1984 Imported Fire Ant Conference Proceedings

PROCEEDINGS

OF THE

1984 IMPORTED

FIRE AN.T CONFERENCE

<~y*¥' Publication of this Proceedings was supported by a grant from the Veterinary Medical Experiment Station, College of Veterinary Medicine, The University of Georgia, Athens, GA.

This publication is the result of a Symposium called and sponsored by the U.S Department of Agriculture, Animal and Plant Health Inspection Service. The opinions, conclusions and recommendations are those of the participants and are advisory to the agency. The papers published herein have been printed as submitted and have not been subjected to review by the agency prior to publication; therefore, the views expressed do not necessarily reflect those of USDA, and no official endorsement should be inferred.

Trade and company names are used in this publication solely to provide specific information. Mention of a trade or company name does not constitute a warranty or an endorsement by the U.S. Department of Agriculture to the exclusion of other products or organizations not mentioned.

-- Proceedings of the 1984 Imported Fire Ant Conference March 27-28, 1984 Gainesville, Florida

Hosted by Florida Department of Agriculture and Consumer Services Division of Plant Industry University of Florida Institute of Food and Agricultural Science

USDA Agricultural Research Service

Prepared by Dr. Michael E. Mispagel Department of Physiology and Pharmacology College of Veterinary Medicine The University of Georgia Athens, Georgia 30602 This Proceedings of the 1984 Fire Ant Conference is dedicated to the memory of the late William F. Buren

with gratitude for his contributions to the science of myrmecology. TABLE OF CONTENTS

Preface to Proceedings--MICHAELE. MISPAGEL,College of Veterinary Medicine, The University of Georgia, Athens, GA ...... 1 Welcoming Remarks--SAL A. ALFIERE,Director, FDACS, Division of Plant Industry, Gainesvill~, FL ...... 2 Bill Buren: Scholar With a Mission--D.L. SHANKLAND,Chairman of the Department of Entomology and Nematology, IFAS, University of Florida, Gainesville, FL ...... 4

Fire Ant Taxonomy--Whither Now?--JAMESTRAGER,University of Florida, Gainesville, FL (Abstract only) 6

Influence of Selected Weedy and Weed-Free Sugarcane Habitats on Diet Composition and Foraging Activity of the Imported Fire Ant--AHMEDD.ALl, T.E. REAGAN,and J.L. FLYNN, Louisiana State University, Baton Rouge, LA (Abstract only) ...... 7 The Imported Fire Ant in Sugarcane Fields in Puerto Rico-- CARLOSCRUZand SANDRACASTRO,University of Puerto Rico, Isabela, PR ...... 8 Imported Fire Ant Resurgence Following Chemical Treatment of Pastures--LOIS WOOD,University of Florida, Gainesville, FL (Abstract only) ...... 10 Studies on the Ecology of Solenopsis in Mato Grosso, Brazil-- JERRYSTIMAC,University of Florida, IFAS, Gainesville, FL (Abst ra cton 1y) ...... 11 Recent Discoveries in Fire Ant Chemistry, Physiology, and Behavior--ROBERT K. VANDERMEER,USDA/ARS,Gainesville, FL. . 12 Current Research Efforts on The Imported Fire Ant at Texas A & M-- S.B. VINSON,Texas A&M University, College Station, TX (Abstract only) ...... 25 Effect of Pro-Drone on the Fire Ant, Solenopsis invicta Buren-- DAVIDBALL, Texas A&M University, College Station,TX (Abst r act 0n1y) ...... '.... 26 Fluorinated Sulfonamides: A New Class of Delayed Action Insecticides--ROBERT K. VANDERMEER,CLIFFORDS. LOFGREN, and DAVID F. WILLIAMS, USDA/ARS,Gainesville, FL ...... 27

Field Trials with Bait Toxicants--HOMER COLLINS, USDA/APHIS/PPQ, Gulfport, MS ...... 40 Chronic Toxicity of a Fire Ant Insecticide in Dogs--DWIGHTB. COULTERandWAYNEA.CROWELL,University of Georgia, Athens, GA (Abstract only) 52 Update on the Registration Status of MK-936--RICHARD A. DYBAS, Merck & Company, Three Bridges, NJ (Abstractonly) 54

An Environmental Risk Assessment of Pro-Drone; Insect Growth Regulator for Controlling the Red Imported Fire Ant (Solenopsis invicta)--DENNIS PAUSTENBACH, Stauffer

Chemical Company, West Port, CT ...... 55

Current Research with Toxic Baits--DAVID F. WILLIAMS, USDAj

ARS, Gainesville, FL ...... 66

Supercooling Studies on Four Species of Fire Ants--OSCAR F. FRANCKE, JAMES C. COKENDOLPHER, and LINDSEY R. POTTS, Texas Tech University, Lubbock, TX (Abstract only) 72

Airborne Venom Dispersal in Solenopsis: Functional Correlates of Insecticidal and Antibiotic Venom Properties-- MARTIN OBIN and ROBERT K. VANDER MEER, USDAjARS, Gainesville, FL ...... 73

1983 Texas Imported Fire Ant Pro-Drone Aerial Application Program--MARK R. TROSTLE, Texas Department of Agriculture, Austin,TX ...... 87

Questions and Answers on Pro Drone Application Program, Texas Department of Agriculture, Austin, TX ...... 88

IFA and Citrus (A videotape presentation)--CLAUDE ADAMS and

CLIFFORD S. LOFGREN, USDAjARS, Gainesville, FL ...... (NA)

Colony Founding Minums: A New Solenopsis invicta caste--JAMES BOSWORTH and R.K. VANDER MEER, USDAjARS, Gainesville, FL .. 92

A Citrus Based Solvent as a RIFA Mound Drench Material--CRAIG SHEPPARD and CYNTHIA A. GATES, University of Georgia,

Tifton, GA ...... 106

Reinfestation Rates of Imported Fire Ants Following Toxic Bait Treatments--CLIFFORD S. LOFGREN and DAVID F. WILLIAMS,

USDAjARS, Gainesville, FL ...... 112

Diurnal and Seasonal Differences in RIFA Roadside Abundance-- DANIEL P. WOJCIK, R.J.BURGESS, and C.M. BLANTON, USDAjARS,

Ga ine svi 11e, FL ...... 120

A Technique for Marking Fire Ants--DANIEL P. WOJCIK,R.J. BURGESS, and C.M. BLANTON, USDAjARS, Gainesville, FL ...... 131

Hematologic and Immunologic Response of Holstein Calves to a Fire Ant Toxicant--DONALD L. EVANS, KAREN L. JACOBSEN, and DORIS M. MILLER, University of Georgia,

Athens, GA ...... 133

Research Highlights: 1983-1984--B.M. GLANCEY, USDAjARS, Gainesville,FL...... 138 Isolation of the Pheromones of Solenopsis invicta and ~. richteri--FRANCISCO ALVAREZ,R.K. VANDERMEER, and CLIFFORDS. LOFGREN,USDA/ARS, Gainesville, FL ...... 162 Economic Impact of Imported Fire Ant in Puerto Rico--JOSE M. RODRIGUEZ,Assistant Secretary of Agriculture, San J uan, PR ...... (NA) Foraging Strategies of Native Ants in Fire Ant Infested Areas of Central Texas--SHERMANA. PHILLIPS, JR., DAVIDM. CLABORN, and DAVEWESTER,Texas Tech University, Lubbock, TX ..... 179

Pro-Drone Update--J. NOELWAGNER,Stauffer Chemical Company, Raleigh, NC ...... (NA) An Update on RO-13-5223 (Logic Fire Ant Insecticide)--LEE BENSON, MAAGAgrochemicals, Vero Beach, FL ...... 197 Accudose Aerosol Injection Methods of Fire Ant Control with Pyrethrins or Dursban--O. GRADYQUERY,Cessco Company, Inc., Charlotte,NC...... 205 Appendix A: Fire Ant Pesticide Fact Sheet--Environmental ProtectionAgency...... 213 Appendix B: Participants in the 1984 Fire Ant Conference .... 226

(NA) - Not available at time of publication Preface to Proceedings

The decision to publish these proceedings was made by the participants of the conference at its conclusion. Many authors submitted abstracts prior to the start of the conference, but formal manuscripts were not requested. Because of the short notice given to authors to prepare contributions to the proceedings, a free format was permitted for this year's proceedings. Authors were requested on a volunteer basis to submit, in camera-ready form, whatever they felt appropriate to effectively convey the information they presented at the conference. The table of contents lists all presentations given during the 1984 Fire

Ant Conference and indicates whether the submission for the proceedings was in abstract form or unavailable at the time of publication rather than a formal manuscript. These submissions have been offered in the spirit of a free ex- change of ideas regarding the imported fire ant and to inform colleagues of the status of current research efforts. In general, these contributions have not been subjected to peer review and, thus, their appearance in these proceedings does not constitute formal publication.

Because of the preliminary nature of many of these data, the authors request that contact be made with them for updated information before data in these proceedings are cited. Where manuscripts have been submitted or are in press, the formal publication should be referenced.

M. E. Mispagel May 1984

1 WELCOMING REMARKS . DR. S. A. ALFIERI, JR. DIRECTOR, DIVISION OF PLANT INDUSTRY FLORIDA DEPARTMENT OF AGRICULTURE AND CONSUMER SERVICES

IMPORTED FIRE ANT CONFERENCE MARCH 27-28, 1984

On behalf of the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, and our Commissioner of Agriculture, Doyle Conner, I welcome you to the 1984 Imported Fire Ant Conference. I trust you will find our facilities adequate here at the Doyle Conner Building. If we can help make your accommodations more suitable during the conference, please do not hesitate to let us know.

The problem of the imported fire ant is one that is common to all of us here, and I see we have representation from the nine affected states and Puerto Rico.

We hear a great deal about what a nuisance the fire ant is to the people who buy the insecticide-bait distributed by the Division of Plant Industry in the state and federal fire ant program.

Besides inflicting very paififul stings, this aggressive ant is an economic pest ot agriculture. The mounds it constructs as it establishes colonies are unsightly and make landscapes difficult to maintain. We most often hear about what a nuisance it is in children's playgrounds, around gardens and landscaped yards, along fences, around farm buildings and in hay fields, as well as reported damage to crops such as soybeans, corn, young citrus...and in some cases injury to young cattle and horses.

The imported fire ant is a regulatory problem as well to all in the infested areas. To certify plants and plant material being moved out of quarantined areas is expensive for nursery owners who must take measures to make sure their export shipments are free from the imported fire ant.

The ant's rapid spread throughout the Southeast has made the name given to it by the Late Dr. William Buren well deserved. Solenopsis invicta does indeed seem inconquerable.

As I looked over the conference program, however, I see research is taking us in a direction of better controls ot Solenopsis invicta. You will hear reports over the next two days on continued investigations into more effective chemical controls and on research into biological control of the imported fire ant. The .Division of Plant Industry is dedicated to furthering this research. For the past three years, we have granted annually $75,000 of the funds from the state/federal imported fire ant program to help support IFAS research in Brazil on biological control. Dr. Jerry Stimac will speak to this subject later this morning.

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r'll be looking forward to hearing more about the discoveries made recently about the biology of the ant itself as well as about efforts to find better controls.

,.vith the array of knowledge represented here, I am certdin this conference will be most informative and productive. Bill Buren: Scholar With A Mission

D.L. Shankland

I want to thank Dr. Nickerson for the opportunity to appear briefly this morning out of respect for a good scientist and a good friend.

There is a great deal we do not know about the imported fire ant, but certainly all we do know, and everything that we will learn about it depends absolutely on the unequivocal identity of that anim~l, with a name to which all relevant information can be tied. It is no surprise to this audience that we owe that knowledge to Bill Buren. But, I believe we owe him a great deal more, ana as chairman of the department in which he ended his career, I feel that debt very keenly.

Bill was born in Kenton, Iowa. He attended Iowa State University where he received the B.A. degree in 1940, and the B.S. in 1943. He did graduate work at Cornell University where he received the Ph.D. in 1953. From 1943 to 1973, he served as a commissioned officer in the U.S. Public Health Service, and retired as Scientist Director of that organization in 1973. During those years he also served on various panels in the National Institutes of Health and was Grants Administration Director for the Communicable Disease Center in Atlanta. He joined the Department of Entomology and Nematology at the university of Florida in 1972, and served as professor of entomology until his death August 13, 1983.

When I think about the imported fire ant problem, and Bill's role in it, I am reminded of an interview of Ernest Hemingway that was described in a book entitled, "Teaching as a Subversive Activity." Hemingway was being interviewed by a young journalist who was trying to get him to say that there is some attribute that a person must have to be a great writer. He had a list, and suggested one attribute after another, and Hemingway disparaged each one in turn. Finally he said, "There is one thing you need; you have to have a built-in, shock-proof crap detector." I believe all good scientists have one of those. Certainly the great ones did. Copernicus had one when he disproved the geocentric theory of the uni- verse. Spallanzani had one when he refuted the theory of spontaneous generation. Lavoisier had one when he disproved the phlogistin theory, and the list could go on and on.

4 5

To get to the truth in the imported fire ant problem, you have to get through an enormous amount of crap, and Bill had a good crap detector. He worked very hard to get at the truth. He pursued his work not only on the precise identity of Solenopsis species and ants that are associated with them, but also toward an understanding of the biology and interactions of these species in the field. without his keen talent for taxonomy, much of the work that has been done on the biology of Solenopsis invicta and other species would have been impossible. Bill pursued this work in the laboratory and the field, and through it all he had a mission. His mission was to disclose the truth as best he could through scientific investigation, and to ensure that evidence and knowledge that had been collected were not ignored in any action taken on the fire ant problem. He pursued this mission vigorously with his colleagues in the department in honest and open debate, and always referred back to the evidence to support his argue- mnts. If he had been dealing with a problem less notable than the imported fire ant, these debates would have gone with no more notice than those that are pursued every day in any scientific community. But Bill perceived that major programs that ignored important evidence and knowledge were being implemented against the fire ant. That the Jim Buck Ross's and Reagan Brown's were prime movers of these programs, and that whatever their motives, the programs were ill conceived and counter productive. Bill had too much integrity to let this go unchallenged. Even though he was not of the temperment to be a public figure, he entered a public debate on the issue with the same unswerving commitment to logic, evidence and science that he used in debating his colleagues. I do not know if Bill was always right in the stands he took, but I do know that he took them with forthright honesty and a towering integrity. It is for his competence as a scientist, and for his consistency in intellectual honesty and integrity that extended from the cloistered academic arena to the public stage that I have the greatest respect for Bill. He brought to us the stuff that great departments are made of. I will miss him. His colleagues will miss him, and science will be better off for his having been here. Fire Ant Taxonomy - Whither Now? James C. Trager

The history of taxonomic work on the genus Solenopsis is reviewed, through the recent efforts of W. F. Buren. Present status of the Buren ant collection and curatorial efforts being carried out by the University of Florida fire ant ecology project are described. The need for, and the possibilities of, future taxonomic work on the genus are discussed.

