterols within sea hares and of their ecological role and origins.

Epidioxysterols are common among marine invertebrates (Min-Hsiung et al., 2007; Mansoor et al., 2006; Aknin et al., 1998; Gunatilaka et al., 1981) and have shown potential as pharmaceutical agents, with significant activity towards various forms of cancer (Iwashima et al., 2002; Gauvin et al., 2000; Sheu et al., 2000). 5α,8α-Epidioxycholest-6-en-3-β-ol (3) (Fig. 2-6) as well, has also shown significant activity towards various forms of cancer (Minh et al., 2004). This study has shown that this compound also has significant activity against the amastigote stage of L. donovani with an IC50 of 4.9 µM, with a therapeutic window of 57.3.

Secondly, it was determined whether D. dolabrifera from the Boca Grande field site on the Coiba Island National Park feed on and prefer cyanobacteria or algae.

I determined the feeding preference of D. dolabrifera to be for the cyanobacterium, L. majuscula. The first experiment conducted was a multiple choice feeding assay in which D. dolabrifera were given a choice between two species of cyanobacteria, a red alga and a green alga. D. dolabrifera significantly preferred feeding on L. majuscula over the other choices (P=0.002, Fig. 3-2). Following this, a no-choice feeding assay showed that D. dolabrifera significantly preferred the L. majuscula to a green alga (P=0.0062, Fig. 3-3). D. dolabrifera is the only sea hare from the sub-family Dolabriferinae known to feed on cyanobacteria (Fig. 1-3).

Thirdly, I wished to evaluate whether sea hares D. dolabrifera and S. striatus were capable of sequestering the dietary peptides isolated from their preferred food L. majuscula and, if so, to determine where these sequestered compounds are stored. The generic mechanism for compound sequestration has been shown to operate in six sea hare species, with thirteen compounds tested (Capper et al., 2005; De Nys et al., 1996; Pennings and Carefoot, 1995; Pennings and Paul, 1993; Paul and Pennings, 1991). Capper et al. (2005) found that in the case of aplysiatoxin-A, B. leachii was excreting the majority of the diet-derived compound rather than storing it in its digestive gland.

I could, however, find no evidence that the D. dolabrifera possessed the generic mechanism for sequestering, although I hesitate to reject this notion entirely, since the mechanism has been verified in six out of seven species of sea hares tested. Future research should be conducted to evaluate the generic sequestration mechanism theory, perhaps using artificial feeding no-choice assays where a variety of secondary metabolites are fed to D. dolabrifera following the methodology used by Pennings and Paul (1993).

The presence of the generic mechanism in the sea hare S. striatus (Capper et al., 2005; Pennings et al., 1996; Pennings and Paul, 1993) was confirmed in the case of the peptides 9401D1 and 9401D2. Furthermore, it stored these peptides in its

digestive gland and other internal organs, rather than in its skin, eggs or fecal mater. In future studies evaluating the generic sequestration mechanism (Pennings and Paul, 1993) in sea hares or other opisthobranch molluscs, I suggest that sea hare S. striatus would act as an effective model for the comparison of compound sequestration in other opisthobranchs.

Finally, L. majuscula, the preferred algal food of D. dolabrifera and S. striatus (Paul and Pennings, 1991) was evaluated for novel and/or bioactive compounds. Through bioassay-guided fractionation, two novel bioactive cyanobacterial peptides were isolated. The compound 9401D1 showed strong activities against

L. donovani with an IC50 of 6.87 µM and a therapeutic window of five and against

P. falciparum with an IC50 of 4.27 µM and a therapeutic window of eight. The compound 9401D2 showed strong activity against P. falciparum with an IC50 of 4.18 µM, with a therapeutic window of seven.

Marine chemical ecology studies have been shown to have important applications in the field of natural products based drug discovery (Matthew et al., 2007; Simmons et al., 2005). There are several examples of potential anticancer agents which have been shown to be sequestered by the sea hare S. striatus (Luesch et al., 2002) including malyngamide A (4) (Paul and Pennings, 1991), lyngbyatoxin A (6) (Capper et al., 2005; Cardellina et al., 1979) and aplysiatoxin (7) (Fig. 4-1) (Kato and Scheuer, 1974). The findings presented in this study provide examples of compounds that are active against the clinically important diseases malaria and leishmaniasis.

