My journey to the ants

TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL. 120, 2009 MY JOURNEY TO THE ANTS RICHARD D. DESHAZO, M.D. JACKSON, MISSISSIPPI ABSTRACT In this paper, I review the strange, unplanned and unexpected journey I have had with Solenopsis invicta, the imported fire ant. Through serendipity, good fortune and repeated invenomation, I have come to count as collaborators a number of entomologists, toxicologists, allergists and immunologists who have guided me on this journey to the ants. We now understand the mechanisms for the cutaneous reactions experienced by 50% of the exposed population stung per year, as well as the immunologic and toxicologic properties of the ants unique venom. Allergen immunotherapy to fire ant extracts has been demonstrated to protect patients from repeat anaphylaxis. Methods have been developed to prevent and treat massive sting attacks on frail elders, including those in residential and medical facilities. The potential beneficial effects of venom components are under investigation. And yes, the journey and the stings continue. Imported fire ants are now ubiquitous in the Southeastern United States and sting up to 50% of the population in their range per year (1). In this paper, I will briefly review the story of how I became involved in research on these insects and some of the information learned along the way. Although I am not the first to use the term, this has been my personal “journey to the ants” (2). Serendipity has allowed me to evolve into a medical entomologist and provided the opportunity to work with a number of wonderful colleagues. The experience has also exposed me to a variety of alcoholic drinks with umbrellas coming out their top. MATERIALS AND METHODS I reviewed the publications on imported fire ants in which I have participated over the years and chose to discuss those I thought might be of greatest interest to clinical climatologists. Correspondence and reprint requests: Richard D. deShazo, MD, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216, Tel: 601-984-5600, Fax: 601-984-5608, E-mail: [email protected] Potential Conflicts of Interest: None disclosed. 85 86 RICHARD D. DESHAZO RESULTS After completing training in clinical immunology and a long stay at Walter Reed Army Medical Center (Figure 1), our family arrived in New Orleans in 1980 to work in the Division of Clinical Immunology at Tulane University. My wife was pregnant with our third child and wanted to return to her southern roots, and my hair never conformed to military standards, anyway. The task at Tulane was to develop a research program centered on lung immunobiology with a focus on the epidemic of opportunistic infections that had suddenly developed in homosexual males in New Orleans. As we were moving into our house, our kids found dirt mounds in the yard and started to play in them (Figure 2). We had never seen these in Maryland. Our two girls were immediately stung by hundreds of red ants and experienced acutely painful skin lesions that burned “like fire.” These lesions developed into pustules that lasted for weeks. Our neighbors, who heard the children crying, came to help and were very familiar with these insects. They told us that fire ants entered the United States through the Port of Mobile, Alabama and had now spread throughout the southeast, including New Orleans, where it was difficult to walk anywhere without encountering them. We learned they had the potential to spread throughout most of the coastal US FIG. 1. Our last hurrah in the Army Medical Corps. The commanding general at Walter Reed (left) congratulates me on my promotion despite my long hair and shaded glasses. My very pregnant wife, Gloria, looks on with glee. She knows we are going back south after 9 years of military life. MY JOURNEY TO THE ANTS 87 FIG. 2. Top. Fire ant mounds common in the coastal United States. Bottom. Fire ants in the process of attacking and stinging a colleague who has disturbed their mound at the base of a landscape timber. Insert. Close up of a fire ant stinging. The ants first bite the host with their powerful mandibles and then insert their stinger with a copulatory movement. (Figure 3). We also learned they were difficult to exterminate. The queen lives far below the surface where workers protect her while she lays eggs to generate new egg-laying progeny. The ants had destroyed many ground nesting bird species and created major problems for farming. Life moved on. Collaborations were established, and