6 Influence of Weedy and Weed-Free Sugarcane Habitats on Diet Composition and Foraging Activity of the Imported Fire Ant (Hymenoptera: Formicidae) by A. D. Ali, T. E. Reagan and J. L. Flynn Department of Entomology, Louisiana State University, Baton Rouge, LA

Submitted to Environmental Entomology 13: (In Press).

ABSTRACT

Imported fire ant (IFA), Solenopsis invicta Buren, diet composition

and foraging activity investigations in selected weedy sugarcane

habitats showed that the most frequent foraging occurred in grass

habitats, which also had the greatest vegetation density and above

ground biomass. Predation observed on 'immature leafhoppers, adult

dipterans, phytophagous insects (collectively), and on foliage

associated fauna was greater (P

weed-free habitats. Sweep net counts of foliage associated arthropods

revealed highest relative abundance in grass (N=828) vs. weed-free

(N=141) habitats. Greatest species diversity (H'=3.464) occurred in

broad1eaf regimes. IFA prey capture efficiency was lower in broadleaf

than in weed-free habitats, possibly due to pollen and nectar foraging,

and was lower in grass habitats likely due to predator satiation. Based

on the results obtained, broadleaves are expected to provide more prey

- 2 per amount dry biomass (g/m ) than grasses or sugarcane alone. These

data show that IFA population levels can be enhanced through judicious

vegetation management which would result in increased ecological

stability of the sugarcane crop protection system.

7 The Imported Fire Ant in Sugarcane Fields in Puerto Rico Carlos Cruz and Sandra Castro Crop Protection Department, UPR Mayaguez Campus

1. Introduction:

The Imported Fire Ant (lFA) Solenopsis invicta was detected for the first time in Puerto Rico during May, 1981 by the late entomologist Dr. William F. Buren. Surveys conducted by APHIS people in Puerto Rico have determined that the Imported Fire Ant is already distributed in the south of the Island from Anasco to Yabucoa. It has been found in roadsides, in plantings of vegetables, making damage to cabbage and cucumber seedlings, in fruits making damage to small trees of avocado and mangoes, and in sugarcane fields in the municipality of Yabucoa. Preliminary studies were conducted in an attempt to determine the spatial distribution, nesting, food habits and the interrelation with other ants or insects in the sugarcane fields in Puerto Rico. 2. Experimental plot:

The experimental plot was selected in a sugarcane field known to be infested with the IFA. It consisted of 35 x 40 m and the sugarcane plants were about 1 m height. The plot was divided into subplots of 4 x 1.52 m (250 subplots). 3. Results:

There were 52 active mounds of S. invicta (81%) and 24 (19%) nests of Pheidole fallax in the experimental plot. The average diameter and height of the IFA mounds was 40.7 and 12 cm, respectively. Average mound per acre was 150 and 69 for S. invicta and P. fallax, respectively. Seventy-eight percent of the IFA mounds were-in the center of the row, associated with the sugarcane stump, while 83 percent of the nests of P. fallax nests were apparently made in abandoned mounds of S. invTcta. Observations made from 8 to 10:00 a.m. during July 8 and 20, August 5, 8, 10 and 12, 1983, indicated that S. invicta was feeding on living specimens of annelids (earthworms), myriapods (millipede), mole crickets,(adults and scarabaeids larvae), some (mostly spiders PhYllO~ha~), an o,er unidentifiedhomopterans, insects.lepidopterans

The greatest number of ants were found on annelids, Phyllophaga and mole crickets. Some small white grubs were found being carried away by S. invicta. Infestation of canes by the sugarcane borer Diatraea - saccharalis was not observed in this sugarcane field. This insect is a serious pest in other sugarcane fields around the island. Larvae of the sugarcane rootstalk weevil, Diaprepes abbreviatus, were buried near S. invicta mounds and were quickly found and killed by the ants. Furthermore, no symptom or damage by this or similar insects

8 9

(Phyllophaga) was observed in the experimental plot or sugarcane fields nearby. No other ant species was observed in the experimental plot. However, two other ant species were found in the sugarcane field around the plot: Paratrechina steinheili and Brachymyrmex heeri. 4. Conclusions:

From this study we can make the following preliminary conclusions: 1) S. invicta is well disseminated in the sugarcane fields in Yabucoa, P.R. 2) It preferred to nest in association with the sugarcane stump. 3) It could be displacing other ant species in the area. 4) It could be favoring aphids and scale insects in the sugarcane fields.

5)Diaprepes) It coul d be and cantrall the sugarcane i ng the white borer grubs(Q. saccharalis (Phyll oPhar . and This is only a preliminary trial and further studies should be conducted to determine the exact role of the imported fire ant in sugarcane fields and other crops in Puerto Rico. Imported Fire Ant Resurgence Following Chemic~l Treotment of Pastures

hy Lois Wood

The red imported fin: ant ([fA), ~olenopsisinvictaBuren, has been reported to be an efficient rolonizer of ecologically disturbed areas. However, the abilitv of ~. Lnv_ictGto colonize disturbed habitats in comparison to native ant species is not clearly defined for a number of different habitGts. Population resurgence pot~ntial of IFA and native ant competitors, particulGrly~. geminata, Conomyrma spp., ond Pheidolc spp. are reported. i\ntpopulGtions in pasture plots treated \vith~lin::< or :u-ndro :lrecomp3red to untreated plots. The role of bro:ldcastapplic3tions of non-specific formicides in the displacement of native ant populations by~. invictG is discussed.

10 Studies on the Eco]ogy of Solenopsis in r!ato Grosso. Brazil

:iV Ji_'rrv Stimac

Research scientists.::It the ~'niv("rsitvof Florid;},in cooperation with scientists from the USDA and E~GRAPA. have established field stations in the t-latoGrosso, Rr.:1ziltostudy the factors that regulate Solenopsis populations in their homeland. Studies have been initiated to discover the roles of biological agents in the dynamics of ~ire ants in Brazil. The feasibility of procurring biological control agents for introduction into the southeastern United States is d iscussecl.

11 Recent Discoveries in Fire Ant Chemistry,~ Physiology, and Behavior Robert K. Vander Meer, Ph.D.

Insects Affecting Man and Animals Laboratory USDA/ARS,Gainesville, Florida 32604

~/ This paper presents the results of preliminary research only and will be published later through established channels with appropriate editing. The paper and data should not be referred to in literature citations until they appear in press. Mention of a proprietary product does not constitute a recommendation by the United States Depart- ment of Agriculture nor does it imply registration under FIFRA as amended.

12 13

ABSTRACT

Recent Discoveries in Fire Ant Chemistry,

Physiology, and Behavior Robert K. Vander Meer, Ph.D., USDA/ARS,Gainesville, Florida

The fire ant, Solenopsis invicta, has a wide array of orientation

mechanisms available to aid in its overall foraging strategy. These include A) light orientati6n, B) positive geotaxis, C) visual-distal

cues, D) territorial markers and E) trail pheromones. Each broadly defined orientation mechanism can be broken down into a hierarchy of

behaviors as illustrated by the trail pheromone complex. It is composed of at least the following sub-categories of behavior-pheromones 1) orientation primer pheromone, 2) orientation pheromone, 3) recruitment pheromone

and 4) aggregation. All of these behaviors are elicited from the contents of the Dufour's gland and are interdependent on each other, both behaviorally and chemically. This hierarchy of trail pheromone behavior provides a mechanism for species specificity between Solenopsis invicta and Solenopsis richteri, previously reported to follow each others trail. They do not exhibit interspecific recruitment. Chemical and behavioral data were presented that demonstrated the occurrence of hybridization between

Solenopsis invicta and Solcnopsis richteri where the 2 species interface in Northern Alabama and Mississippi. This work clearly raises a number

of questions regarding past interpretation of Solenopcis richteri's original displacement and about the future of Solenopsis invicta and

Solenopsis richteri populations. 14

I would like to present the current hypothetical scenario, in simplified form, for fire ant foraging and trail formation. The sequence of behaviors involved in mass-foraging by workers of Solenopsis invicta was outlined by Wilson (Table 1) in 1962. Solitary workers forage away from their nest in irregular looping paths. If a discovered food source is too large the worker returns directly to its nest laying an odor trail. At the nest additional workers are recruited and follow the odor trail.

These workers reinforce the trail when they return to the nest; however, when the food supply is gone, the odor trail is no longer reinforced and it dissipates. We want to address the following primary questions about foraging behavior.

1) Howdo solitary workers find their way back to the nest?

2) What is the behavioral hierarchy of the trail pheromone system?

3) How is Dufour's gland chemistry related to trail pheromone behavioral hierarchy?

The initial trail formation step requires an orientation mechanism, one of which we determined to be (what some people call) "Sun Compass" orientation; however, the light source does not have to be the Sun, it could also be the moon, stars, street lights or any light source. Other orientation mechanisms have also been reported. For example,

1) Positive geotaxis of workers carrying refuse. Translated this means that they orientate in a down hill direction. 2) Workers can orientate to distal-visual cues, which means they can learn from experience a specific path to and from their nest.

(3) Workers can distinguish their nest soil from foreign nest 15

soil . This implies that territorial markers or individual colony odors may playa role in orientation.

4) The trail pheromones. These chemical trails take precedence and dominate other orientation mechanisms once in place. The main point is that there is not just one orientation mechanism. The fire ant has an array of possible orientation mechanisms some of which I am sure have not yet been discovered. Within each orientation mechanism there are most likely subtleties and nuances that at first glance blend together and are not recognized as a composite of several behaviors or sequences of behavior. This point, I think, is well illustrated by our work on the trail pheromone of S. invicta.

Wilson many, many years ago (1959) determined that the Dufour's gland was the source of the trail pheromone. He later ascribed many different behavioral functions to its secretion, such as, attraction, trail following, recruitment, and imigration induction. (Is that how the ant made it to the United States?)

A few years ago we reported the isolation ahd identification of

Z,E-a-farnesene(I) as the major S. invicta trail pheromone along with less active E,E-a-farnesene and 2 homofarnesenes (Fig 1). We have synthesized the 4 homofarnesene isomers and established that they have the Z,E and E,E configuration. This was confirmed by direct comparison with the natural products and by bioassays. (Williams and Vinson (1981) reported Z,Z,Z-allofarnesene as the trail pheromone from their Texas

S. invicta multiple queen colonies.) The standard trail pheromone bioassay we used for the chemical isolation consisted of establishing a bridged worker trail to a food source in such a way that a section of the trail could be removed and 16 replaced with a streak of test material. The worker ants that came in contact with the test material were already following a trail - they were primed toward trail orientation.

Z,E-a-farnesene, the major and most effective S. invicta trail orientation pheromone does not induce trail following when test material is placed among actively foraging workers or when placed next to a colony cell; however, when an equivalent amount of Dufour's gland extract was placed as a spot at the beginning of a component I trail, active trail orientation occurred. The ants required a primer pheromone before they would orientate along the trail. The general term trail pheromone is really composed of orientation primer pheromones, orientation pheromones, and as you can see from Fig. 2 there is more to it than that. A bioassay developed to investigate worker recruitment showed that none of the 4 orientation pheromones individually, or in any of the possible combinations, recruited worker ants. This led to the conclusion that ~omponents responsible for orientation were either mutually exclusive of recruitment components or that unidentified'compounds in combination with the orientation pheromones were required for recruitment.

The recruitment bioassay measures worker response to a point source of test material; however, our results show that both recruitment or attraction, and aggregation occur. To measure just attraction we have developed a V-tube olfactometer that discretly measures attraction. Results with Dufour's gland have shown significant activity down to .01 WE. Details of this olfactometer bioassay will be presented by Mike Glancey. We have at least four bioassayable behaviors attributable to Dufour's gland secretions. Although discussion of full details of our results 17 are not possible within the constraints of this paper, I would like to say that we can duplicate about 85% of Dufour's gland activity in all four behavioral systems using a combination of nine discrete chemical components found in Dufour's gland extract. Interestingly, none of the trail pheromone sub-systems is totally independent of the other behaviorally or chemically. It is 1i ke the 3 musketeers motto HOnefor all and all for oneil.

As you might expect there are more than nine components in Dufour's gland extract and I would not be surprized if to increase activity to 100%, it would be necessary to essentially reconstruct the Dufour's gland GC profile to gain all the subtleties that may be involved. That is by using naturally occuring ratios and amounts of components. We can, of course get greater than 100% Dufour's activity simply by increasing the amount of one or more components. We have been simultaneously working on the trail pheromone system of S. richteri and can also duplicate ~5% of Dufour's gland activity. The following two interesting developments are also related to S. richteri. Our study of a hierarchy of trail pheromone behavior and chemistry provide a unique mechanism for pheromonal species isolation in S. invicta and S. richteri. It has been reported (Table 2) that they follow or orientate to each others trails. As we have found orientation - is not independent of recruitment. Bioassays with Dufour's gland extracts have shown that the two species are not recruited to interspecific Dufour's extracts (Table 3). This provides a mechanism for the separation of the two species in the foraging arena in areas where they interact.

We have chemical evidence that hybridization between S. invicta and

S. richteri is occurring at the interface where they meet in northern 18

Alabama and Mississippi. The chemical criteria used are 1) venom patterns, 2) Dufour's gland components, and 3) cuticular hydrocarbon patterns. The venom of both species was characterized by Murray Blum and collaborators

in the 1970s. S. richteri was easily differentiated from S. invicta by

the absence of C15 alkaloids. However, colonies collected recently that

morphologically looked like S. richteri did not have the right chemistry. The GC patterns of venom alkaloids from S. richteri, S. invicta qnd our proposed hybrid show two things:

A) The hybrid has significant amounts of S. invicta-like C15 alkaloids, which are absent from pure S. richteri.

B) The ratio of the hybrid C13 alkaloids is reversed from that of pure S. richteri.

Our criteria, by the way, for pure S. richteri colonies is the absence of

C15 alkaloid. This criterion is based on Blum's 1973 work and personal

communication with Dr. Blum, who indicated that he never observed C15 alkaloids from the multitude of S. richteri samples he and his collaborators studied. So, regarding venom components the hybrid looks like a

blend of S. richteri and S. invicta.

The problem first came to our attention because of our trail phero- omone investigations and our need for Dufour's gland samples from

S. richteri. Gas chromatograph traces of S. invicta and S. richteri

Dufour's gland extract are very different, with S. richteri having only one major component. This component is found in minute quantities in S. invicta. We have not been able to detect Z,E-a-farnesene in S. richteri.

The S. richteri profile corresponds to Murry Blum's published data of the early 1970s. This is the most dramatic chemical evidence we have so far. The S. invicta contribution to the hybrid dominates, but the 19 pronounced amount of component "C-l" (the single major component in s. richteriDufour's gland extract) clearly differentiates the hybrid from pure S. invicta. Again, morphologically the hybrid looks like S. richteri.

