Harriet L. Wilkes Honors College Honors Theses

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Bioprospecting for useful compounds in the venom of

Robert E. McCurdy Florida Atlantic University,

This paper is posted at DigitalCommons@Florida Atlantic University. http://digitalcommons.fau.edu/wilkes theses/32

Bioprospecting for Useful Compounds

in the Venom of Crematogaster Ants

by

Robert E. McCurdy

A Thesis Submitted to the Faculty of

The Wilkes Honors College in Partial Fulfillment of the Requirements for the Degree of

Bachelor of Arts in Liberal Arts and Sciences

with a Major Concentration in Environmental Science

and a Minor Concentration in Chemistry

Wilkes Honors College of

Florida Atlantic University

Jupiter, Florida

May 2007

Bioprospecting for Useful Compounds in the Venom of Crematogaster Ants

by Robert E. McCurdy

This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. James K. Wetterer, and has been approved by the members of his supervisory committee. It was submitted to the faculty of the Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences.

SUPERVISORY COMMITTEE:

______Dr. James K. Wetterer

______Dr. Eugene Smith

______Dean, Wilkes Honors College

______Date

ii ACKNOWLEDGEMENTS

First of all, I want to thank everyone who contributed to my education. This includes everyone from family, teachers, professors, friends and anyone else who helped get where I am now. All the little or big things that helped me along the way will not go unforgotten. We are all products of our environments and I like to believe that I am a shining example of what an excellent education provides.

Next is my family. You guys have been so great to me. Each one of you has contributed your part to being there when I needed you. Through thick and thin, you were always there for me. Mom, I don’t think I can ever say enough to express my gratitude and love for you. Thanks for believing in me. Paul, you have pushed me and provided me with a role model like no other. Most of all, thanks for being my best friend. Live it up in

NZ and study hard. I’ll see you when I get there. Sarah, I love you for all you are; even when we don’t see eye to eye on everything. Congratulations on being the newest Dr.

McCurdy. Alice, you bring a light to all of our lives like no other. Keep it up. Last, but not least, is my deceased father. Your memory is with me everyday. Thanks for showing me how wonderful life is, especially around the ocean.

To the faculty of the Honors College, it was a real pleasure to learn with all of you. Most especially, I would like to recognize Dr. Wetterer, Dr. Smith, Dr. O’Brien, and

Dr. Moore. Thanks for keeping my interest in the sciences and the environment alive.

Working with each one of you on a personal level has meant a lot to me. Mahalo.

iii

ABSTRACT

Author: Robert E. McCurdy

Title: Bioprospecting for Useful Compounds in the Venom of Crematogaster Ants

Institution: Wilkes Honors College of Florida Atlantic University

Thesis Advisor: Dr. James K. Wetterer

Degree: Bachelor of Arts in Liberal Arts and Sciences

Concentration: Environmental Science and Chemistry

Year: 2007

Bioprospecting, the search for useful compounds in nature, has led to the discovery of many important pharmaceuticals. Most current bioprospecting efforts work with chemicals derived from marine invertebrates and terrestrial plants. I looked for useful compounds in a relatively unstudied source, the venom of Crematogaster ants, using Gas Chromatography-Mass Spectroscopy (GC-MS). Further studies can more accurately identify these chemicals using High Pressure Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR).

iv TABLE OF CONTENTS

Introduction………………………………………………………………………1

Methods…………………………………………………………………………...7 2.1 Study Site.…………………………………………………………………7 2.2 Laboratory…………………………………………………………………9 2.3 Gas Chromatography-Mass Spectroscopy………………………………...9 2.31 Gas Chromatography (GC)……………………………………………..10 2.32 Mass Spectroscopy (MS)……………………………………………….10

Results…………………………………………………………………………...12

Discussion…………………………………………………………………….…14

Works Cited…………………………………………………………………….16

v TABLE OF FIGURES

FIGURES Figure 1 Crematogaster sp. ………………………………………………………...4 Figure 2 Land use map of Abacoa…………………………………………………..8 Figure 3 GC scan of Crematogaster venom……………………………………….13

vi INTRODUCTION

The Secretariat of the Convention on Biological Diversity (CBD) has defined bioprospecting as “the exploration of for commercially valuable genetic and biochemical resources” (Arico and Salpin 2005). Its main goal is to discover compounds that exist in nature with beneficial properties that can be used by humans. Once found and identified, the genetic or biochemical resources can be applied in many different fields, of which drug research is the most notable. Bioprospecting is a field that has received much interest lately as scientists seek out new approaches to curing disease.