we expanded our research to include a large population of hemophiliac patients who had begun to develop the same infections we were seeing in the homosexual males (3). Since New Orleans was the murder capital of the United States at the time, the coroner provided fresh lungs every morning for our lung research. In the Fall, our kids needed a science project, and they decided we would do one on imported fire ants. The kids did a door to door survey of our neighborhood and found that over half of the neighbors were being stung each year. Most developed various cutaneous reactions, and a few experienced anaphylaxis. Since our neighbors reported a variety of cutaneous reactions, we brought some of them into the medical center, stung them and observed the 88 RICHARD D. DESHAZO FIG. 3. Projected habitat of imported fire ants in the continental US. (http://www. ars.usda.gov/Research.docs.htm?docid⫽9165&page⫽1&pf⫽1&cg_id⫽0). reactions under controlled conditions. A hive developed at the sting site within 15 minutes and a pustule appeared by 24 hours in almost everyone who was stung. Some individuals developed not only the hive, but painful, edematous puritic late cutaneous reactions. I had worked on cutaneous hypersensitivity reactions in the Army and fortunately, Mom had one of the best biphasic reactions (4). I biopsied her reactions and those of others and characterized the histopathology in collabora- FIG. 4. Mom had one of the more robust late phase reactions to fire ant stings. MY JOURNEY TO THE ANTS 89 tion with a colleague in Boston (Figure 4). Many were the same IgE mediated, biphasic, late phase allergic responses our group had reported to insulin and pollens. We published the epidemiology and biopsy data in an allergy journal (1). To my surprise, I started getting invitations to speak to medical meetings in Boca Raton, Hilton Head and the Bahamas. They had funny looking drinks with umbrellas at those meetings. To the contrary, the meetings on my immunodeficiency work usually took place in Bethesda, in bad weather. Not even the coffee was free there. I sustained funding on my research assignment and contributed to the literature. By this time, imported fire ants had become the most common cause of anaphylaxis in the Gulf South. A hungry and shorton-funding immunochemist asked me to join him on a grant to study reactions to fire ant venom. To get venom, we tried to milk individual ants with micro-pipettes (Figure 5). This worked, but took too much time, and we kept getting stung. So, we collected thousands of ants by digging up their huge mounds on the levees around New Orleans and putting the mounds into garbage cans filled with water. The ants floated to the top, were scooped up and blenderized into extracts. The extracts had adequate venom protein for use in research studies and in immunotherapy to protect patients from anaphylaxis (Figure 6) (5). Fire ant venom is unique among insects as it is composed of only 5% protein, while 95% is piperdeine alkaloid. The alkaloids have been demonstrated by others to be used for communication, signaling and defense by fire ants. We found that the protein content, rich in phospholipase, was responsible for the allergic reactions, and the piperdeine alkaloids were responsible for necrotic sterile pustules that oc- FIG. 5. A drop of venom at the top of a fire ant stinger. (Source: US Department of Agriculture) 90 RICHARD D. DESHAZO FIG. 6. RAST inhibition curves demonstrating the venom content of whole body fire ant extracts made in the laboratory. (From reference (3). Used with permission). curred at the site of most stings (Table 1). This led to more publications in allergy journals and more drinks with umbrellas in them. I had little success getting my research on what had come to be known as the acquired immunodeficiency syndrome published in the New England Journal. On a lark, I sent the NEJM a manuscript on fire ant work I had planned to submit to an allergy journal. They accepted it with minimal revision (6). I got invited to more exotic locations to give more talks but kept going to Bethesda in the winter for the AIDS work. After nine years of very hard work in the laboratory at Tulane, I was offered a department chair job in our native Alabama at the new TABLE 1 Components of Imported Fire Ant Venomⴱ⫹ Protein AA/kd % Venom Protein Sol i 1 148/37 2–4 Sol i 2 Sol i 3 Sol i 4 119/26 212/24 117/13 50–75 15–25 8–10 Comment Phospholipase, homologous to that in the wasp; some lesser cross