These results brought up an interesting question. Table 3. demonstrated species isolation in the foraging arena by the lack of interspecific recruitment activity. As Table 4 illustrates, both species respond to hybrid Dufour's gland extract and the hybrid responds to

Dufour's gland extracts from both S. invicta and S. richteri. The hybrid can go either way. These results provide behavioral evidence for hybridization. Similar combinations of S. invicta and S. richteri cuticular hydrocarbon patterns were observed for the hybrid. Cuticular hydrocarbons have been shown to be diagnostic of the species. Our results make it clear that anyone working with S. richteri is obligated to check the venom pattern to verify the purity of the colony. Some questions raised by the work are as follows: 1. The Demise of the Original S. richteri Population: Outcompeted Behaviorally and/or Genetically? 2. What is the Distribution of Pure and Hybrid S. richteri?

3. Is the Hybrid Reproductive? 4. Is the Current S. invicta Population Genetically Pure? 5. Does a Similar Phenomenon Occur in South America?

6. Is the Hybrid More Cold Hardy?

7. What Morphological Features Distinguish the Hybrid from Pure S. richteri?

At next year's meeting we should have answers to some of these questions and more questions to be answered. 20

Table 1. Sequence of Behaviors Involved in Mass-Foraging by Workers

of Solenopsisinvicta (after Wilson, 1962)

1) Hunting by solitary workers who move away from next in

irregular looping paths.

2) Worker locates food source and inspects it. 3) If food source is large, worker returns to nest laying an odor trail.

4) Nextjadditional workers are recruited and follow odor trail to food.

5) Additional foragers reinforce trail. 6) When food supply is gone, odor trail dissipates. 21

Table 2. Combined Trail Pheromone Species Specificity Results

Numbers refer to references and a

Source of positive response from test species

Trail pheromone extract S. invicta S. richteri S. geminata S. xyloni

S. invicta 1,2,3 *,2,3 3

S. richteri *,2,3 *,2,3 * * * S. geminata 1,2,3 2,3

S. xyloni 1 *,2 1,2,3 1,2,3 l. E. O. Wilson, Anim. Behav. lQ, 134 (1962). Dufour's gland extract.

2. M. R. Barlin, M. S. Blum, and J. M. Brand, J. Insect Physiol ~, 839 (1976). Dufour's gland extract. 3. D. P. Jouvenaz, C. S. Lofgren, D. A. Carlson, and W. A. Banks,

The Florida Entomologist, &1, 244 (1978). Purified whole ant extracts. * Reference 1 did not test 22

Table 3. Percent Response to Dufour's Gland Extract

Dufour's Gland

TEST SPECIES S. invicta S. richteri

S. invicta 100.0 14.4

S. richteri 13.3 100. 0

Table 4. Percent Response to Dufour's Gland Extract

Dufour's Gland

TEST SPECIES S. invicta S. richteri Hybrid

S. invicta 100.0 14.4 93.5

S. richteri 13.3 100.0 107.5

Hybrid 91.1 113.6 100.0 Fig 1. Structures of S. invicta Trail Orientation Pheromones

I. Z, E-a-FARNESENE II. EI E-ex-FARN ESENE

N W

III. Z, E- HOMOFARNESENE IV. E,E- HOMOFARNESENE Fig 2. Trail Pheromone Behavioral and Chemical Sub-Systems

Orientation Primor

Orientation

TRAIL PHEROMONE Recruitment

Aggregation N .j::>

Unknown Current Research Efforts on the Imported Fire Ant at Texas A&M. S. B. Vinson

An overview of the current research projects on the Imported Fire Ant were presented. The Texas program is presently emphasising the genetics, food and bait distribution and preference, caste polyethism, development of polygyny and monogyny, ecological interactions and reproduction physiology, as major thrust areas of research. In addition, the program is involved with various cooperaters in examining the effectivness of products for fire ant control.

25 .".3~Tr;ACT

The ;::f:'e~-;o ,< "',.-,,-:C,-,,::e c:: the Fire Ant, "r:Jlenopsis invictcc Buren

David::. ,.,

;,1ature ,~()2.o"ie3 8:' c':,.-" :meo '"ere treated with Pro-Drone and the hiseolO,;ico.l e :':-e~'ts 'to ,':e n;>een' S ovaries were examiHed at monthly ir:tervo.ls. -:'"e o-"~,ries s',u,;etl c.n overall reduction in size and the individu~2. 8~~ri~L8s .~re ~t.ro~hied with the surroundingsquamous epi thelh,..:: s:1O'.;i,,~ :]li.'ht h:iDercronhy. The oocytes decreased in size and n~~ber su~p,estin~ reabsorbtion. All treated queens died within 6 months. Relatively little iifference was seen in the number of eggs produced by treated and control queens. However, treated colonies began producing female reDroducti~e brood showin~ apparent morpholo~ical and histological anomalies i~dico.ti,,~ tho.t the reproductive co.pabilities of the fire ant were severely affected.

26 Fluorinated Sulfonamides: A NewClass of Delayed Action Insecticides

Robert K. Vander Meer, Ph.D.; Cliff Lofgren, Ph.D.; and Dave Williams, Ph.D.USDA/ARS,Gainesville, Florida

~ This paper presents the results of preliminary research only and will be published later through established channels with appropriate editing. The paper and data should not be referred to in literature citations until they appear in press. Mention of a proprietary product does not constitute a recommendation by the United States Depart- ment of Agriculture nor does it imply registration under FIFRA as amended.

27 28

ABSTRACT

Fluorinated Sulfonamides: A New Class of Delayed Action Insecticides

Robert K. Vander Meer, Ph.D.; Cliff Lofgren, Ph.D.; and Dave Williams, Ph.D., USDA/ARS, Gainesville, Florida

A new class of delayed action toxicant with the generalized formula

RfS02NRIR2were presented. Rf is a fluoroaliphatic radical and RI and R2 Can be any chemically compatible groups. Structure activity-relationships showed that activity was lost if the steric bulk of RI or R2 was great

(i.e. Rl=H, R2=t-Butyl). Unsaturated hydrocarbon substituents gave fast kill if the unsaturation was directly attached to the nitrogen; however, if a methylene group was placed between the nitrogen and double bond, delayed activity was observed. Mono-alcohol substituents showed delayed activity, but diols were inactive. Polyether substituents, either hydrogen or methyl-end capped, all showed similar delayed activity. The

C8Fl7 fluorcarbon radical yielded the best activity, and it was demonstrated that both the fluorocarbon and sulfone groups are essential to the activity of this class of comnound. Both water soluble and oil soluble members of the class make them versatile in several different control situations and for several different types of insect pests. 29

We would like to introduce a new class of fire ant toxicant that has only-been alluded to at past meetings. All of the compounds presented were obtained from 3-M Company, St. Paul, Minnesota. The results are from our primary screening bioassay. The general bioassay procedure is outlined in Table I. In most of the examples the length of the bioassay was extended to 21 days, and unless otherwise specified the samples were dissolved in soy bean oil.

All of the toxicants fit into the general structure RfS02A, where Rf is a fluoroaliphatic radical and A is any structurally compatible residue. Most of the compounds fall into the sulfonamide general formula,

RfS02NR1R2' where Rl and R2 are any compatible structures. The activity of these compounds and their structure-activity relationships can be illustrated by keeping Rf constant and varying Rl and R2' Rememberthat for fire ant control we want delayed action over as wide a range of activity as possible.

Table II illustrates the simplest examples where Rl and R2 ; Hare alkyl groups. We see excellent delayed activity in almost all of the compounds in this series. However, when the steric bulk of the alkyl group is increased to t-butyl (10713) all activity is lost. Interestingly, the diethyl (10707) and the dOdecyl (29777) both show delayed action.

When the substituent is unsaturated (Table III), we found that if the unsaturation was directly attached to the nitrogen (10717 and 10715) fast kill at 1.0% was observed. However, if a methylene group was placed between the nitrogen and unsaturation delayed action resulted. Compound10710 looks especially good!

If Rl Or R2 are monoalcohol groups (Table IV), delayed action results. Increasing the length of the N-alkyl group (29782, 29754, 29765) did not 30

appreciably change the delayed toxicity. However, 2 hydroxy1s on 1 group In (10732) or one each on R1 and R2 (10731) resulted in no activity. contrast the ester (29771) had delayed activity (Table IV). We also tested a number of polyethers, some ending in a hydrogen

(29753, 29773, 29772) and others capped with a methyl group (10749 and 29769). In both cases the compounds exhibited delayed toxicity (Table V). Addition of any other functionality, with the exception of 10733 and 2977S (Table VI), gave non-toxic compounds. For example the epoxide (10705), amine (29761), amide (10706) and phosphate (29752) were inactive.

To look at the other end of the molecule we kept -S02NH2 constant and varied the fluorocarbon radical. Table VII shows the results of decreasing the fluorocarbon chain length. Below Rf = C6F13there is no activity except for Rf = CF3' which may be due to fumigant action and not ingestion. Based on these results we feel that Rf = CSF17 or C6F13 provides the best activity. We have demonstrated the importance of both the fluorocarbon and sulfone part of the molecule by the inactivity of compounds 10721 and 10739 (Table VIII). All previous compounds were oil soluble, but the generality of this class of toxicants is due to the water solubility of certain members of the group. Table IX illustrates the excellent results obtained from several sulfonic acids and salts formulated in honey-water.

This general class of compounds have also been shown to be effective against cockroaches, flies and mosquitoes. Fluoronated su1fonamides and related compounds have a lot of potential and we look forward to following their progress in the pesticide field. 31

Table 1. Primary Screening Method for Evaluation of Chemicals as Bait Toxicants Against the Imported Fire Ant 1. Three reps of 20 worker ants placed in 30ml cups for 14 days.

2. Test chemicals dissolved in soybean oil or honey-water (1:1). 3. Toxic solution offered to ants on cotton swabs for 24 hrs. 4. Toxic solution removed from cups, ants remain w/o food for 24 hrs. 5. Cotton swabs saturated with soybean oil placed in cups for remainder

of test period. 6. Knockdown and mortality counts recorded at intervals of 1,2,3, 6, 8, 10, and 14 days. 7. Preliminary tests with all chemicals conducted at 1.0%.

8. Chemicals giving >89% kill are retested at 1.0, 0.1 and 0.01%

or until the minimum concentration giving >89% kill is determined.

9. Delayed kill is defined as <15%kill after day 1 and >85% kill after

day 14 at any dosage. Promising toxicants have delayed kill over a 10-fold or greater range of concentrations. Table II. Primary Screening Results for Alkyl Substituted Sulfonamides.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21 0.01 0 0 0 3 7 7 10 20 23 29759 C8F17S02NH2 0.1 0 0 0 2 33 77 92 95 98 H 1.0 43 85 98 100 I 29758 0.01 0 0 2 3 7 7 7 23 40 C8F17S02NCH3 0.1 0 0 7 88 97 98 100 1.0 17 93 100 H I 29757 0.01 0 0 0 2 2 10 22 50 C8F17S02NC2H5 a 0.1 0 0 2 80 97 97 98 98 100 H 1.0 25 100 I 10712 0.01 2 2 2 2 2 3 5 27 65 C8F17S02NCH(CH3)2 0.1 a 0 10 75 93 98 100 1.0 83 97 100 w H N I 10713 1.0 C8F17S02NC(CH3)3 a 0 0 0 0 a 5 0.01 0 0 0 2 5 10 20 50 60 10707 C8F17S02N(C2H5)2 0.1 0 7 13 78 92 98 100 H 1.0 30 100 I 29777 C8F 17S02NC12H25 0.01 0 0 0 5 5 5 7 13 17 0.1 0 0 0 0 2 2 20 50 80 1.0 0 2 3 78 97 100 Table III. Primary Screening Results for Unsaturated Su1fonamides.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

yH3 10717 0.01 0 0 0 8 8 13 25 37 57 CsF17S02NCH=CH2 0.1 0 7 33 77 90 92 100 1.0 100 ¥ 10710 0.01 2 2 2 2 2 2 12 37 75 -C8F17S02NCH2CH=CH2 0.1 3 3 3 48 60 78 93 98 100 1.0 13 53 80 100 T2H5 10709 0.01 5 5 5 5 5 5 15 15 30 C8F17S02NCH2C=CH 0.1 2 2 2 2 3 5 43 73 87 1.0 0 0 0 2 45 60 90 93 100

10715 w -C8F17S02NC6HS 0.01 2 2 2 2 2 2 3 12 27 w (recrysta1ized 0.1 0 0 0 18 63 87 90 95 98 linear isomer) 1.0 88 92 93 97 98 98 98 98 100

Y2HS 29767 0.01 0 0 0 0 0 3 3 3 3 , C8F17S02NCH2C6H5 0.1 0 0 0 2 2 3 8 18 42 1.0 0 0 0 2 42 83 100 Table IV. Primary Screening Results for Mono- and Oi Alcohol Substituted Sulfonamides.

CONC. PERCENT MORTALITY AT SPECIFIED OAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

r2H5 29782 0.01 0 0 2 2 2 2 2 3 C8F17S02NC2H40H 2 0.1 0 0 0 0 2 2 8 40 60 1.0 0 0 0 45 67 88 98 100 r4H9 29754 0.01 0 0 0 0 0 0 0 0 2 C8F 17S02NC2H 4 OH 0.1 0 2 2 3 3 3 25 48 78 1.0 0 0 0 0 0 40 92 98 100 h2H25 29765 0.01 0 0 0 0 0 0 0 0 0 C8F17S02NC2H40H 0.1 0 0 0 0 0 0 2 3 15 1.0 0 0 0 0 2 32 77 88 100 fH3 29756 0.01 0 2 5 8 8 8 C8F17S02NC4H80H 0 10 13 0.1 0 0 0 2 5 30 75 85 92 w 1.0 0 2 10 83 85 95 100 "'" 10731 C8F17S02N(C2H40H)2 1.0 0 0 0 2 5 10 35

yH3 10732 1.0 0 0 0 2 2 2 5 C8F17S02NCH2fH-rH2 OH OH

0 hH5 II 29771 0.01 0 0 0 0 0 8 10 13 18 C8F17S02NC2H40CC17H35 0.1 2 2 2 7 10 17 20 32 58 1.0 0 0 2 87 98 98 98 98 98 Table V. Primary Screening Results for Polyether Substituted Sulfonamides. CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

Y2H5 29753 0.01 0 0 0 0 3 7 7 13 20 C8F17S02N(C2H40)3H 0.1 0 0 0 0 0 0 3 7 27 1.0 0 0 2 52 87 98 99 100 Y2H5 29773 0.01 0 0 0 0 0 0 0 0 0 C8F17S02NC2H40(C3H60)8H 0.1 0 0 0 0 0 0 0 3 17 1.0 0 0 0 3 5 23 37 45 60 Y4H9 29772 0.01 0 0 2 2 2 2 2 2 2 C8F17S02NC2H40(C3H608H 0.1 0 0 0 0 0 3 3 15 45 1.0 0 2 2 2 2 40 87 97 100 T2H5 10749 0.01 2 3 3 3 3 5 8 C8F17S02N(C2H40)7CH3 0.1 5 5 8 15 20 23 40 w 1.0 2 5 5 38 57 80 88 U1 Y2H5 29769 0.01 0 0 0 3 7 7 10 13 17 C8F17S02N(C2H40)17CH3 0.1 0 0 0 2 3 5 10 25 48 1.0 0 0 32 100 Table VI. Bioassay Results for Oddball Nitrogen Substituents.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

Y2H5 10733 0.01 0 0 0 0 3 3 5 23 47 C8F17S02NC2H4 Cl 0.1 0 0 2 22 83 95 97 100 1.0 57 87 98 100

29778 0.01 2 3 3 8 8 S 13 15 lS C8F17S02N(N1- 0.1 0 2 3 10 10 52 67 SO S8 1.0 0 2 17 92 100 yH3/\ 10705 1.0 2 2 2 3 8 15 30 C8F17S02NCH2CH-CH2

w 29761 1.0 2 2 2 2 2 2 10 "" C8F17S02NC2H4NH2

0 yH3 II 10706 1.0 0 0 0 0 3 5 10 C8F17S02N-C2H4CNH2

Y2H5 Pi 29752 2 3 - 3 3 3 3 10 CSF17S02NC2H40P(OH)2 1.0 Table VII. Effects of Decreas i ng the F1uorocarbon Chai n Length.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

10703 0.01 0 0 0 3 3 5 7 8 17 CF3S02NH2 0.1 3 3 3 5 5 5 17 27 58 1.0 2 8 18 33 42 50 67 73 82

10744 CzF5S02NH2 1.0 3 17 22 35 40 45 50

10745 1.0 0 0 0 3 3 3 5 C4F9S02NH2 10702 0.01 0 0 0 2 2 2 2 5 12 C6F13S02NH2 0.1 3 7 7 7 7 17 63 77 92 1.0 0 3 30 67 75 87 95 98 100

29759 0.01 0 0 0 3 7 7 10 20 23 C8F17S02NH2 0.1 0 0 0 2 33 77 92 95 98 1.0 43 85 98 100 "w Table VIII. Illustration of Fluorocarbon and Sulfone Activity Requiements.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 8 10 14 17 21

29759 0.01 0 0 0 3 7 7 10 20 23 C8F17S02NH2 0.1 0 0 0 2 33 77 92 95 98 1.0 43 85 98 100

10721 1.0 5 7 8 12 12 12 12 C8H17S02NH2 2 3 5 13 110754 CFlH2S02NH2 1.0 0 0 0

R 2 2 2 2 10739 Cl15CNH2 1.0 0 0 0

(X;w Table IX. Primary Screening Results for Several Sulfonic Acids and Salts.