The finding that compounds present in the cyanobacterial food source are sequestered by the sea hare is similar to the well known example of the sea hare D. auricularia and the dolastatins. Dolastatin-10 (1) (Fig. 1-1) was first isolated from D. auricularia (Pettit et al., 1987) and later discovered in its cyanobacterial food source (Luesch et al., 2001). Dolastatin-10 is a potent microtubule inhibitor (Simmons et al., 2005), with significant inhibition of P. falciparum (Fennell et al., 2003). The results presented in this study are the first example in which

compounds with significant activity against parasites responsible for tropical disease have been found in both the sea hare S. striatus and its cyanobacterial food source. These results provide additional evidence that chemical ecological studies of sea hares and their cyanobacterial food sources not only provide insight into the relationships between the invertebrate and its food sources but may also facilitate the search for compounds with activity against tropical diseases which are responsible for enormous human suffering, particularly in the developing world (Gelb and Hol, 2002).

The first step in drug development is natural products drug discovery, where scientists isolate compounds with activity towards such diseases. Through my work with the Panama ICBG, a biodiscovery group, two novel cyanobacterial peptides were isolated. Compound 9401D1 has significant activity against L. donovani, which is the cause of American visceral leishmanisis. Compounds 9401D1 and 9401D2 have significant activity against P. falciparum the chloroquine resistant and most fatal form of malaria.

Suggestions for Future Research It would be of great interest to return to the Boca Grande field site in the Coiba National Park in Panama to conduct further research. A possible follow up research study could be to carry out a feeding experiment in which the novel compounds 9401D1 and 9401D2 would be fed in artificial feeding assay to D. dolabrifera and S. striatus in order to evaluate whether the compounds act as feeding stimulants or deterrents. In addition, it would be interesting to evaluate the susceptibility of these sea hares to fish, comparing the palatability of whole live animals, digestive gland extracts, and skin extracts of D. dolabrifera and S. striatus containing the metabolites against those who do not contain the metabolites. As well, isolation of a greater mass of peptides 9401D1 and 9401D2 from L. majuscula would be useful for further studies of their antiplasmodial and antimonial properties.

Appendix A: NMR data of α5,α8-epidioxycholest-6-en-3-β-ol

13C

Appendix B: MS data for the L. majuscula compounds

Peptide 9401D1

Peptide 9401D2

List of References

ABOURRICHE, A., CHARROUF, M., CHAIB, N., BENNAMARA, A., BONTEMPS, N. and FRANCISCO, C. 2000. Isolation and bioactivities of epidioxysterol from the tunicate Cynthia savignyi. Farmaco 55:492-494.

AGUAYO, A., KRAUT, E. H., MOORE, D. F., HOFF, P. M., WRIGHT, J. J. and JONES, D. 2000. Phase II study of dolastatin 10 administered intravenously every 21 days to patients (Pts) with metastatic colorectal cancer (CRC). Abst: 1127, presented at the American Society of Clinical Oncology (ASCO) Annual Meeting. New Orleans, Louisiana. May 20th-23rd, 2007.

AKNIN, M., VIRACAOUDIN, I., FAURE, R. and GAYDOU, E.-M. 1998. 5α-8α- Epidioxycholest-6-en-3-β-ol from three cone snails of the Indian Ocean. Journal of the American Oil Chemists' Society 75:1679-1681.

AVILA, C., BALLESTEROS, M., CIMINO, G., CRISPINO, A., GAVAGNIN, M. and SODANO, G. 1990. Biosynthetic origin and anatomical distribution of the main secondary metabolites in the nudibranch mollusc Doris verrucosa. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 97:363-368.

BECERRO, M. A., STARMER, J. A. and PAUL, V. J. 2006. Chemical defenses of cryptic and aposematic gastropterid molluscs feeding on their host sponge Dysidea granulosa. Journal of Chemical Ecology 32:1491-1500.

BEHRENS, D. W. 2005. Nudibranch behavior. New World Publications, Jacksonville, Florida. 176 pp.

BEHRENS, D. W. and HERMOSILLO, A. 2005. Eastern pacific nudibranchs: A guide to the opisthobranchs from Alaska to Central America. Sea Challengers, Monterey, California. 133 pp.

BENKENDORFF, K., DAVIS, A. and BREMNER, J. 2001. Chemical defense in the egg masses of benthic invertebrates: An assessment of antibacterial activity in 39 mollusks and 4 polychaetes. Journal of Invertebrate Pathology 78:109- 118.

BJORKMAN, A. and BHATTARAI, A. 2005. Public health impact of drug resistant Plasmodium falciparum malaria. Acta Tropica 94:163-169.

BROCKS, J. J., LOGAN, G. A., BUICK, R. and SUMMONS, R. E. 1999. Archean molecular fossils and the early rise of eukaryotes. Science 285:1033-1036.