reactivity to yellow jacket venom carbohydrate Unique in fire ants, the major allergen Some homology with antigen 5 wasp venom protein Unique in insects Non-protein Components: 95% insoluble alkaline 2-methyl-6-n-alkyl and n-alkenyl piperadines: Solenopsins, isosolenopsins, dihydrosolenopsins. ⴱ 0.04 – 0.11 ml of venom/sting, 10 –100mg protein per sting. ⫹ Fire ant venom is cross-reactive with that of C. vittatus, the common striped scorpion. MY JOURNEY TO THE ANTS 91 medical school in Mobile, the fire ant epicenter. My research shifted from basic immunobiology to clinical research. The fire ants followed me. The ants had continued to adapt to their North American environment, expand their habitat and had begun to invade homes during dry weather (7). We heard about indoor fire ant attacks on frail elders in nursing homes and in one of our university hospitals where fire ant intrusion had occured (8). We found that the majority of individuals who died after fire ant attacks succumbed to heart failure (9, 10). Collaborations were developed with a series of entomologists to work out the medical entomology of these intrusions and resulted in a series of publications on fire ant entomology and pest control management in health care facilities (11, 12). After giving some more talks about fire ants in nice locations, we moved to Mississippi for a second chair job nine years later. We found that the US Department of Agriculture had released a species of fly imported from South America in south Mississippi in an experiment to control the ants. It lays its eggs in the heads of the ants. When the eggs hatch, the ants’ heads fall off (Figure 7). An Ole Miss toxicologist had heard of our work, and we developed a collaboration with him and interested graduate students. By this time, the venom proteins had been sequenced. That allowed the Natural Products Unit in the School of Pharmacy on our Oxford campus synthesized the alkaloids (Table 1). We then investigated the antibacterial and cytotoxic properties of alkaloids Solenopsin A, B, and C in collaboration with a doctoral student (Figure 8). Subsequently, the ability to inhibit nitric oxide synthetase activity and to cause direct cardiotoxic, convulsant, and respiratory depressant activities were demonstrated in vivo (Figure 9) (13–15). Especially interesting was the strong inhibition of myocardial contractility by the solenopsins (Figure 10). We postulated that this activity of the alkaloids could explain the heart failure that we reported in patients after massive invenomation (16). We also expanded our work on control of fire ants and treatment of reactions, began to FIG. 7. A florid fly hovers over a fire ant waiting to lay its eggs in the fire ant’s head (left). Fire ants in various phases of loosing their heads after florid flys’ eggs hatch (right). (Source: US Department of Agriculture). 92 RICHARD D. DESHAZO FIG. 8. Features of fire ant venom (Source: Robin Rockhold, PhD). look at the antimicrobial activities of venoms and developed an interest in other stinging ants (17, 18). Then we got invited to Ponte Vedra to join the American Clinical and Climatologic Association. The Association had some of those umbrella drinks and gave us a chance to review our fire ant work. Maybe all those fire ants we blenderized over the years would be pleased to know they were remembered before such an august group. FIG. 9. Some properties of fire ant venom alkaloids (Source: Robin Rockhold, PhD). MY JOURNEY TO THE ANTS 93 FIG. 10. Effects of alkaloids on rat myocardial contractility in vivo (Source: Adapted from reference 5). DISCUSSION Fire ants pose increasing peril to fauna, flora and agriculture in the coastal United States and Puerto Rico, where active infestation has already occurred. Their habitat will continue to expand. Invenomization has been associated with cutaneous and systemic allergic reactions, acute coronary syndromes, cerebrovascular accidents and seizures in humans. No method to stop the expansion of the range of these insects has yet been identified. Since there will be increasing contact between these insects and humans, we were anxious to understand the mechanisms for the clinical reactions that occur after invenomization. We now understand that the 5% of the venom that is protein is responsbile for the IgE-mediated, immediate and late phase reactions, reactions that can be prevented or attenuated by venom immunotherapy. Extracts for this therapy can be easily and inexpensively prepared from live fire ants and contain adequate quantities of fire ant venom to induce a protective immune response. The other 95% of the venom is composed of piperadine alkaloids, with a variety of cytotoxic and antibacterial activities. Some are strong inhibitors of nitric acid synthetase. Hopefully, the information we have learned about these fascinating insects will contribute to further research on treatment of reactions to invenomization and identify medical applications for venom components, including antimicrobial activities now under investigation. 