CONC. PERCENTMORTALITYATSPECIFIED DAYS NUMBER STRUCTURE % 1 2 3 6 S 10 14 17 21

50950 1.0 2 2 23 87 100 CSF17S0l H I 10700 1.0 2 37 73 98 100 CSF17S02NNa rH3 10701 1.0 62 97 100 C8F 17S02NNa + 10 47 95 100 10750 CgF17S0; N(C2H5)4 1.0

10727 1.0 0 37 62 95 100 . C8F17S03H w <.0 10728 1.0 15 77 95 100 C6F13S03H 1983 Field Trials ,,'1th Baj t Toxicants

For Presentation At:

1984 Imported Fire Ant Conference

March 27-28, 1984

Gainesville, Florida

By: HanerL. Collins

Imported Fire Ant Station

USDA,AHlIS, Pl'Q ,

Guli'p::Jrt, Mississippi

'Ihis report documents the results of research only. Mention of a pesticide or proprietary product does not constitute a recommendation for use or endorsement by the USDA, nor does it imply registration under FIFR!\ as amended.

40 41

ABSTRACT

1983 Field Trials w.l.thBait Toxicants

Haner L. Collins

Imported Fire Ant Station

USDA, APHIS, FPQ

Gulf'port, Mississippi

Large scale field trials with bait toxicants applied by aircraft were

conducted in 3 states during the 1983 season. Results obtained in the Spring series of trials indicated that the pregelled defatted corn formulation of

RO 13-5223 (Logic (R» provided F5r% colony mortality 12 weeks after applica-

tion. Amdro (R) provided 'Off/,colony mortality versus 75% with the .on% pre-

gelled defatted corn formulation of MK-936 (Affirm (R». Other formulations of both Logic (R) and Affirm (R) were somewhat less effective in producing

colony mortality; however, all treatments drastically reduced brood produc-

tion and pretre1j.tment popp.lation indices.

Preliminary results of a seriei:i of trials initiated in the Fall with

these same products indicated that applications made in October may not be as

effective as Spring applications.

Efficacy of two Prodrone treatments in Kendall and Kerr County, Texas

'is being moDitored. Two preliminary post treatment population surveys have

been completed to date. 10 weeks after the second application, a 62% reduc-

tion in the pretreatment population index was noted. Additional surveys will

be conducted througj'lout the 1984 season. 42

For th". ;':3.,i H'Vcra.] yearf" our primary re]€: ill IFA research hap been to conduct larg~ scale efficacy trials ~'ith bait toxicants ~'hich are in the Experi-! mental Use PErmit (EUP) stage of developnent. Since agency policy relative to fire ant cOTJtrol is directed toward area-wide population suppression, it is nece1 sary that WEhave efficacy data based on aerial application under progra'Jl condi-I tions. Two s~ts of trials were initiated in 1983. The Spring series has been canpleted, bu-- only preliminary data is available for the Fall series.

In addition to our ElJP trials, we are monitoring the effects of the

500,000 acrE Prodrone progra11 in Kendall and Kerr County, Texas. This project is incomplete at the present time and only prelimina.ry data is available.

In the Spring of ]933 (Slide) we evaluated two formulations of MK-936

(AffinnR) a'1Q two fo:rmulations of RO 13-5223 (WgicR). Amdro was used as a standard. P.ll formulations were applied by USDAaircraft at each test site.

Test sites were located on airport properties at lDngview, Texas; Shreveport, lDuisiana; and Albany, Georgia. Each test plot was approximately 150 acres in size. Evaluation procedures described elsewhere (Slide) were used to determine efficac~' of each product under evaluation. 43

S P R I N G 1 983 T EST S

A. J REA T MEN T S (MA Y 3 - 1L 1 9 8 3)

1. MK - 9 3 6 .0 11 % P GD 2. MK - 9 3 6 .0 0 5 5 % P GD 3. R 0 1 3 - 5 2 2 3 1 .0 % P GD 4. R0 1 3 - 5 2 2 3 1 .0 % E C G

5. A M D R 0

6. UN T R"E ATE D C HECK

B. T EST SIT E S. (A I R P 0 R T S)

1. LON GV I E W, T E X A S 2. S H REV E P 0 RT. LOU I S I A NA 3. ALBANY, GEORGIA 44

EVALUATION PROCEDURES

HARLAN ET AL. 1981. SOUTHWESTERN ENTOMOL. 6:150-157

BAN KSET A L. 1983. F LOR I DA E NT 0 MOL 0 GIS T. 66:172-181

POPULATION INDEX SCALE

LOFGREN & WILLIAMS. 1982. JOUR. ECON. ENT. 75:798-803 45

\'.'ne"EVET the &-~:ee}'. p:>::-t.treat.!OC'nt resu1ts frcc', all sjtc~, wer" ('cr..bined and averaged (Slide), we found that .Amdroprovided 93% colo!\y mortality with a 9&% reduction in the population index. Other fonnulations were less effective in providi.ni; colony mortality, but were higj11y effective in reducing the population index. Brood production was severely decreased by all treatments.

The second (Slide) and final evaluation was conducted 12 wecl"..safter treatment and indicated t.'1at RO 13-5223 applied as a 1%pregelled defatted corn fomulation was sligj1tly more effective than the other treatments in this series of trials. However, all treatment were considered hi@11y effective since a C)fffror more reduction in population index was obtained. Only a small percentage of surviving colonies were producing brood at this time. Absence of brood is a strong indication that a colony will eventually die because of its inabilitJ' to repopulate. S PR I NG 1 983

6 WEEKS POST TREATMENT

%COLONY % CHA NGEl N % C0 LON I E S FOR MUL A T ION MORTALITY POP UL A T ION I NDE X WITHBROOD

AMDRO 9 3 - 9 8 1 9

MK-936 .011% PGD 5 7 - 9 4 0.6 oj::> 0")

MK-936 .OQ55% PGD 4 4 -91 0 . 3

RO 13-5223 1 % P GD 4 1 - 92 0

RO 13.-5223 1% ECG 3 3 - 88 0

UNTREATED CHECK 2 8 -27 9 3 S P R I N G 1 9 8 3

12 WEEKS POST TREATMENT

% COLONY % C H A N GEl N % CO LON I E S FOR M U L A T ION MORTALITY POP U L A T ION I.ND E X -WITH BROOD

RO 13-5223 l%PGD 8 7 - 9 9 0

Af1DRO 8 6 - 97 1 5 ~J

MK-936 .011% PGD 7 5 - 96 1 . 2

R013-5223 l%ECG 7 4 - 9 6 4 . 0

MK-936 .0055% PGD 6 1 - 9 6 0 . 6

UNTREATED CHECK 2 8 - 3 3 9 4 48

I Our Fall series of trials (Slide) was essentially identical to t.~e Spring

I series. Two formulations of both MK-936 and RO 13-5223 were applied to three test sites in Texas, Alabama. and Georgia. Treatments were applied by USDA I aircraft between October 18 and October Z7.

A preliminary evaluation conducted 6 weeks af'ter treatment (Slide) indi- I cated that very little colony mortality had occUITed at that time. However,

I brood production as well as the population index was reduced by most treatments. A final evaluation of this series of tests is planned for late May. As previously mentioned, we are m:mitoring the effects

of the Kendall/Kerr I County, Texas Prodrone treatment. A pretreatment survey of 65 efficacy sites

(plots) v:itl'.i.T1the treated area (Slide) and 25 sites in an adjacent untreated

I county was completed in June, 1983. We have canpleted two preliminary post-

treatment evaluations to date (Slide). The first evaluation was made 12 weeks I after the first application (September) mereas the second evaluation was canpleted 10 weeks after the second application (Novenber). These should be con-

sidered very preliminary data since the slow and subtle effects of Prodrone are well docunented. Althoug11 rniniJnal colony mortality was evident during the second evaluation (Novanber), a 62% reduction in the population index was recorded versus a 19}breduction in the untreated checks.

I Our third evaluation is scheduled for the week of April 23. By that time,

we should have a much better indication of the final results of this treatment. I 49

FALL 1983 TESTS

A.- TREATMENTS (OCT 18-27,1983)

1. MK - 9 3 6 . 011 % P G D (1 A )

2. MK - 9 3 6 .011% P G D (3 C ) 3. R 0 1 3 - 5 2 2 3 1% PGD 4. R 0 1 3 - 5 2 2 3 1% ECG 5. A M D R 0 6. U N T REA TED CHECK

B. T EST SIT E S (A I R P 0 R T S )

1. T Y l E R, T E XA S 2. M0 N T G0 MER Y, ALAB AMA 3. VA l DOS T A, GE 0 R G I A FALL 1983 PRE L I MI.oNARY RES UL T S - 6 WEEKS P0 ST T REA T MENT AVERAGE OF 3 SITES EXCEPT AS NOTED

FORMULATION % COLONY % CHANGE IN % COLONIES MORTALITY POPULATION INDEX WITH BROOD MK - 936 (1 A ) 2 9 - 8 5 0 . 7

MK- 936 (C 3 ) * 1 4 - 8 0 0

tT1 AMDRO 3 5 - 5 7 5 5 a

R013-5223 ECG 6 -57 2 0

R013-5223 PGD 3 - 5 5 27

UNTREATED CHECK 3 + 1 7 9 8

it NOD AT A FOR T YL E R, T E XAS. TABLE 1. PreliminaryResults of Two ProdroneApplicationsin Kendall and Kerr County, Texas

Pretreatment IFA Population Status of IFA Population at the Indicated Post-Treat Interval (June 1983) Mean (+SD)% Reduction in No. Mean (1SD)% Change in Pretreat8ent of Active Nests Population Index Mean No. Nests 12 Wks. After 10 Wks. After 12 Wks. After 10 Wk.. After Per Efficacy Mean Population 1st Appl. 2nd Appl. 1st Appl. 2nd Appl. Location Site Index/Site (Sep 83) (Nov 83) (Sep 83) (Nov 83)

Ker,dall &. Kerr Co. (..ithin treated iU'ea) 8.8 . 165.0 58(129) 37(135) -72 (125) -62(+39)

Bcxa.r Co. 16.3 . 321. 8 . (25) (Untreated) 33 24(121) -41 (125) -19(+30)- CHRONIC TOXICITY OF A FIRE ANT INSECTICIDE* IN DOGS.

DB Coulter and WACrowell Dept of Physiol & Pharm (DBC) and Dept of Path (WAC), Col Vet Med, The Univ of Georgia, Athens, GA 30602.

Fifteen dogs (mean body wt 21kg, mean age 2.5 yr) were assigned to five groups. Two in each group were fed the insecticide each week. One dog in each group served as a control.

Assigned dosages were:

Gp I 0.6 gmjkgjwk bait ( 5 mgjkgjwk) 1 male 1.1 gajkgjwk bait (10 mgjkgjwk) 1 male

Gp II & III 2.3 gmjkgjwk bait (20 mgjkgjwk) 1 male, 1 female 4.5 gmjkgjwk bait (40 mgjkgjwk) 1 male, 1 female

GP IV & V 5.7 gmjkgjwk bait (50 mgjkgjwk) 2 males, 2 females Animals were checked for clinical abnormalities of the integument, neuralmuscular, cardiovascular, hemolymph, respiratory, alimentary, urinary, and reproductive systems. One male dog fed 40 mgjkgjwk died after 29 wks and one male fed 50 mgjkgjwk died after 4 wks. The dogs were ataxic and had opisthotonous prior to death. Lesions at necropsy did not implicate any particular body system. Blood chemistries, and electrodiagnostic procedures have not revealed prodromal signs of toxicity. Two treated dogs have had transient ataxia of the hind limbs and another two have highly abnormal sperm. Two control dogs have had some abnormal sperm. Three of the dogs on the higher dose have remained apparently healthy after 30 wks of ingesting the insecticide. No beneficial aspects, eg, less parasites, of feeding the insecticide have been ob~erved. The following variables in the indicated systems were monitored: Urinary System Respiratory System Polyuria Nasal Discharge Polydipsia Coughing Blood Chemistries Dyspnea Dysuria Radiographs Lung Compliance Reproductive System Airw~y Resistance Estrus Cycle Mating Pregnancy Hemolym~h.SystemBloodC emistries Evaluate Sperm Cell Counts Testicle Size LymphNode Size

52 53

Alimentary System Cardiovascular System Appetite Electrocardiograph Body Weight Blood Pressures Diarrhea Rectal Temperature Neuralmuscular System Internal Parasites Reflexes Weakness Integumentary System Nerve Conduction Times Paras ites Electromyographs Inflammation Brainstem Auditory Evoked Responses Photosensitivity Impedance Audiometry Effluvium Behavior

*AMDRO AC 217,300 Trademark of American Cyanamid Co. TI1 Affirm (KK-936) Fire Ant Insecticide: A Novel Natural Product Insecticide for Control of the Red Imported Fire Ant (Solenopsis invicta)

Richard A. Dybas Merck Sharp & Dohme Research Laboratories Three Bridges, N. J. 08887

AffirmTI1 (MK-936) fire ant insecticide is a novel insecticide bait under development for control of the red imported fire ant, Solenopsis invicta Buren. Affirm which contains 0.011% active ingredient avermectin BI' a macrocyclic lactone natural product isolated from the soil microorganis~ Streptomyces avermitilis, is formulated on pregel defatted corn grits with soybean oil as a fire ant attractant. This product under the code number MK-936 bas been extensively tested since 1981 by USDA and university researchers in all nine states with known RIFA infestation and found to be highly effective when applied at a rate of 1.0 pound of bait per acre. One pound of bait contains 50 mg active ingredient.