While drug companies dedicate valuable time and resources uncovering the pathologies of diseases so as to design new drugs to counter the effects, there may be a compound already in existence that an organism has been using for some time for this particular purpose. Therefore, research dealing with the discovery and properties of novel compounds in nature warrants more attention.

What constitutes bioprospecting in international law is not yet undefined. A few countries have defined it in laws governing biodiversity but diverge in what steps to bringing a product to market actually encompass bioprospecting. To simplify matters,

Arico and Salpin (2005) listed the steps:

• Systematic search, collection, gathering or sampling of biological

resources for purposes of commercial or industrial exploitation;

• Screening, isolation, characterization of commercially useful

compounds;

• Testing and trials; and

1 • Further application and development of the compounds for

commercial purposes, including large-scale collection,

development of mass culture techniques, and conduct of trials for

approved commercial sale.

Sought after compounds could be identified by indiscriminately testing natural compounds from any organism, but chances of getting a promising candidate compound could be daunting. Instead, scientists begin their searches by seeking out organisms that have displayed desirable attributes. In the case of the rosy periwinkle (Catharanaus roseus), generations of people had been using this flower to treat diabetes in Madagascar

(Gept 2004). When researchers took a closer look in the 1950s, they found a compound, which is later marketed as the drug Vincristine that helped cure thousands of childhood leukemia cases (Gept 2004). Here is an example of local knowledge of the environment leading to a treatment whose benefit to humanity is practically immeasurable.

With so many potential compounds and organisms out there, how should scientists proceed with bioprospecting efforts. Like in the case of the rose periwinkle, traditional knowledge of plants can prove to be helpful. But in today’s modern societies, intimate knowledge of natural remedies have been lost; so instead, science should turn to organisms that demonstrate unique chemical solutions to environmental pressures. For this study, the unique properties of venom are investigated further.

Bioactive compounds from ants are exploited by a variety of other in nature. Many birds rub ants on their feathers, a behavior called “anting,” apparently to help control parasites (Revis and Waller 2004). The chemicals found in ants are thought to retard plumage deterioration by helping control microbe and fungal growth. Anting

2 creates better fitness for the birds, as well as making them more attractive to potential

mates. In another case, the skin of certain poisonous frogs and toads contain poisonous

compounds known as pumiliotoxins, apparently sequestered from ants and other

in their diets (Jones et al. 1999). The frogs are able to utilize the chemicals created by the ants for defense for their own purpose of avoiding predation.

Humans have only recently begun to tap into the potential of ant venoms. One research group has focused on engineering a more potent baculovirus for pest control on crops (Szolajska et al. 2004). Natural baculoviruses have proven to be effective, but slow acting insecticides. The lag time between infection and death has ruled out baculoviruses as a viable control method because significant damage can be inflicted on the crops before the die. By engineering the genetic material coding for poneratoxin, a neurotoxin from the ant Paraponera clavata, into the baculoviruses, the viruses kill the pests at a much quicker rate (Szolajska et al. 2004). The group has shown that the baculoviruses with the recombinant DNA are both a quick-acting and wide spectrum insecticide suitable for commercial use.

These instances represent how chemicals from ant venom can be utilized for a multitude of applications. They also show that further studies can uncover even more useful compounds from the potent mix of chemicals that ants use to protect themselves

from their enemies.

Ant venoms are shown to be more structurally diverse than both bee and

venoms (Blum 1992). They have also been shown to have many biochemical pathways

that are notable. As Blum (1992) reported:

3 The poison gland products of selected ant venoms are

cytotoxic and hemolytic against a variety of red blood cells.

Some venoms are potently bactericidal and fungicidal

against a variety of microorganisms. Venoms of Solenopsis

and Monomorium species are highly effective contact

insecticides and repellents as well. In addition, these

secretions can also inhibit enzymes (e.g., Na+ ATPase),

uncouple oxidative phosphorylation, reduce mitochondrial

respiration, and release histamine from mast cells.

With all these possible pathways, it becomes apparent that more attention needs to be paid to the potential of the compounds contained in ant venom.