94 RICHARD D. DESHAZO ACKNOWLEDGEMENTS I would like to thank my closest collaborators on this work, Brian Butcher, PhD (Tulane), HF Dvorak, MD (Harvard), WA Banks, MD (USDA), David F. Williams, PhD (USDA), Stephen Kemp, MD (UMC), and Robin Rockhold, PhD (UMC), for their hard work and support. Leigh Wright. BA (UMC) assisted with the production of this manuscript. I would also like to thank the millions of fire ants who have given their last full measure of venom. REFERENCES 1. deShazo RD, Griffing C, Kwan TH, et al. Dermal hypersensitivity reactions to imported fire ants. J Allergy Clin Immunol 1984;74(6):841–7. 2. Holldobler B, Wilson OW. Journey to the Ants. A Story of Scientific Exploration. Bellnap Press, Cambridge, MA, 1994. 3. deShazo RD, Daul CB, Andes WA, et al. A longitudinal immunologic evaluation of hemophiliac patients. Blood 1985;66(4):993– 8. 4. deShazo RD, Levinson AI, Dvorak HF, et al. The late phase skin reaction: Evidence for activation of the coagulation system in an IgE-dependent reaction in man. J Immunol 1979;122(2):692– 8. 5. Butcher BT, deShazo RD, Ortiz AA, et al. Superiority of Solenopsis invicta venom to whole-body extract in RAST for diagnosis of imported fire ant allergy. Int Arch Allergy App Immunol 1988;85:458 – 61. 6. deShazo RD, Butcher BT, Banks WA. Reactions to the stings of the imported fire ant. New Eng J Med 1990;323(7):462– 6. 7. deShazo RD, Banks WA. Medical consequences of multiple fire ant stings occurring indoors. J Allergy Clin Immunol 1994;93(5):847–50. 8. Kemp SF, deShazo RD, Moffitt JE, et al. Expanding habitat of the imported fire ant (Solenopsis invicta): A public health concern. J Allergy Clin Immunol 2000; 105(4):683–91. 9. deShazo RD, SF Kemp, deShazo MD, et al. Fire ant attacks on patients in nursing homes: An increasing problem. Am J Med 2004;116(12):843– 6. 10. Rupp MR, deShazo RD. Indoor fire ant sting attacks: A risk for frail elders. Am J Med Sci 2006;331(3):134 – 8. 11. Goddard J, Jarratt J, deShazo RD. Recommendations for prevention and management of fire ant infestation of health care facilities. So Med J 2002;95(6):627– 33. 12. Williams DF, deShazo RD. Biological control of fire ants: An update on new techniques. Ann Allergy Asthma Immunol 2004;93:15–22. 13. Yi GB, McClendon WD, Desaiah D, et al. The fire ant venom alkaloid, Isosolenopsin A, a potent and selective inhibitor of neuronal nitric oxide synthase. International J Toxicol 2003;22:81– 6. 14. McClendon WD, Yi GB, Desaiah D, et al. Selective inhibition of neuronal nitric oxide synthase by venom alkaloids from the imported fire ant (Solenopsis Invicta). J Invest Med 2003;51:S281. 15. Howell G, Butler J, deShazo RD, et al. Cardiodepressant and neurologic actions of Solenopsis invicta (Imported Fire Ant) venom alkaloids. Ann Allergy Asthma Immunol 2005;94(3):380 – 6. 16. Yates A, deShazo RD, Butler J, et al. Piperidine alkaloids from solenopsis invicta (red imported fire ant) venom exert systemic toxic effect in rats. Ann Allergy Asthma Immunol 2005;94(1):187. MY JOURNEY TO THE ANTS 95 17. Klotz JH, deShazo RD, Frishman AM, et al. Adverse reactions to ants other than imported fire ants. Annals Allergy Asthma Immunol 2005;95(5):418 –25. 18. White HDW. Evaluation of Antimicrobial Potential of Synthetic Solenopsis invicta (Red Imported Fire Ant) Venom Alkaloids. Doctoral thesis, Department of Microbiology, University of Mississippi June 2006. DISCUSSION Barondess, New York: Dr. deShazo, that was a marvelous talk. I noticed, as you pointed out, that the fire ants were largely confined to the red regions, and I noticed that there were very few in the blue regions; and putting that together with this last part about the decapitating fly, I wondered if you would like to draw anything broader out of your observations? Deshazo, Jackson: Well, it is very difficult for me to do since I may be the only Democrat I know in the entire state of Mississippi. However, there probably is some relationship.