O~_H r""~ ~!~~I H <;:H3 H~ -H o..~~ ~ k

a -Component R . Ct HI I 80% b-Component R. CH, '20%

AVERMECTIN St MK-936

Affirm is a slow-acting stomach insecticide which is extremely active against the red imported fire ant when fed in a bait at the recommended rate. Affirm is toxic to fire ant workers. However, laboratory and field trials have indicated that Affirm is most effective against the RIFA queen and either kills the queen or prevents egg production. As a consequence of Affirm treatment, further development of colony brood population ceases. Death of the treated colony occurs gradually since the primary effect of Affirm is on the viability of the fire ant queen rather than high acute toxicity for workers. In large acreage field trials, Affirm 0.011% fire ant insecticide applied aerially or with ground driven equipment has provided in excess of 75% colony mortality, greater than 90% reduction in population indices, and essentially complete elimination of worker brood in treated colonies.

54 19R4 Fire Ant Conference Gainesvill~, FIorina

AN ENVIRONMENTAL RISK ASSESSMENT OF PRO-DRONETM INSECT GROWTH REGULATOR FOR CONTROLLING THE RED IMPORTED FIRE ANT (SOLENOPSIS INVICTA) Dennis Paustenbach, Ph.D., Environmental Toxicologist, Environmental Services Department, Stauffer Chemical Company, Westport CT

There has been a great neal of research aimed at developing pesticides which pose minimal hazard to non-target organisms and the environment. Ideally, such compounds should have a unique mode of action, low application rate, and be similar to naturally occurring substances. Pro-Drone is an insect growth regulator used to control fire ants (Solenopsis invicta) which meets these criteria. This paper describes the results of toxicity tests, environmental fate studies and the risk assessment which have been conducted prior to marketing this insect growth regulator.

MV-678 is the active ingredient in Pro-Drone and is an analog of the natural insect juvenile hormones. It does not exhibit direct toxic effects in insects or in animals because it's mode of action involves alteration of the insect endocrine system which has no similarity to that of mammals. MV-678 mimics the action of the natural insect juvenile hormones by altering fire ant caste differentiation by producing less workers and more nonviable sexuals. The disappearance of workers leads to a decline in food gathering, brood tending, colony maintenance, and eventual death of colonies.

MV-678 exhibits very low acute toxicity. The acute oral LD50 to rats is greater than 5,000 mg/kg and the acute dermal LD50 to rabbits is greater than 2,000 mg/kg. MV-67R is a mild skin irritant and is a non-irritant to the eyes. Mutagenicity tests have shown that MV-678 is negative in the Ames assay and in the forward mutation transformation carcinogenicity assay. MV-678 did not demonstrate teratogenetic potential in tests conducted in rats and rabbits.

Environmental toxicity tests have demonstrated that MV-678 has no adverse effects on nontarget organisms that have been tested. No deleterious effects were observed on approximately 30 species of freshwater aquatic invertebrates in ponds treated with MV-678 at 10 times the normal application rate. The acute 96 hr. LC')O for stickleback fish and bluegill sunfish was greater than 100 ppm. The dietary LD50 on bobwhite qllail was greater than 2,000 ppm. A feed-through sturly on chickens has indicAted that MV-678 had no deleterious effects on the foorl intake and body weigllt of the animals. Environmental fate studies indicate that complete degradation of MV-678 to carbon dioxide and water occllrs rapirlly as a result of physical and chemical processes (photolysis and hydrolysis) as well as by microbial degradation. Field tests have shown thAt due to the manner in which MV-678 is formulated, no significant quantities of Pro-Drone are taken up by any ant species except the red imported fire ant. Consequently, tests and observations indicate that due to the formulation, Pro- Drone does not present a potential to adversely affect other ants or insects.

The results of a risk analysis show that the unlikely uptake of the Pro-Drone by non-target organisms, as well as the very low application rate of 4.R grams of MV-67H per acre of land, combined with its apparent lack of toxicity, makes the risk to mammals, avian, and aquatic species very low. Based on the available data, the widespread Ilse of Pro-Drone for the control of fire ants should present no unreasonable risk to man or the environment.

55 56

PRO-DRONET" PRO-DRONET". Insect Growth Regulator Active Ingredient . CH30

. 1-(8-methoxy-4.8-dimethynonyl) -4-(1-methylethyl) benzene Bait Formulation

For Control of the Red Imported Fire Ant . 1.2%active ingredient . 98.8%corn grits and soybean oil III . Light yellow granules

1 2

ADVANTAGESOF INSECT GROWTH REGULATORS . Unique mode of action (not a toxicant) . Interferes with natural biological processes in a non-toxic manner

How Pro-Drone Works . Insect does not develop "resistance" . Unique mode of action . Not a poison but an insect growth regulator . Species specific . Shifts population from workers to non workers . 12-16weeks for maximum effects . Environmentallysafe . Fire ant colony dies of starvation . Low application rate 3 4

INSECT JUVENILE HORMONES (JH) AND PRO-DRONE . .' .. 0 ~OCMJ JH I ~ ~o/ Naturally occunlng Insect Estradiol ) I I!! Juvenile~ (Mammalian) ~.( JH II

I I I:: JH III ~o/ CHO~ J 110/~ PRO-DRONE MV~7B (AI-3-36206) Proetaglancln (Insect JH Analog) (Mammalian) CH~~

5 6 57

FIELD EXPERIENCEWITH PRO-DRONE LARGE AREA FIELD TRIALS WITH PRO-DRONETM . Pro-Dronewasdeveloped in the early seventies . 400,000 acre trial conducted in Kerr and . Field tests by USDA began in 1976 Kendall Counties, Texas in 1983 (Spring and Fall) . Field trials by USDA, TDA and Stauffer on six, 1000acre plots conducted in 1981 . 140,000 acre trial in Chambers and and 1982 Jefferson Counties, Texas in Fall 1983 and . Two applications gives 83-93%control Spring 1984 . No adverseenvironmental effects have been noted . 40,000 acre trial in Jefferson County, Texas in Spring 1984

1 8

PRO-DRONET" HOW MUCH PRO-DRONE IS APPLIED?

Application . 1/5 of an ounce (a.i.) spread over 43,560 ft2 . 0.88 pounds per acre

. 1/5 ounce of active ingredient . 4.8 grams in 7.72 million grams of pasture grass (4" deep) . By air or ground equipment . 1070granules on a 6000 ft2 lawn (120' x SO')

9 10

PRO-DRONET" TOXICITY STUDIES IN NUMEROUS SPECIES Toxicology . LDSO . Acute oral greater than 5000 mg/kg - Cows - Rabbits -Mice . Acute dermal greater than 2000 mg/kg - Chickens -Pigs -Quail . Non irritating to eyes - Ducks - Horses - Rats . Not a mutagen - Dogs . Lackscarcinogenic potential . Not a teratogen

11 12 58

ACUTE STUDIES ACUTE STUDIES (Technical MV-678) (Formulation 1o2G) . OralLDso(Rat):> 5000 mg/kg (Class IV) . OralLDso (Rat): > 5000 mg/kg (IV) . Dermal LDso(Rabbit):> 2000 mg/kg (Class IV) . Dermal LDso(Rabbit):> 2000 mg/kg . Irritation (Rabbit) (IV) - Skin: Moderate (Class III) . Irritation (Rabbit) - Eye: Non-irritating (Class IV) - Skin: Mild (III) . Inhalation LCso(Rat): > 4.5 mg/L (Class IV) - Eye: Non-irritating (IV) Summary: Non-toxic acutely Summary: Non-toxic acutely

13 14

RELATIVE ACUTE TOXICITY GENETIC TOXICOLOGY TESTS OF PRO-DRONE Ames Test Substance LDso(mglkg) Category . Detectsgene mutations at Histidine Locus Potassium Mouse Lymphoma Assay (MLA) 10 . Detects gene mutations at Thymidine Kinase Locus Cyanide Extremely toxic . Detects chromosome aberrations and sister Lead 100 Very toxic chromatid changes (SCE) Chinese Hamster Ovary (CHO) Assay 1500 Aspirin Moderately toxic . Detects gene mutation at Hypoxanthine- Ethanol 2000 Moderately toxic phospho ribosyl-transferase (HGPRT) locus . Detects chromosome aberrations and SCE Table salt 3000 Slightly toxic Cell 'n'ansformatlon (BALB/3T3) Pro-Drone 5000 Practically non-toxic . Predicts carcinogenic potential by measuring loss of I contact inhibition 15 16

PRO- DRONE LACKS RESULTS OF IN VITRO TESTS CARCINOGENICPOTENTIAL (MV-678) MLA MLA CHO CHO . Not mutagenic in standard tests Ames (Gene) (Chromosomal) (Gene) (SCE) MTA . No evidence for carcinogenic hazard Non-Act. Neg. Neg. Neg. NT NT Neg. - Negative in BALB/3T3test Act. Neg. Neg. Neg. Neg. Pos: - - No similarityto knowncarcinogens

NT ; Not Tested - Insecthormonesandanalogsare 'Very Weak not carcinogenic - Negligibleexposure potential

17 18 59

SUBCHRONIC STUDIES SUMMARY OF 1ST SUBCHRONIC OF MV-678 RAT STUDY (MV-678)

. Rat (2 studies; 90 days) . Biological adaptive response noted at 50, 200 and 800 mg/kg . Dog (1 study; 90 days) . No degenerative target organ toxicity at levels up to and including 800 mg/kg

. Minimal clinical chemistry effects suggesting hepatic dysfunction at high doses

19 20

SUMMARY OF 2ND SUBCHRONIC DESCRIPTIONOF SUBCHRONIC RAT STUDY (MV-678) DOG STUDY (MV-678) . No significant effects on clinical chemistry, body weights or food consumption at any METHODS:3-month duration dose Oral Gavage BeagleDog . No treatment-related effects noted at 2 and 10 mg/kg/day Doselevels Numberof (mg/kg/day) Dogs/Sex . Changes in liver weight and cell size 0 5 observed at 30 and 200 mg/kg/day 10 5 . No degenerative changes in any treatment 100 5 group 1000 5

21 22

SUBCHRONIC TOXICOLOGY DESCRIPTIONOF ENZYME INDUCTION STUDYIN RATS No Effect Level (Rat) = 30 mg/kg/day @ 30 day (MV-678) 3-day Screen: (Liver Wt) . Animals were treated for 3 days and sacrificed 3D-day Screen: . Animals were treated for 30 days No Effect Level (Rat) = 10 mg/kg/day @ 90 day . Sacrificesoccurredat 30days.45 days. and 60 days (Liver Wt) allowing for a 15and a 30 day recovery period Data Collected: . Liver weights and histopathology No Effect Level (Dog) = 40 mg/kg/day @ 30 day . Hepatic microsomal protein content and enzyme assays: - Cytochrome P-450 contents (Liver Wt) - NADPH-cytochrome P-450 reductase - Ethylmorphine N-demethylase - Aniline hydroxylase

23 24 60

RESULTSOF ENZYME INDUCTION INTERPRETING THE ENZYME STUDYIN DOGS (MV-678) INDUCTION STUDIES (MV-678) Significant effects of dose level 10 mg/kg/day . Increase in 2 of 4 microsomal enzyme parameters . Reversible stimulation of microsomal liver Significant effects of dose level 200 mg/kg/day enzymes . Increase in 3 of 4 microsomal enzyme parameters . An increase in smooth endoplasmic . Increased incidence of very mild hepatocellular reticulum and liver cell size hypertrophy Significant effects of dose level 800 mg/kg/day . A fully reversible process . Increased liver weight . Induction is an adaptive response . Increased microsomal protein content . Enzyme inducers include phenobarbital, . Increase in all 4 microsomal enzyme parameters tolbutamide, codeine, estradiol, . Increased incidence of mild hepatocellular hypertrophy testosterone, phenylbutazone. phenacetin

25 26

EFFECTS ON OFFSPRING (TERATOLOGY) CONCLUSIONS FROM TETRATOGENICllYSTUDIES Species Doses(mg/kg/day) Results (MV-678)

Rat (rodent) 0,40,1000 No . Maternal no effect level (rat) - 40 mg/kg effects . Maternal no effect level(rabbit) - 50 mg/kg Rabbit (non-rodent) 0,10,50,200 No . Not teratogenic or embryotoxic at anydose Effects in either species

27 28

DESCRIPTIONOFA METABOLISM SUMMARYOF MV-678TESTING STUDY WITH MV-678IN RATS. Methods: Acute Toxicity: Essentially non-toxic TestSubstance : Labeled MV-678 Subchronic Toxicity: No degenerative target Species (Strain) : Rat (Sprague-Dawley) organ effects Route of Administration: Oral Gavage and i.v.(Low dose) Teratogenicity: Negative in 2 species Dose Levels : 20, 200 mg/kg Treatment Duration: Single Dose Mutagenicity: Negative in 5 in vitro tests Data Collected : Radioactive content of plasma. urine, feces, tissues, expired air Carcinogenicity: No indication of gena- : Metabolite identification toxicity basedon structure .To be started 7/1/84 or in vitro test results

29 30 61

AQUATIC TOXICOLOGY AQUATIC TOXICOLOGY (VERTEBRATES) (INVERTEBRATES)

Species Exposure (96 hr) Results Species Resulla Meaning Daphnia LCso =1 ppm Insignificant Rainbow Trout 100 ppm (nominal) No hazard (2.4 mg/l actual) effects Mysid Shrimp LCso =3.0 ppm Insignificant Bluegill Sunfish 500 ppm (nominal) No hazard effects (6.2 mg/l actual) Planktonic invert. No adverse Stickleback Fish 100 ppm (nominal) No Nektonic invert. effect at Insignificant (2.4 mg/l actual) effects Benthic invert.} 10x application hazard

31 32

NEGLIGIBLE RISK TO PONDS RISK TO AQUATICSPECIES AND STREAMS "WORSTCASE SCENARIO" Assume: 1 acre lake (6" deep) . Doesn't leach from bait 1 acre drainage basin 100% Runoff . Essentially non-toxic to fish 4.8 g/acre application Estimated Env. Cone. (EEC) . Quickly degrades in sunlight EEC = (Lb AI/Acre) (Size of Basin) (% Runoff) . Poor water solubility (Water Area) (Depth) (62.4 Lb/fP)