There are over 11,000 known species of ants and to speed the discovery of a useful compound, I focused on the ants in the Crematogaster, a group known to contain alkaloids, a class of nitrogenous compounds that includes many important drugs: e.g., quinine, codeine and morphine. In recent years there have been a good deal of interest in discovering the venom constituents in the venom of the highly evolved Figure 1. Crematogaster sp. Source of data from www.antweb.org. Crematogaster ant. Crematogaster ants are unique in the way they apply their venom to their enemies. They hold their gasters, or abdomen, over their heads when they are threatened. This posture allows them to attack in a 360° radius (Longino 1993). In addition to this adaptation, their venom is also more

4 evolved than other groups. The venom is applied topically by wiping its

spatula-like device on the skin of its victim instead of injecting it like other more

primitive ants do (Kugler 1978). The evolution of such advanced defenses indicates the

utilization of novel compounds that warrant more consideration.

In one study, researchers isolated fourteen new and three previously discovered

long-chain derivatives in three species of Crematogaster ants from Papua New Guinea

(Leclercq et. al 1997). The (E,E)-cross conjugated dienones are transformed into highly

electrophilic toxins by an enzyme upon release from the Dufour’s gland. Leclerq and

colleagues (1997) correlated this data with three previous studies of theirs dealing with

European species and determined that long-chain contact poisons may be a distinguishing

characteristic of the venoms of this ant genus (Daloze et al. 1987, Pasteels et al. 1989,

Daloze et al. 1991).

Two follow up studies confirmed the venom secretions in the Dufour’s gland

from a Brazilian Crematogaster ant contained the same class of cross-conjugated dienone

compounds (Daloze et al. 1998, Leclerq et al. 2000). The presence of these compounds in

a New World species suggests that they are common to all Crematogaster species. In

addition to those findings, Leclerq and colleagues (2000) also found furanocembroid diterpenes, which were not found in the Crematogaster ants from Papua New Guinea and

Europe. The follow up studies come to the conclusion that the venom constituents are more diversified than originally thought. Since the furanocembroids have only ever been found in two C. brevispinosa subspecies, there is evidence that the venom constituents could be used as a chemotaxonomic indicator (Leclerq 2000).

5 With the diversity of the venom constituents for Crematogaster confirmed, the need to look to for more novel compounds in unstudied species arises. In Florida, there are eleven known Crematogaster spp. (J. Wetterer, personal communication). Two species, C. ashmeadi and C. atkinsoni, have been identified within the Abacoa Greenway, a series of preserves located within a housing development in Jupiter, Florida (Wetterer and Moore 2004).

6 METHODS

Study Site

The Abacoa Greenway is 250-acres of linked preserves located within an extensive housing development in Jupiter, FL. The idea of the preserve is to protect a diminishing scrubby flatwoods pine ecosystem, and create a pathway within suburban sprawl to allow genetic and species migration. The Greenway is dominated by mature slash (Pinus ellioti Engelman) and saw palmetto (Serena repens Bartram).

Many ecologically important and protected reside in the Greenway, including the gopher tortoise (Gopherus polyphemus), the eastern indigo snake (Drymarchon corais) and the scrub jay (Aphelocoma coerulescens) (Blubaugh 2006). Wetterer and Moore

(2004) conducted a survey of the ants found in a portion of the Greenway (Fig. 2, the black arrow) by baiting the entrance of gopher tortoise burrows. Species of note that were documented include Solenopsis invicta, the invasive red fire ant, and two species in the genus Crematogaster, C. atkinson and C. ashmeadi. C. ashmeadi is known to be the

“dominant ant species of arboreal ant community inhabiting the longleaf pine forests of the southeastern United States (Baldacci &Tschinkel 1999).”

7

Figure 2. Land use map of Abacoa. Source of data from www.abacoa.com.

For this study I located a colony of Crematogaster ants in a separate strand of scrub pines in the Greenway (Fig. 1, red arrow). The ants were found in the decaying wood of a dead scrub pine. The colony was found inhabiting the holes left by boll

8 weevils. The ants were confirmed as an undescribed species found in the scrub pines of

South Florida and the Florida Keys (M. Deyrup, personal communication). I collected

~200 specimens and brought them back to the lab for chemical analysis.

Laboratory

The ants were perturbed until they exhibited defensive posturing (placing their abdomens over their heads) indicative of venom release (Longino 2003). The ants were then dropped into methanol for extraction of the venom constituents. The methanol and ants were put on an agitator for 5 minutes and 1 mL of the resulting methanol solution was put in a glass vial and the methanol was blown off using pure nitrogen gas (N2). The

extracted chemicals were left on the bottom of the vial as a white residue. The chemicals

were then dissolved in 1 mL of methylene chloride.