BROWER, L. P. and GLAZIER, S. C. 1975. Localization of heart poisons in the Monarch Butterfly. Science 188:19-25.

BRYCESON, A., BERMAN, J., GUTTERIDGE, W. and KARBWANG, J. 2007. Three new drugs for leishmaniasis: The story of liposomal amphorericin, miltefosine and paromomycin, in 100 year anniversary meeting of the Royal Society of Tropical Medicine and Hygiene. London, England. September 13th-15th, 2007.

BUCKNER, F. S., VERLINDE, C. L., LA FLAMME, A. C. and VAN VOORHIS, W. C. 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrobial Agents and Chemotherapy 40:2592-2597.

BURJA, A. M., BANAIGS, B., ABOU-MANSOUR, E., BURGESS, G. J. and WRIGHT, P. C. 2001. Marine cyanobacteria - a prolific source of natural products. Tetrahedron 57:9347-9377.

CALDERÓN, A. I., ROMERO, L. I., ORTEGA-BARRÍA, E., BRUN, R., CORREA, M. D. and GUPTA, M. P. 2006. Evaluation of larvicidal and in vitro antiparasitic activities of plants in a biodiversity plot in the Altos de Campana National Park, Panama. Pharmaceutical Biology 44:487-498.

CAMACHO-GARCÍA, Y., GOSLINER, T. M. and VALDÉS, Á. 2005. Guía de campo de las babosas marinas del Pacífico Este Tropical/Field guide to the sea slugs of the Tropical Eastern Pacific. California Academy of Sciences, San Francisco, California. 129 pp.

CAPPER, A., CRUZ-RIVERA, E., PAUL, V. J. and TIBBETTS, I. 2006a. Chemical deterrence of a marine cyanobacterium against sympatric and non- sympatric consumers. Hydrobiologia 553:319-326.

CAPPER, A., TIBBETTS, I., O'NEIL, J. and SHAW, G. 2005. The fate of Lyngbya majuscula toxins in three potential consumers. Journal of Chemical Ecology 31:1594-1606.

CAPPER, A., TIBBETTS, I. R., O'NEIL, J. M. and SHAW, G. R. 2006b. Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula and resultant growth rates in two species of opisthobranch mollusc. Journal of Experimental Marine Biology and Ecology 331:133-144.

CAPSON, T. L. 2007. Biodiscovery research in Panama: Linking science, technology, human health, and conservation in the host-country context, pp., in S. Carrizosa and P. McGuire (eds.). Legal and scientific implications of bioprospecting contracts: A handbook for policymakers. The ABS Project (IUCN-Environmental Law Centre), in press.

CARDELLINA, J. H., 2ND, MARNER, F. J. and MOORE, R. E. 1979. Seaweed dermatitis: Structure of lyngbyatoxin A. Science 204:193-195.

CAREFOOT, T. H. 1987. Aplysia - Its biology and ecology. Oceanography and Marine Biology 25:167-284.

CARMARGO, L. B. and LANGONI, H. 2006. Impact of leishmaniasis on public health. Journal of Venomous Animals and Toxins Including Tropical Diseases 12:527-548.

CIAVATTA, M. L., GAVAGNIN, M., PULITI, R. and CIMINO, G. 1996. Dolabriferol: A new polypropionate from the skin of the anaspidean mollusc Dolabrifera dolabfrifera. Tetrahedron 52:121831-121838.

CORBETT, Y., HERRERA, L., GONZALEZ, J., CUBILLA, L., CAPSON, T. L., COLEY, P. D., KURSAR, T. A., ROMERO, L. I. and ORTEGA-BARRÍA, E. 2004. A novel DNA- based microfluorimetric method to evaluate antimalarial drug activity. American Journal of Tropical Medicine and Hygiene 70:119-124.

CRUZ-RIVERA, E. and PAUL, V. 2006. Feeding by coral reef mesograzers: Algae or cyanobacteria? Coral Reefs 25:617-627.

CRUZ-RIVERA, E. and PAUL, V. J. 2007. Chemical deterrence of a cyanobacterial metabolite against generalized and specialized grazers. Journal of Chemical Ecology 33:213-217.

DE NYS, R., STEINBERG, P. D., ROGERS, C. N., CHARLTON, T. S. and DUNCAN, M. W. 1996. Quantitative variation of secondary metabolites in the sea hare Aplysia parvula and its host plant Delisea pulchra. Marine Ecology Progress Series 130:135-146.

DRUGS FOR NEGLECTED DISEASES. 2003. Neglected Diseases, http://www.dndi.org/cms/public_html/insidecategoryListing.asp?CategoryId =89 date accessed November 4th, 2007.