EEC = 0.0074 ppm = 7.4 ppb

33 34

AQUATIC RISK POSSIBLE AVIAN TOXICOLOGY CRITERIA ACTION Species Exposure Results EEC> 1/10 Acute LCso Candidate for Bobwhite lDso-single dose No effects at restricted use (any fish) Quail 2150 mg/kgkJay EEC> 1/10 Acute LC10 Labeling for that species Bobwhite LDso-fivedaysin feed No effects at (most sensitive species) may be required Quail 5000 ppm Mallard No effects at Less Risk No restrictions LDso-five days in feed Duck 5000 ppm

Chickens 90 days in feed No effects at 2500 mg/kgkJay

35 36 62

NEGLIGIBLE RISK NEGLIGIBLE RISK TO DOMESTIC TO BIRDS BIRDS (CHICKENS)

. Random foragers . No adverse effects in 2 year feeding study . Birds prefer particles larger than 10 mg (Pro-Drone weighs 1.6 mg) . No effect on the eggs . Bait rapidly gathered by fire ants . No bioaccumulation . Essentially non-toxic to birds

37 38

NEGLIGIBLE RISK TO LARGE PRO-DRONET. ANIMALS (COWS) No adverse effects on wildlife . No toxic effects were seen in cows fed Fish large amounts of Pro-Drone for 2 years . Stickle back Aquatic organisms . Blue gillsunfish . Dragon flies . Rainbow trout . Cows showed no progressive increase in . Mayflies tissue levels (doesn't bioaccumulate) Birds . Divingbeetles . Bobwhitequail . Plankton . Mallardduck . Cows delivered healthy offspring . Chickens

39 40

PRO-DRONETMRESIDUES \ PRO-DRONET" -... 1/ Maximumtheoretical residues: DegradedbySUnllg:,. ..,...... (Assumes no pickup by fire ants and no degradation) /1 ,\ Water 0.004 parts per million Soil 0.008 parts per million Forage 0.620 parts per million

. Actual residues are significantlyless . No residues detected at 0.05 parts per million Undetectable In water in forage or soil

41 42 63

DEGRADATION OF PRO-DRONE BY SUNLIGHT SOIL CH30 MOBILITY /1\ Formulated material is not mobile in soil Soil (900F) Soil (700 F) Water (650 F) / ~ \ t1l2 =15 hrs. t'/2=30 hrs. t1l2=6 days

43 44

LEACHABILITY IN WATER ENVIRONMENTAL DEGRADATION OF PRO-DRONE

CH30~ Lessthan3% of bait formulation ~ ~ OH will leach into water emethYlatlon YdrOIYI" OH-- over 48 hours. HO ,. CH30 ,. ~ .1 ~ .1 OH .. .. OH FormIc Acid 0101 Acetic AcId ~ Epoxldes ~ CO2 + H2O_Acetic Acid

45 46

DEGRADATIONOF MV-678 PROPOSEDBIOTRANSFORMATION DUE TO SOIL METABOLISM PATHWAYS FOR PRO-DRONE

' "" c ~~. ~~ f : \ ¥ \ ..MV,," i" \ . ..eo> '" - \ ~~ ,-po...d ~~-,. -r~~ I \ . 0".....,.«..- .-\ --~~~ ~~ ~ - \ ~ .. \ / " ~ .. ?;P'..:~:---'-' ~~~ L~~ "../.. {-.{.:"''''''-- /" +- -'-'-'-. .~/ - .:-=-==-==~ :J .ON ,, w_. An"T".,~, ~~. ~~ 47 48 64

LACKS ENVIRONMENTAL ENVIRONMENTAL FATE PERSISTENCE UNDER FIELD CONDITIONS . Low to moderate solubility In fat A.Over500samplesof soli, water, silt and vegetation, as well as representativeinsects . Low to moderate bioconcentration factor and wildlife were collected by USDA In Kerr and Kendall Counties, Texas . Very low water solubility . MV-678was not detected in any of these samplesat the levelof 0.01ppm . Rapidly degraded by light B. Residueprogram has been conducted by . Rapidly converted by soil and tissue Stauffer In 11crops and grasses enzymes to water soluble compounds . MV-678was not detected even at 2x application rate at the level of 0.05ppm 49 50

NO ANTICIPATED RISK TO HUMANS

PHYTOTOXICITY . Noresiduesin foods

None observed at specified application rate . Essentially non-toxic to mammals, birds and fish 1 j . Lacks carcinogenic potential

. No health problems or irritation in manufacturers, researchers or applicators i

51 521 i PRO-DRONET. NATIVE ANTS APPLICATOR EXPOSURE DATA

Average Potenllal Exposure to MV-678, mg/day Oil loving, seed gathering and sugar loving types j Dermal' Inhalation' Total Dose . Loader 0.697 0.058 0.755 . Fire ants usually are predominant in infested areas Pilot 0.249 0.023 0.272 . Field observations do not suggest lessening of native ant population Pilot 0.0193 0.010 0.0293 Field Observer 0.00153 0.009 0.Q105 J 'Assuming 1% dermal absorption 'Assuming 100%pulmonary absorption I 53 54 J 65

IMPACT ON NON-TARGETANTS IMPACTON TEXAS A&M AND TEXAS TECH OTHER NON-TARGETINSECTS UNIVERSITIES A. Kendall, Kerr and Bandera Counties, Texas A. Kendall, Kerr and Bandera Counties, Tex.. . 200 pitfall traps monitored each month for 12 consecutive months . Over 500 environmental, wildlifeand insect . 10 different geographical/acations samples werecollectedand assayed by USDA . 30 different ant species (5 families) have been identified B. Jefferson and Chambers Counties, Tex.. . Study by Texas Tech will identify and qualitatively . Scavenging rate studies evaluate the presence of many different types of . Qualitative assessment of impact on native ants arthropods before and after treatment C. College Slatlon, Tex.. . Study objective is to evaluate the effect on subterranean ants B. Jefferson and Chambers Counties, Texas . 180 bait stations in 20 different areas were used . Qualitative evaluation of uptake by other insects . 6 months after treatment, no significant effects noted in any species (10 identified) and birds following treatment (Texas Tech)

55 56

NEGLIGIBLE RISK TO BEES STUDIES TO IDENTIFY POTENTIALHAZARD TO BEES. . Bees don't forage from ground - 99% lands on soil . Evaluate the uptake of Pro-DroneT. by bees . Bees are attracted to suger under field conditions using pollen traps - Pro-Drone is soaked in soybean oil . Evaluate the toxicity of Pro-DroneT. if it . Bees carry particles like pollen (40pm) reaches the hive using colony traps - Pro-Drone is much larger (1000pm) . Evaluate the toxic and IGR effects of . limited availabilityof bait MV-678 in bees under lab conditions - Bait rapidly gathered by fire ants 'Grant awarded to Texas Tech University

57 58

PRO-DRONET" MECHANISM OF ACTION OF MV-678IN FIRE ANTS Pro-Drone controls the red imported . Small decrease in egg production probably due to hormonal effects fire ant with minimalrisk to fish, . long term impairment of reproductive potential is wildlife, and the environment. apparently due to atrophy of the ovaries. reduction in the number of epithelial cells lining the ovarioles. and reduction in the number of oocytes

. Only females rather than male workers are produced in colonies treated with MV-678 (validated. in part, in field observations)

. The queen usually dies within 6 months of treatment (single or multiple queen colonies)

59 60 Current Research with Toxic Baits

David F. Williams~

Insects Affecting Man and Animals Research Laboratory USDA-ARS Gainesville, Florida 32604

The number of chemicals evaluated by the USDAas bait toxicants against the red imported fire ant (Solenopsis invid:a Buren) from 1958 through June, 1983 was 7,435. Eighty-six percent of these were not effective. Eight percent gave rapid kill of workers and were therefore not usable as bait toxicants. The remaining 6% of the total number exhibited delayed toxicity and were therefore further tested as bait toxicants. Of these compounds, 5% showed delayed action over 1 to

9-fold dosage range while less than 1% showed delayed activity over 10 to 99-fold dosage range. Only two compounds mirex and avermectin Bl (effects on queens only) showed delayed action over a lOa-fold dosage range. Of the total 7,435 chemicals screened, approximately 60 have actually reached the field testing stage. In this group, only three chemicals have been commercially developed for public use, mirex, Amdro@ (American Cyanamid Company)and Pro-Drone@ (Stauffer Chemical Company). Mirex, as most everyone knows, is no longer available for fire ant control, having had its registrations cancelled by the Environmental

Protection Agency in 1978. Twoother chemicals that are very promising

~ This paper presents the results of preliminary research only and will be published later through established channels with appropriate editing. Therefore, the paper and data should not be referred to in literature citations until it appears in publication. Mention of a proprietary product does not constitute a recommendation by the USDA.

66 67

as bait toxicants and ones which the companies have applied for conditional

registrations are Affirm@ (alias avermectin Bl)' (MK-936) (M-895) by Merck and Company, and Logic@ (alias R013-5223) by Maag Agrochemica1s Company. For the present, we have decreased laboratory and field screening tests of chemicals. Large numbers of chemicals are no longer being screened as bait toxicants because we have now made available several chemical tools to the public for use for control of fire ants. We are still testing several insect growth regulators and two of these look very promising in laboratory tests and will probably be tested in small field tests soon.

The results of field tests with the f1uoronated su1fonamides from

3M Company are shown in Tables 1 and 2 and although some of the results are erratic, a few of these compounds showed promise. The first field test was conducted at Lake City, Florida in 1982 (Table 1). The Chemical

AI3-29759 gave excellent control of fire ants at 6 and 12 weeks post- treatment while AI3-29758 gave 99%control at 6 weeks, but decreased to 73%after 12 weeks. In the second field test (Quitman, Georgia, 1982),

AI3-29757 looked very good giving 90 and 96% control after 15 and 24 weeks respectively. The third field test (Table 3) was conducted at Homervil1e, Georgia in 1983. In addition to the original three f1uoro- nated su1fonamides (AI3-29757, 29758, and 29759), several other promising ones were evaluated. Because of dry conditions during the evaluation periods the results overall did not look very good; however,

AI3-10702 and 29759 did perform the best giving 90 and 80% control respectively, after 18 weeks posttreatment. Additional field tests are planned with the most promising of the f1uoronated su1fonamides in 1984 and hopefully the best chemical can be selected for large scale field tests in late 1984 and early 1985. 68

Research on the use of house fly and/or eye gnat pupae as bait-

carriers of chemicals for controlling the fire ant is continuing. A major problem of all current toxic baits used for fire ant control is

that they rely on the chemical being dissolved in soybean oil, which is

the food attractant. Unfortunately, soybean oil is also highly attractive to other nontarget ant species, many of which are very beneficial. We presented a new approach to this dilemma in our report at the IFA

Conference in Starkville, Mississippi in 1983. By using a new carrier,

fly pupae, which is attractive to fire ants but not to many other ants, we hoped to protect nontarget ants, which will not feed on the pupae. After several promising tests with Amdroand fenoxycarb (Maag R013-5223) in the laboratory and in the field, we decided to continue with this research. An update of recent field results is shown in Table 3. The results clearly indicate that house fly pupae and eye gnat pupae treated with fenoxycarb (Maag R013-5223) offer promise as attractant-carriers containing toxicants for controlling the red imported fire ant. Al- though the results after 8 and 16 weeks posttreatment with all the various combinations did not give as good control as the standard (fenoxycarb bait using soybean oil on pregel defatted corn grits), lower application rates were used and therefore the grams of active ingredients per acre were much smaller. Additignal field tests of this research are underway. Also, publication of this information will be

forthcoming soon. Table 1. Effects of granular soybean oil baits containing t,uoronated sulfonamides on populations of the red imported fire ant in Florida and Georgia.-

Toxicant Application Mean % reduction in population Chemical concn (%) rate index after wks indicated AI3-No. In SBO gAllA 6 12 15 24

Lake City, FL 1982

29757 1.0 1. 56 83 73 29758 1.0 1.42 99 84 29759 1.0 1.36 81 75 29759 2.5 3.40 95 91 CHECK 0 0

m Quitman, GA1982 I.D 29757 1.0 1. 61 90 96 29758 1.0 1. 53 71 62 29759 1.0 1. 61 85 82 10702 1.0 1. 61 81 73 CHECK 13 9

~ Avg. of three l-acre plots. Baits consisted of 70%pregel defatted corn grits impregnated with 30% of the SBO - toxicant solution. ~ Evaluations in this test were not conducted at 6 and 12 wks. Also, the results of the first 3 chemicals are only for one plot while the results of 10702 were an average of 2 plots. The reason for this is that the other plots were plowed under by the landowner. Table 2. Continued effects of granular soybean oil baits containing fluoronateg/sulfonamides on populations of the red imported fire ant in Homervi11e, Georgia 1983.-

Toxicant Application Mean% reduction in population Chemical concn (%) rate index after wks indicated AI3-No. in SBO g Al/A 6 12 18 10702 1.0 1. 28 75 67 90 10707 1.0 1.43 30 37 45 10710 1.0 1.36 30 51 42 10712 1.0 1.29 49 52 42 10733 1.0 1.47 60 75 59 29757 1.0 1.50 81 52 74 29758 1.0 1.44 40 46 58 29759 1.0 1.46 80 64 80 -...J CHECK 16 46 30 0

~ Avg. of three l-acre plots. Baits consisted of 70%pregel defatted corn grits impregnated with 30%of the SBO- toxicant solution. 71

Table 3. Effects of house fly pupae and eye gnat pupae baits containing fenoxycarb (Maag RO 13-5223) on populations of RIFA. Alachua County, FL, June, 1983. (Avg. of three l-acre plots.)

Bait Application rate Mean % reduction in population treatment 1bs/ A index after indicated weeks 8 16

HFP (6%) 0.125 84 68 0.25 85 75 0.5 84 69

HFP (1 2%) 0.125 81 73 0.25 81 85 0.5 91 81

HFP (18%) 0.125 92 76 0.25 83 87 0.5 88 79

EGP (6%) 0.125 87 58 0.5 82 76

Logic (Std.) 1.0 92 96

Amdro (Std.) 1.0 93 84

CHECK 29 22

~/ Pupae were dipped in acetone - toxicant solution for 24 hours, washed in clean acetone for 5 sec and air dried for 24 hrs. HFP = housefly pupae; EGP = eye gnat pupae. Figures in ( ) equals concentration of acetone solution of fenoxycarb into which pupae were dipped. SUPERCOOLING STUDIES ON FOUR SPECIES OF FIRE fu~TS

Oscar F. Francke, James C. Cokendolpher and Lindsey R. Potts Departments of Biological Sciences and Entomology Texas Tech University, Lubbock, Texas 79409

ABSTRACT.--The phenomenon of cooling water below the incipient freezing point (OoC) is termed supercooling. . The freezing point of water can be depressed by mixing it with substances which have a lower freezing point (such as alcohol or ethylene glycol) and are thus known as "anti-freezes", or by eliminating any impurities in the water which might act as ice nucleators. Supercooling is an adaptive physiological response found in many animals which occur in environments subject to freezing temperatures; and insects found in temperate zones often have seasonally depressed supercooling points to protect them from potentially injurious winter temperatures. Although there are many possible injurious effects from cold, a reliable indicator of survivability by insects in cold temperatures is their supercooling point. Winter-kill has been cited in the literature as an important factor limiting the northward spread of fire ants in general, and of imported fire ants in particular. The implication being that despite some protection from cold temperatures received by the ants from moving deep underground where temperatures are not as cold as at the surface and actually seldom fall below freezing, fire ants are thought to have limited or no ability to supercool. We determined the supercooling points of worker larvae, worker pupae, minor workers, medium workers, major workers, reproductive larvae, and male and female pupae and adults in four species of fire ants: Solenopsis aurea Wheeler, Solenopsis geminata (Fabricius), Solenopsis invicta Buren, and Solenopsis xyloni McCook. The worker caste has a slightly lower supercooling point than the reproductive caste in the four species. Within each species adults have a higher supercooling temperature (-7oC to -lZoC) than immatures, and larvae0 have a higher0 supercooling temperature (-SoC to -16°C) than pupae (-16 C to -24 C).