Gas Chromatography-Mass Spectroscopy (GC-MS)

GC-MS technology is a powerful tool that chemists use to characterize unknown

samples. There are two parts to this technique, which identifies individual components in

a solution. First, the gas chromatography vaporizes the sample and then separates out the

individual components of the solution based on their interaction with a long capillary

column, usually around 30m in length. After separation, the individual molecules are

sent to the mass spectrometer. Here the gas molecules that have eluted off the column are

converted into ions before separated based on mass/charge ratio by the mass analyzer.

For this experiment, an Agilent 6000 series instrument was used as well as its

9 corresponding software laboratory. It was programmed from 70°C to 200°C at 15°C/min and from 200°C to 275°C at 5°C/min.

Gas Chromatography

Gas chromatography begins when the sample is injected into an oven that vaporizes the sample. The vaporized sample is then introduced into a column filled with, in this case, dimethylpolysiloxane, which is a silicon based organic polymer, by way of a chemically inert gas like helium (He). As the sample travels through the column, it interacts with the dimethylpolysiloxane, which retains the sample in the column; this is considered the stationary phase. As the temperature increases within the column, different sized molecules stop the interacting with the column and move freely through it.

When molecules are no longer being held in the stationary phase, they enter the mobile phase and elute out of the column and into the mass spectrometer. The abundance of each molecule is measured and plotted against the retention time in the column.

Mass Spectroscopy

After being separated by gas chromatography, the components of the solution are further characterized by mass spectroscopy. There are three parts to the method: ion source, mass analyzer and detector. First, the molecule is subjected to an ion source, in this case electron impact (EI), which is most suitable for the volatile samples coming off a GC column. EI subjects the sample to a high energy electron beam (70 eV). The electron usually has enough force when impacting the molecule to remove another electron. The result is a charged ion that is unstable and prone to decay into smaller

10 fragments, one being charged and the other not. The uncharged fragments are discarded while the charged ions are sent into the mass analyzer. The Agilent 6000 series GC-MS

uses a quadrapole mass filter. The quadrapole operates by changing the voltage being run through four magnets, which only allows particular ions with the proper mass to charge ratio (m/z) through to the detector. The voltages scan through a range of Rf/DC voltages

that correlate with separate m/z ratios. The detector is called an electron multiplier and

operates by creating a cascade of electrons in a cone, amplifying the signal. The

instrument correlates the scanning voltage with the signal from the detector to determine

the m/z ratio and abundance of the fragment coming out of the machine.

11 RESULTS

The GC-MS data exhibited many peaks, but most were minor components (Fig.

3). Starting at 23:08 and ending with 24:37, there were a series of five peaks that were of

interest. The peaks were of high abundance and had molecular weights between 356 and

552 daltons. The corresponding mass spectra for these peaks yielded no positive

identification within the machine’s library (<90%). The (E,E)-cross conjugated dienones could most likely be identified within the peaks at 23:08-24:37 minutes.

12

Figure 3. GC scan of Crematogaster ants, Retention time (min) vs. Intensity (abundance).

13 DISCUSSION

The GC-MS data was inconclusive in identifying (E,E)-cross conjugated dienones, the common venom constituents in Crematogaster spp. The main reason for the inability to distinguish this group of chemicals in the venom is the lack of mass spectra for the group in the software laboratory. The reported peaks, which suggested a molecular weight of 356-552 daltons, are consistent with previously published data

(Leclerq et al. 1997).

Further testing could easily identify the expected (E,E)-cross conjugated dienones. First, better sampling techniques might yield fewer interferent chemicals. By immersing the entire ant in methanol, cuticular chemicals contaminated the sample. If the venom glands and the Dufour’s gland were isolated through dissection, there is a better chance of isolating and identifying the target group of chemicals. With High-Powered

Liquid Chromatography (HP-LC), further separation of the compounds could be attained.

From there, the structure of individual venom components could be determined with

Nuclear Magnetic Resonance (NMR) technology.

The study of the chemicals in the venom of Crematogaster spp. illustrates the need to protect our natural resources. The potential of a unique chemical in its venom may have been lost within the rapid growth of suburbia in South Florida. The benefits of putting aside natural lands cannot be fully calculated or realized until we have put more effort and research into natural world around us. The conservation of our natural resources is extremely important to help preserve biodiversity. With intact biodiversity,

14 there is an increased chance that one day we will be saving human lives with an undiscovered natural compound or genetic resource.

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16 Gept, P. 2004. Who owns biodiversity, and how should its owners be compensated?

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18