DRUGS FOR NEGLECTED DISEASES. 2004. DNDi: 'The gap is growing: More resources needed now for neglected diseases', http://www.accessmed- msf.org/prod/publications.asp?scntid=111120041526439&contenttype=PA RA& date accessed November 4th, 2007.

ERIXON, N., DEMARTINI, L. and WRIGHT, W. 1999. Dissociation between sensitization and learning-related neuromodulation in an aplysiid species. The Journal of Comparative Neurology 408:506-514.

FENNELL, B. J., CAROLAN, S., PETTIT, G. R. and BELL, A. 2003. Effects of the antimitotic natural product dolastatin 10, and related peptides, on the human malarial parasite Plasmodium falciparum. Journal of Antimicrobial Chemotherapy 51:833-841.

FUJIKI, H., IKEGAMI, K., HAKII, H., SUGANUMA, M., YAMAIZUMI, Z., YAMAZATO, K., MOORE, R. E. and SUGIMURA, T. 1985. A blue-green alga from Okinawa

contains aplysiatoxins, the third class of tumor promoters. Japanese Journal of Cancer Research 76:257-259.

GALLIMORE, W. A., GALARIO, D. L., LACY, C., ZHU, Y. and SCHEUER, P. J. 2000. Two complex proline esters from the sea hare Stylocheilus longicauda. Journal of Natural Products 63:1022-1026.

GARCIA-PICHEL, F. 1998. Solar ultraviolet and the evolutionary history of cyanobacteria. Origins of Life and Evolution of Biospheres 28:321-347.

GARSON, M. J. 2001. Ecological perspectives on marine natural product biosynthesis, pp. 71-114, in J. B. McClintock and B. J. Baker (eds.). Marine Chemical Ecology. CRC Press, Boca Raton, Florida.

GAUVIN, A., SMADJA, J., AKNIN, M., FAURE, R. and GAYDOU, E.-M. 2000. Isolation of bioactive 5α,8α-epidioxy sterols from the marine sponge Luffariella cf. variabilis. Canadian Journal of Chemistry 78:986-992.

GAVAGNIN, M., CARBONE, M., NAPPO, M., MOLLO, E., ROUSSIS, V. and CIMINO, G. 2005. First chemical study of anaspidean Syphonota geographica: Structure of degraded sterols aplykurodinone-1 and -2. Tetrahedron 61:617-621.

GELB, M. H. and HOL, W. G. J. 2002. Drugs to combat tropical protozoan parasites. Science 297:343-344.

GERWICK, W. H. 2007. Year 09 of the Panama ICBG Program: Successes and New Challenges as we Prepare for the Competitive Renewal, in International Cooperative Biodiversity Groups Annual Meeting. Panama City, Panama. June 20th, 2007.

GERWICK, W. H., TAN, L. T. and SITACHITTA, N. 2001. The Alkaloids. In G. A. Cordell (ed.). Academic Press, San Diego, California. 75-184 pp.

GHAZALI, S. R. 2006. Displays of defense: Behavioral differences in antagonist avoidance in four opisthobranch mollusks. Report. University of California in Berkeley, Berkeley, California. 16 pp.

GINSBURG, D. W. and PAUL, V. J. 2001. Chemical defenses in the sea hare Aplysia parvula: Importance of diet and sequestration of algal secondary metabolites. Marine Ecology Progress Series 215:261-274.

GUNATILAKA, A. A. L., GOPICHAND, Y., SCHMITZ, F. J. and DJERASSI, C. 1981. Minor and trace sterols in marine invertebrates. 26. Isolation and structure elucidation of nine new 5α,8α-epidioxy sterols from four marine organisms. Journal of Organic Chemistry 46:3860-3866.

HERMOSILLO, A., BEHRENS, D. W. and RÍOS JARA, E. 2006. Opistobranquios de México: Guía de babosas marinas del Pacífico, Golfo de California y las islas oceánicas. Comisión National para el Conocimiento y Uso de la Biodiversidad (CONABIO), Guadalajara, Mexico. 135 pp.

HOFFMAN, M. A., BLESSING, J. and LENTZ, S. 2003. A phase II trial of dolastatin-10 in recurrent platinum-sensitive ovarian carcinoma: A gynecologic oncology group study. Gynecologic Oncology 89:95-98.

IIJIMA, R., KISUGI, J. and YAMAZAKI, M. 2003. A novel antimicrobial peptide from the sea hare Dolabella auricularia. Developmental & Comparative Immunology 27:305-311.