I 72 I Airborne Venom Dispersal in i;olcno/,:;i:;: Functional Correlates of Insecticidal and Antibiotic Venom Properties Martin S. Obin

University of Florida Department of Zoology Gainesville, Florida 32611 Robert K. Vander Meer

USDA/ARS Insects Affecting Man and Animals Research Laboratory P.O. Box 14565 Gainesville, Florida 32604

73 74

ABSTRACT

Venom Dispersal in SoZcno~JiG: Functional Correlates of Insecticidal and Antibiotic Properties of Venom Alkaloids

Martin Obin and Robert K. Vander Meer, Ph.D., USDAjARS, Gainesville, Florida

.c;olcrlOp;;i;; invicta, :;. f./r!i'!;,:r>';", and,. ::"If1:"JI,J[.,[exhibit venom extrusion

and "gaster-flagging" when they encounter heterospecifics in the foraging arena. Behavioral and gas-liquid chromatographic studies demonstrated

that in addition to wiping venom on antagonists, S. invicta repels them via airborne droplets released during "gaster-flagging". The size of the observable droplets vary from 150mmto an aerosol-like 2a~m particle.

Data suggest that this same aerosol venom dispersal mechanism is used in applying much smaller particles of the antibiotic venom to S. invicta

'brood. Carefully controlled chemical analysis of brood for worker venom alkaloids gave 0.9 - 1.1 ngjimmature. Our discoveries of new antagonistic and beneficial uses for fire ant venom alkaloids help further elucidate their adaptive significance to the colony. 75

Airborne Venom Dispersal in SoZenopsis: Functional Correlates of Insecticidal and Antibiotic Venom Properties Martin S. Obin

University of Florida Department of Zoology Gainesville, Florida 32611 Robert K. Vander Meer

USDA/ARS Insects Affecting Man and Animals Research Laboratory P.O. Box 14565 Gainesville, Florida 32604

SoZenopsis invicta Buren (Myrmicinae) is an indigenous South American ant species that has attained introduced pest status in the southeastern United States. It is perhaps best knownfor the large mounds it constructs and for the painful sting from which it derives its common name - the fire ant. As with other hymenopteran venoms, research has focused on the effects of fire ant venom in vertebrate models. However, as early as 1958, Blum et al. recognized the insecticidal and antibiotic properties of the alkaloids that constitute over 95% of the fire ant venom (see also Jouvenaz et al., 1972). We were interested in the adaptive significance of these venom characteristics, particularly with respect to "gaster flagging" behavior (Adams and Traniello, 1981) observed in S. invicta colonies by ourselves and others (Bhatkar et al., 1972). During "gaster

flagging" workers raise and vibrate the gaster while extruding the sting.

We hypothesized that during gaster flagging, ::. invi,'01 workers dispersed

venom through the air. However, in S. invicta young workers tend brood, while older workers do most of the foraging (Mirenda and Vinson, 1981;

cf wilson, 1978), and we had observed gaster flagging by both worker

types. It therefore occurred to us that the function of airborne venom 76 dispersal might be different in brood tending and foraging contexts. Venomdirected to the brood or the surrounding brood chamber would reduce the likelihood of microbial infection, while venom directed at heterospecifics encountered in the foraging arena would function as a repellent.

Our hypotheses concerning venom dispersal and its adaptive significance were based on the following combination of existing studies and personal observation.

A. During careful observations of confrontations between S. invicta and foragers of both Pheidole dentata (Myrmicinae) and Camponotus flo~idensis

(Formicinae) we had noted violent withdrawal, antenna dragging and other grooming behavior on the part of ants that approached gaster flagging S. invicta from the rear, but did not actually contact the fire ant's sting. This strongly suggested that venomwas being dispersed through the air. Moreover, other Myrmicines including species of Solenopsis are known to wipe repellent venom on antagonists as part of either raiding (Holdobb1er 1973; Blum et al., 1980), or interference competition strategies (reviewed in Adamsand Traniel10, 1981).

B. Antimicrobial exocrine products are not without precedent in ants

(Maschwitz et al., 1970). Indeed, Wilson (1971) has suggested that the evolution of antibiotic exocrine products was crucial to the development of subterranean existence among ants. To test our hypotheses, we initiated studies designed to (1) more fully describe gaster-flagging by S. invicta, (2) determine whether venom was dispersed through the air by gaster flagging during heterospecific confrontations and (3) determine whether worker-derived venom was present on the surface of the brood. 77

Study 1: Analysis of Gaster Flagging Behavior Methods

Ants tested were members of monogynous queen-right colonies collected four to six months previously and maintained at the USDAFire Ant Project Laboratories, Gainesville, Florida. Colonies contained more than 10,000 individuals and included immatures at all developmental stages. Colonies were maintained in plastic petri dish cells with Caston~ floors at 26-27°C on a diet of honey-water, fly pupae and hard-boiled egg. These brood chambers were placed in Fluo~coated plastic trays that served as foraging arenas. Ant behavior in the brood chamber was observed in three different

:;. ii/Ot>I,! colonies under ambient and fluorescent 1ight during a total of 260 minutes. Confrontation behavior was observed following introduction of individual s. invicta foragers or brood tenders into the foraging tray of heterospecific (5. geminata) colonies (N = 11 trials). Data were collected for 10 minutes post introduction. Interspecific interactions in the petri dish arenas were observed under a dissecting microscope fitted with an ocular micrometer. These interactions were also filmed with a Fujica ZC1000 Single-8 camera fitted with a Tamron

Tele-Macro lens (1:1). Results

We recognized three distinct types of gaster flagging in s. invicta, hereafter referred to as the "headstand", "aggressive waggle" and "brood fl ag". The former two were exhibited only by workers during interspecific encounters.

Interspecific encounters. Both brood tenders and foragers exhibited 78

the "headstand" and/or "aggressive waggle" during the arena encounters.

While performing the headstand, workers straighten the rear legs, elevate the petiole 70-90 degrees to the substrate and extrude the sting. They then vibrate the gaster almost imperceptibly in the vertical plane. A drop of venom up to 0.2mmin diameter appears at the tip of the sting within several seconds. The antennae are elevated to a position parallel

with the substrate as the ant moves its head slowly from side to side.

A foreleg tap (0.2-1.5 taps/second) was noted throughout or during portions of all "headstand" sequences observed. The "headstand" was displayed by workers in response to initial contact with a heterospecific or in response to grappling or other agonistic interaction between nestmate(s) and heterospecifics occuring within 2.0-3.0cm. Individual Solenopsis of bath species often gaster flagged in the headstand position immediately next to a grappling pair.

"Aggressive waggling" is distinguished from "the headstand" by three criteri a. First, the gaster is elevated no more than 45 degrees. In addition, although the sting is extruded, no venom droplet is secreted during this behavior. However, when "aggressive waggling" follows or is interrupted by "headstanding", venommay be present at the sting tip. Furthermore, gaster vibration during "aggressive waggling" is much more vigorous (and thus recognizable), and ants can be observed waggling laterally as well as vertically.

During the arena encounters, we observed :iol('llOr)~:t~:workers "waggling" at heterospecifics subsequent to initial contact and brief "headstanding".

We also observed seven instances in which workers brandishing extruded stings and venom droplets "waggled" at heteraspecifics held by the mandibles of an aggressing ant. In six of these cases, ants that "waggled" 79

at immobilized heterospecifics did so after "headstanding" within 0.5cm

of the antagonists for over 60 seconds. However, in one instance, an

s. invicta worker with previously-extruded venom visible at the tip of

the sting rapidly approached to within 1.Ocm of a pinioned S. geminata worker. She then turned, positioned her extruded sting directly over the head of the immobilized ant and "waggled" for several seconds before walking away.

Gaster Flagging in the Brood Chamber. The "brood flag" combines the less vigorous gaster vibration of the "headstand" and the reduced vertical deflection of the gaster observed during the "aggressive waggle". We noted nine instances of such behavior. In no instances were venom droplets observed at the tip of the sting, nor were workers exhibiting this behavior observed feeding or grooming, or carrying immatures.

Study 2: Airborne Venom Dispersal by Foragers Methods

Two experiments were performed. In the first, encounters in petri dish arenas were staged between groups of 8-12 :;. inv,>LIl and :;. (lcminalAI workers. Whenalarmed individuals assumed the headstand posture and began gaster flagging, a TLCplate impregnated with Iodoplatinate Reagent (Touchstone and Dobbins, 1978) was held 1-2cm from the tip of the sting while the ant was prodded from the rear and side with forceps. (Iodoplatinate turns from red to either white or blue in the presence of

alkaloids.) An impregnated plate was discarded whenever it touched an

ant, the arena surf"ace or forceps that had contacted either. The size

and pattern of venom droplets on the TLC plate were analyzed under the

dissecting microscope. [Tests with alkaloid standards indicated that as

little as 5ngjspot (minimumspot diameter = O.02mm) could be visualized

by this method.] 80

In the second experiment, lone S. invictaforagers were introduced into petri dishes housing 10-12 S. geminata workers. During confrontations the S. invicta intruder would invariably perform an "aggressive waggle".

S. geminataworkers making their initial approach to the introduced

S. invicta worker were occasionally repelled from a distance of up to

1.5cm. Eight of these were immediately collected and swirled in hexane (250~1) for 30 seconds. An internal standard was added to this sample which was subsequently analyzed for the presence of species-specific - S. invictapiperidine alkaloids (Brand and B1um1 1972) by gas liquid chromatography (DB-1) fused silica capillary column (J. & W. Scientific, Inc.), 15mx 0.32mm, sp1it1ess; Varian 3700, F1D, temperature program = 150 - 200 at 2°C/minute; DB-225 fused silica capillary column (J. &W.

Scientific, Inc.), 30m x 0.26mm, split1ess; Varian 3700, FID, 200°C isothermal). Results

Analysis of six TLC plates revealed from 4-11 venom drop1ets/mm2. The droplets can be assigned to two discrete size classes: (1) diameter = 0.08-0.l3mm, and (2) diameter = 0.02-0.03mm. The larger droplets constituted from 9.9% (1 of 11) to 100% (4 of 4) of all venom droplets noted in a sampling area. No ordered pattern of droplets was apparent.

Species-specific S. invicta venom alkaloids were recovered from the cuticle of S. geminata workers visibly repelled by gaster flagging. Total venom present in the aggregate sample (N=8 ants) was between 3.5 and 4.0 micrograms.

Study 3: Worker-Derived Venom on the Brood Methods

We froze a brood cell containing workers and brood and then separ- ated the brood from brood tenders in an air 5tream (Stringer et al., 1972). 81

The brood sample (N = 8,000 immatures) was examined to insure that workers were absent and then rinsed for 1 minute in 2:1 chloroform/methanol (5ml). This rinse was divided into 2 equal subsamples, each of which was reduced to 25~1 under a stream of nitrogen and resuspended in 500~1

hexane. Alkaloids were isolated and purified by a) washing 3 times with

0.5N sulfuric acid (200~1), b) separation of the aqueous phase followed

by basification with 1.5N potassium hydroxide (250~1) and c) extraction of the alkaloids from the basified aqueous phase 3 times with hexane (200~1). Internal standard was added to the hexane extract prior to

GLCanalysis (as above).

We also quantified the percent alkaloids extracted from whole workers by our rinse method, and then quantified the alkaloid contents of 5 extirpated pupal poison glands. In addition, we lined a section of the apparatus used to separate brood from brood tenders with filter paper and conducted another brood/worker separation. The filter paper was then cut into strips, extracted with hexane under vacuum and analyzed by GLC for the presence of venom alkaloids. Results

The two brood rinse subsamples yielded 1.128ng alkaloids/immature and 0.917ng alkaloids/immature. Whenapplied to s. invicta adults, our rinse technique extracted less than 0.002% of the total available alkaloids from the surface and/or poison sacs. We assumed that all 4,000 immatures per subsample contained the amount of alkaloids measured in the extirpated pupal sacs (= 257ng/pupa), and that our technique extracted 0.002% of these alkaloids - that is to say, approximately 5pg/immature. This figure represents 0.48% and 0.55% of the total alkaloid content in our two rinse samples. Venom alkaloids quantified in 2 samples of 82

filter paper lining the ant sorting device were present at 13.0 and

16.3pg/immature. Thus, between 1.25 and 1.48% of the venom in our brood rinses was potentially a result of contamination from worker venom extruded during the sorting process. Total venom on the brood surface excluding the maximum possible contamination from poison glands of workers and immatures was calculated to be 1.036 and 0.896ng/immature, respectively.

Discussion

Our data indicate that venom is dispersed through the air during gaster flagging by S. invicta (and presumably by congenerics). This is to our knowledge the first evidence of airborrae~ directional dispersal of venom droplets by the sting apparatus of a hymenopteran. The venom dispersed by gaster flagging visibly repels heterospecifics and presumably functions as an antiseptic in the brood chamber. Selection pressures contributing to the evolution and maintenance of insecticidal and antibiotic ant secretions may be especially intense in tropical and sub- tropical habitats since these habitats 1) support a great diversity and number of potentially predacious and/or competitive ant species and

2) provide especially favorable conditions for microbial growth. While other myrmicine ants rely on repellents produced in the poison gland, their antibacterial secretion - phenyl acetk .acid - is derived fr.om the metap1eura1 gland (Maschwitz et a1. 1970). At present, no study of the metapleural gland in any S()leno!)::?:uspecies §~ available. Data presented here strongly suggest, however, that ttleproduction and dispersal of antibiotic exocrine materials in SolenopsiB spp. is a function of the poison gland and sting apparatus. This findj~9 in turn argues in favor of an alternative role for the metapleural gland in fire ant ecology. 83

Behavioral observations suggest that the "headstand" behavior is used by workers to pump large quantities of venom from the poison reservoir into the sting. These large quantities function as airborne repellents when directed at heterospecifics during aggressive waggling. The size of venom droplets visualized by Iodoplatinate (Study #2) indicates that whole venom drops secreted during "headstanding" (diameter = O.lmm) may be "flung" from the sting tip during "waggling". These large drops are usually accompanied by smaller droplets (diameter = 0.02 - 0.03mm). Data indicate that as much as 500 nanograms of venommay be dispersed during each bout of "waggling". Since they must defend the brood from raiding ants or other predacious arthropods that reach the brood chamber, it was not surprising that brood tenders exhibited the full repetoire of confrontation gaster flagging behavior observed in foragers (i.e., the "headstand" and "aggressive waggle"). That alkaloids on the brood surface actually convey enhanced protection from microorganisms has not been determined, but the small quantity of venom present argues strongly in favor of an aerosol or fine droplet method of application involving droplets with diameters much less than

20jlm. Recall that our method of visualizing 0olcnopsis venom alkaloids permitted detection of no less than 5.0ng venom/spot - a spot 20-30jlm in diameter. If one assumes that the lng of venom on the surface of immatures is evenly distributed, one must conclude that drops much smaller than 20jlm are dispersed by brood tenders. We propose that by refraining from "headstanding", brood tenders can dispense smaller droplets, and as a consequence, smaller amounts of venom. Such a capacity is especially important when one considers the potentially harmful effects of a concentrated venomdrop on nonscleritized immatures. 84

Other investigators (Mirenda and Vinson, 1981) have reported stridulation by S. invicta brood tenders. Like gaster flagging, stridulation requires both gaster elevation as well as vibration. However, our observations indicate that unlike gaster flagging, stridulation involves no sting extrusion, is marked by more vigorous gaster vibration, and always occurs in conjunction with direct brood care (i.e., grooming, feeding and transporting of brood).