IWASHIMA, M., TERADA, I., IGUCHI, K. and YAMORI, T. 2002. New biologically active marine sesquiterpenoid and steroid from the Okinawan sponge of the genus Axinyssa. Chemical & Pharmaceutical Bulletin 50:1286-1289.

JAMBOU, R., LEGRAND, E., NIANG, M., LIM, P., VOLNEY, B., EKALA, M. T., BOUCHIER, C., ESTERRE, P., FANDEUR, T. and MERCEREAU-PUIJALON, O. 2005. Resistance of Plasmodium falciparum field isolated to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366:1960- 1963.

JIMÉNEZ, C., EMILIO, C., LUIS, R. and RIGUERA, R. 1986. Epidioxy sterols from the tunicates Dendrodoa grossularia and Ascidiella aspersa and the gastropoda Aplysia depilans and Aplysia punctata. Journal of Natural Products 49:905-909.

JIMÉNEZ, C., QUIÑOÁ, E., RIGUERA, R., VILALTA, R. and QUINTELA, J. M. 1989. The dietary origin of epidioxy steroids in Actinia equina. A carbon -14 incorporation experiment. Journal of Natural Products 52:619-622.

JOHNSON, P. M., KICKLIGHTER, C. E., SCHMIDT, M., KAMIO, M., YANG, H., ELKIN, D., MICHEL, W. C., TAI, P. C. and DERBY, C. D. 2006. Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica. The Journal of Experimental Marine Biology 209:78-88.

JOHNSON, P. M. and WILLOWS, A. O. D. 1999. Defense in sea hares (Gastropoda, Opisthobranchia, Anaspidea): Multiple layers of protection from egg to adult. Marine and Freshwater Behaviour and Physiology 32:147-180.

KAHLOS, K., KAGAS, L. and HILTUNEN, R. 1989. Ergosterol peroxide, an active compound from Inonotus radiatus. Planta Medica 55:389-390.

KATO, Y. and SCHEUER, P. J. 1974. Aplysiatoxin and debromoaplysiatoxin, constituents of the marine mollusk Stylocheilus longicauda (Quoy and Gaimard, 1824). Journal of American Chemical Society 96:2245-2246.

KAY, A. 1979. Hawaiian marine shells. Bernice Pauahi Bishop Museum Press, Honolulu, Hawaii. 653 pp.

KICKLIGHTER, C. E. and DERBY, C. D. 2006. Multiple components in ink of the sea hare Aplysia californica are aversive to the sea anemone Anthopleura sola. Journal of Experimental Marine Biology and Ecology 334:256-268.

KICKLIGHTER, C. E., SHABANI, S., JOHNSON, P. M. and DERBY, C. D. 2005. Sea hares use novel antipredatory chemical defenses. Current Biology 15:549- 554.

KINDLER, H. L., TOTHY, P. K., WOLFF, R., MCCORMACK, R. A., ABBRUZZESE, J. L., MANI, S., WADE-OLIVER, K. T. and VOKES, E. E. 2005. Phase II trials of dolastatin-10 in advanced pancreaticobiliary cancers. Investigational New Drugs 23:489-493.

KLUSSMANN-KOLB, A. 2003. Phylogeny of the Aplysiidae (Gastropoda, Opisthobranchia) with new aspects of the evolution of seahares. Zoologica Scripta 33:439-462.

KRUG, L. M., MILLER, V. A., KALEMKERIAN, G. P., KRAUT, M. J., NG, K. K., HEELAN, R. T., PIZZO, B. A., PEREZ, W., MCCLEAN, N. and KRIS, M. G. 2000. Phase II study of dolastatin-10 in patients with advanced non-small-cell lung cancer. Annals Oncology 11:227-228.

KURSAR, T. A., CABALLERO-GEORGE, C. C., CAPSON, T. L., CUBILLA-RIOS, L., GERWICK, W. H., GUPTA, M. P., IBAÑEZ, A., LININGTON, R. G., MCPHAIL, K. L., ORTEGA-BARRIA, E., ROMERO, L. I., SOLIS, P. N. and COLEY, P. D. 2006. Securing economic benefits and promoting conservation through bioprospecting. Bioscience 56:1005-1012.

LUESCH, H., HARRIGAN, G. G., GOETZ, G. and HORGEN, F. D. 2002. The cyanobacterial origin of potent anticancer agents originally isolated from sea hares. Current Medicinal Chemistry 9:1791-1806.

LUESCH, H., MOORE, R. E., PAUL, V. J., MOOBERRY, S. L. and CORBETT, T. H. 2001. Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1. Journal of Natural Products 64:907-910.

MANSOOR, T. A., LEE, Y. M., HONG, J., LEE, C. O., IM, K. S. and JUNG, J. H. 2006. 5,6:8,9-Diepoxy and other cytotoxic sterols from the marine sponge Homaxinella sp. Journal of Natural Products 69:131-134.

MARGOLIN, K., LONGMATE, J., SYNOLD, T. W., GANDARA, D. R., WEBER, J., GONZALEZ, R., JOHANSEN, M. J., NEWMAN, R., BARATTA, T. and DOROSHOW, J. H. 2001. Dolastatin-10 in metastatic melanoma: A phase II and

pharmokinetic trial of the California Cancer Consortium. Investigational New Drugs 19:335-340.

MARINESCO, S., DURAN, K. and WRIGHT, W. 2003. Evolution of learning in three aplysiid species: Differences in heterosynaptic plasticity contrast with conservation in serotonergic pathways. Journal of Physiology 550:241- 253.

MATTHEW, S., ROSS, C., ROCCA, J. R., PAUL, V. J. and LUESCH, H. 2007. Lyngbyastatin 4, a dolastatin 13 analogue with elastase and chymotrypsin inhibitory activity from the marine cyanobacterium Lyngbya confervoides. Journal of Natural Products 70:124-127.

MCCLINTOCK, J. B. and BAKER, B. J. (eds.). 2001. Marine Chemical Ecology. CRC Press, Boca Raton, Florida. 634 pp.

MIN-HSIUNG, P., YU-TING, H., CHI-I, C., CHI-TANG, H. and BONNIE, S. P. 2007. Apoptotic-inducing epidioxysterols identified in hard clam (Meretrix lusoria). Food Chemistry 102:788-795.

MINH, C. V., KIEM, P. V., HUONG, L. M. and KIM, Y. H. 2004. Cytotoxic constituents of Diadema setosum. Archives of Pharmacal Research 27:734-737.

MIYAMOTO, T., HONDA, M., SUGIYAMA, S., HIGUCHI, R. and KOMORI, T. 1988. Studies on the constituents of marine opisthobranchia. III. Isolation and structure of two 5,8α-epidioxysterols and a cholesteryl ester mixture from the albumen gland of Aplysia juliana. Liebigs Annalen der Chimie 6:589- 592.

MONKS, A., SCUDIERO, D., SKEHAN, P., SHOEMAKER, R., PAULL, K., VISTICA, D., HOSE, C., LANGLEY, J., CRONISE, P., VAIGRO-WOLFF, A., GRAY-GOODRICH, M., CAMPBELL, H., MAYO, J. and BOYD, M. 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. Journal of the National Cancer Institute 83:757-766.

MORAN, M. 2005. A breakthrough in R&D for neglected diseases: New ways to get the drugs we need. PLoS Medicine 2:0828-0832.

NAGLE, D. G., CAMACHO, F. T. and PAUL, V. J. 1998. Dietary preferences of the opisthobranch mollusc Stylocheilus longicauda for secondary metabolites produced by the tropical cyanobacterium Lyngbya majuscula. Marine Biology 132:267-273.

NAGLE, D. G. and PAUL, V. J. 1998. Chemical defense of a marine cyanobacterial bloom. Journal of Experimental Biology and Ecology 225:29-38.

NAGLE, D. G., PAUL, V. J. and ROBERTS, M. A. 1996. Ypaoamide, a new broadly acting feeding deterrent from the marine cyanobacterium Lyngbya majuscula. Tetrahedron 37:6263-6266.

PAUL, V. J., CRUZ-RIVERA, E. and THACKER, R. W. 2001. Chemical mediation of macroalgal-herbivore interactions: Ecological and evolutionary perspectives, pp. 227-265, in J. McClintock and W. Baker (eds.). Marine Chemical Ecology. CRC Press, Boca Raton, Florida.

PAUL, V. J., NELSON, S. G. and SANGER, H. R. 1990. Feeding preferences of adult and juvenile rabbitfish Siganus argenteus in relation to chemical defenses of tropical seaweeds. Marine Ecology Progress Series 60:23-34.

PAUL, V. J. and PENNINGS, S. C. 1991. Diet-derived chemical defenses in the sea hare Stylocheilus longicauda (Quoy et Gaimard 1824). Journal of Experimental Marine Biology and Ecology 151:227-243.

PAWLIK, J. R., KERNAN, M. R., MOLINSKI, T. F., HARPER, M. K. and FAULKNER, D. J. 1988. Defensive chemicals of the spanisch dancer nudibranch Hexabranchus sanguineus and its egg ribbons: Macrolides derived from a sponge diet. Journal of Experimental Marine Biology and Ecology 119:99- 109.

PENNINGS, S. C. 1994. Interspecific variation in chemical defenses in the sea hares (Opisthobranchia: Anaspidea). Journal of Experimental Marine Biology and Ecology 180:203-219.

PENNINGS, S. C. and CAREFOOT, T. H. 1995. Post-ingestive consequences of consuming secondary metabolites in sea hares (Gastropoda: Opisthobranchia). Comparative Biochemistry and Physiology Part C: Comparative Pharmacology and Toxicology 111:249-256.

PENNINGS, S. C., NADEAU, M. T. and PAUL, V. J. 1993. Selectivity and growth of the generalist herbivore Dolabella auricularia feeding upon complementary resources. Ecology 74:879-890.

PENNINGS, S. C., NASTISCH, S. and PAUL, V. 2001. Vulnerability of sea hares to fish predators: Importance of diet and fish species. Coral Reefs 20:320- 324.

PENNINGS, S. C. and PAUL, V. J. 1993. Sequestration of dietary secondary metabolites by three species of sea hares: Location, specificity and dynamics. Marine Biology 117:535-546.

PENNINGS, S. C., PAUL, V. J., DUNBAR, D. C., HAMANN, M. T., LUMBANG, W. A. and NOVACK, B. 1999. Unpalatable compounds in the marine gastropod Dolabella auricularia: Distribution and effect of diet. Journal of Chemical Ecology 25:735-755.

PENNINGS, S. C., WEISS, A. M. and PAUL, V. J. 1996. Secondary metabolites of the cyanobacterium Microcoleus lyngbyaceus and the sea hare Stylocheilus Iongicauda: Palatability and toxicity. Marine Biology 126:735-743.

PEREZ, E. A., HILLMAN, D. W., FISHKIN, P. A., KROOK, J. E., TAN, W. W., KURIAKOSE, P. A., ALBERTS, S. R. and DAKHIL, S. R. 2005. Phase II trial of dolastatin-10 in patients with advanced breast cancer. Investigational New Drugs 23:257-261.

PETTIT, G. R., KAMANO, Y., HERALD, C. L., TUINMAN, A. A., BOETTNER, F. E., KIZA, H., SCHMIDT, J. M., BACZYNSKYJ, L., TOMER, K. B. and BONTEMS, R. J. 1987. The isolation and structure of a remarkable marine animal antineoplastic constituent: Dolastatin 10. Journal of the American Chemical Society 109:6883-6995.

PITOT, H. C., FRYTAK, S., CROGHAM, G. A., HILLMAN, D. W., REID, J. M. and ARNES, M. 2002. Phase II study of dolastain-10 in patients with advanced renal cell carcinoma. Abst: 2409, presented at the American Society of Clinical Oncology (ASCO) Annual Meeting. Orlando, Florida. May 18th-21st, 2002.

PRINCE, J. S. and JOHNSON, P. M. 2006. Ultrastructural comparison of Aplysia and Dolabrifera ink glands suggests cellular sites of anti-predator protein production and algal pigment processing. Journal of Molluscan Studies 72:349-357.

RAMOS, L. J., ROCAFORT, J. L. L. and MILLER, M. W. 1995. Behavior patterns of the aplysiid gastropod Bursatella leachii in its natural habitat and in the laboratory. Neurobiology of Learning and Memory 63:246-259.

REID, W. V., LAIRD, S. A., GÁMEZ, R., SITTENFELD, A., JANZEN, D. H., GOLLIN, M. A. and C., J. 1993. A new lease on life, pp. 1-52, in W. V. Reid, S. A. Laird, C. A. Meyer, R. Gámez, A. Sittenfeld, D. H. Janzen, M. A. Gollin and C. Juma (eds.). Biodiversity prospecting: Using genetic resources for sustainable development. World Resources Institute, Washington, DC.

ROGERS, C. N., STEINBERG, P. D. and DE NYS, R. 1995. Factors associated with oligophagy in two species of sea hares (Mollusca: Anaspidea). Journal of Experimental Marine Biology and Ecology 192:47-73.

RUDMAN, W. B. 1999. Stylocheilus striatus (Quoy & Gaimard, 1832), [in] Sea Slug Forum. http://www.seaslugforum.net/factsheet.cfm?base=stylstri date accessed August 11th, 2007.

RUDMAN, W. B. 2002a. Comment on ink gland in Stylocheilus striatus by Angela Capper, [in] Sea Slug Forum. http://www.seaslugforum.net/display.cfm?id=7872 date accessed August 11th, 2007.

RUDMAN, W. B. 2002b. Comment on observing sea slugs in aquaria by Sarah Bredensteiner, [in] Sea Slug Forum. http://www.seaslugforum.net/showall.cfm?base=captivi2 date accessed July 19th, 2007.

RUDMAN, W. B. 2005. Petalifera petalifera (Rang, 1828), [in] Sea Slug Forum. http://www.seaslugforum.net/factsheet.cfm?base=petapeta date accessed August 10th, 2007.

SERA, Y., ADACHI, K. and SHIZURI, Y. 1999. A new epidioxy sterol as an antifouling substance from a Palauan marine sponge, Lendenfeldia chondrodes. Journal of Natural Products 62:152-154.

SHEU, J. H., CHANG, K. and DUH, C. Y. 2000. A cytotoxic 5α,8α-epidioxysterol from a soft coral Sinularia species. Journal of Natural Products 63:149-151.

SIMMONS, T. L., ANDRIANASOLO, E., MCPHAIL, K., FLATT, P. and GERWICK, W. H. 2005. Marine natural products as anticancer drugs. Molecular Cancer Therapeutics 4:333-342.

STALLARD, M. O. and FAULKNER, D. J. 1974. Chemical constituents of the digestive gland of the sea hare Aplysia californica. I. Importance of diet. Comparative Biochemistry and Physiology 49B:25-35.

THACKER, R. W., NAGLE, D. G. and PAUL, V. J. 1997. Effects of repeated exposures to marine cyanobacterial secondary metabolites on feeding by juvenile rabbitfish and parrotfish. Marine Ecology Progress Series 147:21- 29.

THOMPSON, J. E., WALKER, R. P., WRATTEN, S. J. and FAULKNER, D. J. 1982. A chemical defense mechanism for the nudibranch Cadlina luteomarginata. Tetrahedron 38:1865-1873.

VAISHAMPAYAN, U., GLODE, M., DU, W., KRAFT, A., HUDES, G., WRIGHT, J. and HUSSAIN, M. 2000. Phase II study of dolastatin-10 in patients with hormone-refractory metastatic prostate adenocarcinoma. Clinical Cancer Research 6:4205-4208.

VAN DE LOOSDRECHT, A. A., BEELEN, R. H. J., OSSENKOPPELE, G. J., BROEKHOVEN, M. G. and LANGENHUIJSEN, M. M. A. C. 1994. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. Journal of Immunological Methods 174:311-320.

WASTON, M. 1973. Midgut gland toxins of Hawaiian sea hares-1.Isolation and preliminary toxicological observations. Toxicon 2:569-267.

WATERBURY, J. B., WATSON, S. W., GUILLARD, R. R. L. and BAND, L. E. 1979. Widespread occurrence of unicellular, marine, planktonic, cyanobacterium. Nature 277:293-294.

WHITTON, B. A. and POTTS, M. 1982. The biology of cyanobacteria. In N. G. Carr and B. A. Whitton (eds.). Blackwell Science Publishing, Oxford, England.

WINKLET, A., GALLIMORE, W. A. and SCHEUER, P. J. 2000. Malygamides O and P from the Sea Hare Stylocheilus longicauda. Journal of natural products 63:1422-1424.

WORLD HEALTH ORGANIZATION. 2006. Facts on ACTs: January 2006 update, http://www.rbm.who.int/cmc_upload/0/000/015/364/RBMInfosheet_9.htm date accessed November 5th, 2007.

WORLD HEALTH ORGANIZATION. 2007a. Leishmania: The disease and its epidemiology, http://www.who.int/leishmaniasis/disease_epidemiology/en/index.html date accessed November 4th, 2007.

WORLD HEALTH ORGANIZATION. 2007b. Leishmania: The magnitude of the problem, http://www.who.int/leishmaniasis/burden/magnitude/burden_magnitude/en/ index.html date accessed November 5th, 2007.

WORLD HEALTH ORGANIZATION. 2007c. Malaria Fact Sheet N°94, http://www.who.int/mediacentre/factsheets/fs094/en/index.html date accessed November 4th, 2007.

WORLD HEALTH ORGANIZATION. 2007d. Research on Leishmaniasis, http://www.who.int/leishmaniasis/research/en/ date accessed November 4th, 2007.

WRIGHT, W. 1998. Evolution of nonassociative learning: Behavioral analysis of a phylogenetic lesion. Neurobiology of Learning and Memory 69:326-337.

WYLIE, C. R. and PAUL, V. J. 1988. Feeding preferences of the surgeonfish Zebrasoma flavescens in relation to chemical defenses of tropical algae. Marine Ecology Progress Series 45:23-32.