Mass recruitment to resources by pheromones has been intensely studied in S. invicta (Vander Meer, 1983). The correlation between possession of mass recruitment systems and possession of interference repellents in ants has been pointed out by Holdobbler (1978). Holdobbler (1982) concludes that the "competitive behavioral repertoire of a species during foraging is as important to an understanding of foraging biology as the behavioral scale of foraging techniques". I suggest that this observation may be of value to i;ol('Yl()IJ::i~;control programs, especially those utilizing baits. As an eXiJlllple, we cite the work of Adal1lsand

Trani ell 0 (1981) with "4onomoriwn minimwn. They report that the prob- ability of interspecific interference at baits rose from 5 to 100%when baits were too large for retrieval by a lone forager, but not large enough to elicit full, avid recruitment, thereby insuring adequate chemical defense of the resource by repellent venom. 85

REFERENCESCITED

Adams, C. S. and J. F. A. Traniello. 1981. Chemical Interference

Competion by Vonomonium minimum (Hymenoptera: Formicidae).

Oecologia 51:265-270. Bhatkar, A., W. H. Whitcomb,W. F. Buren, P. Callahan and T. Carlysle.

1972. Confrontation behavior between Lasiu3 Nconiger (Hymenoptera:

Formicidae) and the imported fire ant. Environmental Entomology,

1: 275-279.

Blum, M. S., J. R. Walker, P. S. Callahan and A. F. Novak. 1958.

Chemical, insecticidal and antibiotic properties of fire ant

venom. Science, 128:306-307.

Blum, M. S., T. H. Jones, B. Holdobbler, H. M. Fales and T. Jaouni, 1980.

Alkaloidal venom mace: offensive by a thief ant. Naturwissen-

schaften, 67:144-145.

Brand, J. M., and M. S. Blum. 1973. Biochemical evolution in fire

ant venoms. Insect Biochem., 3:45-51.

Holdobbler, B. 1973. Chemische Stategie beim Nahrungserwerb der Diebameise

(Solenopsis fugax Latr.) und der Pharaoameise (Vonomorium pharaonis L.)

Oeco1ogia, 11 :371-380.

Jouvenaz, D. P., M. S. Blum and J. G. MacConnell. 1972. Antibacterial

activity of venom alkaloids from the imported fire ant, ,';o/er!O!'{;7::;

':UOi,-!d Buren. I\ntimicrob. Agents Chemother., 2:291.

Maschwitz, U., K. Boob and H. Schilldknecht. 1970. Ein beitrag zur

funktion der metathoracaldruse der ameisen. J. InsectPhysiol.,

16:387-404. 86

Mirenda, J. T. and S. B. Vinson. Division of labour and specification

of castes in the red imported fire ant .';01.>//°1'::(::i'll'i../,.[ Buren.

Anim. Behav., 29:410-420.

Stringer, C. E., B. M. Glancey, C. C:,Craig and B. B. Martin. 1972. Air separation of different castes of the imported fire ant. J. Econ. Entomol., 65:872-873.

Touchstone, J. C. and M. F. Dobbins. 1978. Practice of Thin Layer

Chromatography. New York. John Wiley and Sons.

Vander Meer, R. K. 1983. Semiochemicals and the red imported fire ant

(Solenopsis invicta Buren) (Hymenoptera: Formicidae). Flori da Ent., 66:139-161.

Wilson, E. O. 1962. Chemical communication amongworkers of the fire ant Solenopsis saevissima (F. Smith). 1. The organization of mass-foraging. Anim. Behav., 10:134-147. 1971. The Insect Societies. Cambridge. Harvard University Press. - 1978. Division of labor in fire ants based on physical castes. J. Kans. Ent. Soc., 51:615-636. TITLE: 1983 TEXAS IMPORTED FIRE ANT PRO-DRONE AERIAL APPLICATION PROGRAM

AUTHOR: MARK R. TROSTLE

The 1983 program in Texas was a cooperative program between the United States Depart- ment of Agriculture/Animal Plant Health and Inspection Service/Plant Pest Quarantine, Texas Department of Agriculture and Kendall and Kerr Counties. The program was established to evaluate the percent control achieved on a large scale application of Pro- Drone. Pro-Drone is a insect growth regulator discovered by the USDA and developed and marketed by Stauffer Chemical Company. The Kendall and Kerr Counties area was chosen for the following reasons: 1) the area met the label guidelines of pasture, rangeland, turf grass and non-agricultural land; 2) the area was located on the western movement of the imported fire ant, and 3) the two counties had submitted written request for aerial treatment programs. The overall pre-treatment levels of IFA in the counties averaged 30 mounds per acre. There were 65 quarter-acre efficacy plots established in the treatment area and 25 quarter-acre plots established outside the treatment area as check sites. Prior to tLeatment, there were test sites established to monitor soil, water. vegetation, insects and wildlife. These samples will be run by the National Environmental Monitoring Laboratory at Gulfport, Mississippi. Texas Tech University will monitor native ants and other insects to document the number of insects before treatment and after treatment. The first treatment started on June 10 and was completed on June 20. This treatment used 8 Gruman Ag Cats and 4-Turbine Air Tractors. During this time, there was 449,920 acres treated at a rate of .88 Ibs per acre. The program involved 23 TDA and 7 USDA/APHIS/PPQ personnel. The personnel duties ranged from Supervision of the program, supervision of loading of aircraft, surveying area prior and during treatment and monitoring of environmental criteria. The program cost per acre for the spring was $1.94 or a total of $874,986.41. The second application started on Sept 26 and was completed on October 1st. This treatment used the same 8 Gruman Ag Cats and 4-Turbine Air Tractors. There were 451,894 acres treated at a rate of .88 Ibs per acre. The program involved 28 TDA and 7 USDA/APHIS/PPQ personnel. These government personnel duties remained the same as previously mentioned. TDA also hired_8 part-time personnel to assist Inspectors in evaluation of Pro-Drone displacement and also to ensure that efficacy sites were treated with Pro-Drone. The program cost per acre was $1.56 or a total of $707,139.28. The two applications of Pro-Drone cost $3.50 per acre. On September 15, 1983 I received a report from Joe Ford, Lab- oratory Director of the National Environmental Monitoring Lab, and of 74 soil, 42 sediment and 66 vegetation (grass), all were negative for Pro-Drone. The results on the mammals, birds, insects and water have not been completed yet. Approximately 12 weeks after the first application, the first evaluation of Kendall and Kerr Counties took place. The reason for this time space is due to the action of the Pro-Drone. Pro- Drone effects the colony by causing a shift in the brood and as there are no worker brood being developed, there's no workers to maintain the colony. Pro-Drone takes 12-16 weeks for any results to occur in the colony. The first evaluation showed a mean reduction in number of active nest of 58%. The second evaluation, 10 weeks after second application showed a 37% mean reduction in number of active nests. This data furnished by Horner Collins, USDA/APHIS. These results should be considered preliminary since this'product is known for its slow and subtle effects upon IFA colonies. During the second evaluation, it was apparent that most colonies within the treated area contained much less brood than colonies outside the treated area. These plots will be evaluated 3 times during 1984. The evaluation times will be spring, summer and fall.

87 \ 1)1:PAI ~'r;YIEN'r - ~()F J\(;I ~I(I Jl~rl II{I~ i ~fh QUESTIONS AND ANSWERS ON PRO...DRONE APPLICATION PROGRAM

In response to the serious Texas A & M and Texas Tech projects. problem created by the red University that our most An important part of the imported fire ant in both rural promising long-range overall fire ant program is a and urban areas in Texas, the solution will be found through demonstration project to limit Texas Department of such biological approaches further spread of the fire ant in Agriculture has worked with as development of a sterile Texas. This project will use university researchers, ant, discovery of a natural the insect growth regulator farmers, the Speaker's Select predator, etc., the Depart- called Pro-Drone. to treat Committee on Fire Ants, and ment's efforts to control fire Texas' westernmost boundary others to. develop a reason- ants include increased of the fire ant infestation, able and realistic program funding for research on such which is in Kerr and Kendall aimed at controlling the fire biological controls, as well as Counties. The following ant. It is clear that a method to providing information to local questions and answers are completely eradicate the fire county officials on the most intended to provide the public ant simply does not exist at current treatments available, with complete information this time. Because TDA and the funding of a limited about the treatment program. agrees with researchers at number of demonstration

1. What areas in Kendall and necessary to reduce as much (Kendall: 249-2131: Kerr: Kerr Counties will be reinfestation as possible 257-7093). treated? from outside the application area. Previous test applica- 4. What is Pro-Drone? As shown in the diagram tions have been limited to below, all of Kendall County small areas of 200 to 1,000 Pro-Drone is a commercially will be treated except the acres. Although some available pesticide which has cities of Boerne, Comfort, the reinfestation on treated land been tested by the manu- city lake and the river. may occur from fire ants on facturer and approved by the Approximately 30% of Kerr the property of people who Environmental Protection County will be treated, do not want their land Agency (Reg. No. 476-2211) including the area north of included in the application for use on the red imported the Guadalupe River from the program, TDA is en- fire ant by broadcasti ng on eastern county line to just couraging those indiyiduals pasture and range grass, west of Ingram, excluding the to treat the fire ants on their turf. and nonagricultural cities of Center Point, property with other available land. Pro-Drone is a modified Kerrville and Ingram. methods. insect hormone that acts as an insect growth regulator. It 3. Who should I notify if I DO is produced as a bait, consist- Kerr NOT wish my property to be ing of defatted corn grits with treated? soybean oil and the active ingredient added. Call the County Judges' offices in either Kendall or 5. Is Pro-Drone a poison? Kerr County, as appropriate, 2. Why is such a large area to be or go by their office in No. Pro-Drone is not a treated? person. Leave your name. poison. ;ests performed by address, phone number and the manufacturer which were Treatment of a large area is the location of the land revicNed and approved by 88 89

the Environmental Protec- approved the results of these when the soil temperature IS tion agency have shown no tests in its registration above 60° F. and the ants are adverse effects in mammals, procedure. actively gathering food. The birds or fish. No detectable dew will be allowed to residues were noted in those 8. How does Pro-Drone work to evaporate to prevent any animals exposed to Pro- control the Red Imported Fire granules from sticking to the Drone at the recommended Ant? grass. Pro-Drone will not be application rate (4.8 grams of applied if heavy rainfall is the active ingredient per Pro-Drone is collected by expected within 4-6 hours, or acre). Its method of worker fire ants who carry it if winds are greater than 10 application further adds to its into the colony for food miles per hour. The spring safety because it is not used supply. When eaten, Pro- application will be evaluated, as a liquid spray which can be Drone disrupts the natural and if it is satisfactory, the inhaled. Pro-Drone has also hormone system of the ant second treatment will be been shown to be biode- larvae, causing larvae that made in the Fall of 1983. gradable and non-persistent would normally hatch into in the environment. It has a workers to develop instead soil half-life of 68 hours. It is into sexuals that cannot mate 11. Why is the aerial application also readily degraded by successfully. Over a period of method of treatment to be sunlight. several months, the dis- used? appearance of worker ants 6. Who developed Pro-Drone leads to a decline in food Aerial application is the most for use in controlling fire gathering for the colony and logical treatment method for ants? the eventual death of the large areas of land because it colony. is physically impossible and The United States Depart- economically unfeasible for ment of Agriculture de- 9. How effective is Pro-Drone? farmers and ranchers to treat veloped the modified insect hundreds of acres of their hormone (now called Pro- Based on experience on test property by mound to mound Drone) in 1974. Stauffer plots of 200 to 1,000 acres appl ication. Fu rthermore, Chemical Company acquired each in Valdosta, Georgia aerial application requires the rights to produce the and in Brazoria, Jefferson, that less material be used compound in 1978. Austin, and Trinity Counties since coverage is uniform. in Texas, Pro-Drone has Pro-Drone was designed 7. What tests hav,e been been over 90% effective in specifically to be broadcast conducted to determine the controlling the red imported through aerial application. safety and effectiveness of fire ant after the second Pro-Drone? application. Pro-drone is designed to be applied twice 12. Why is TDA using Pro-Drone during the year, either in in this project instead of Efficacy (effectiveness) tests other available treatments? on Pro-Drone were con- Spring-Fall or Fall-Spring. It ducted jointly by the Texas has no residual effects and Department of Agriculture consequently, does not 1. Most importantly, because and the United States prevent reinfestation. Pro-Drone is not a poison Department of Agriculture on and has essentially no test plots of 200 to 1,000 10. How will the Texas Depart- toxic effects on mammals, acres in Valdosta, Georgia ment of Agriculture use Pro- birds, and fish, it is more and in Brazoria, Jefferson, Drone to control the fire ant? desi rable than certai n Austin, and Trinity Counties pesticides which have in Texas. Over the past five TDA will apply Pro-Drone by known harmful' side years, the manufacturer and aerial application outside effects. the USDA have conducted incorporated and residential tests to evaluate the efficacy, areas to pastures, range 2. Test plots have shown Pro- the environmental effects, grass, turf and nonagri- Drone to be more effective mammalian toxicology, and culturalland using calibrated than many other registered the subjective human effects equipment. It will be applied compounds in controlling which might be associated at a rate of .88 Ibs. per acre fire ants. with the use of Pro-Drone (less than one pound per The EPA reviewed and acre) The bait will be applied 3. The land use patterns in 90

Kendall and Kerr Counties Section 104.2 of that same per acre in Kendall County are precisely those for report states that the use of and 15-20 mounds per acre in which Pro-Drone was Pro-Drone will not pose a Kerr County. USDA recom- approved: range grass, significant risk to nontarget mends treatment when pastures, turf, and non- aquatic and terrestrial infestation reaches an agricultural land. organisms because of its low average of 10 mounds per toxicological hazard to acre. 13. Will Pro-Drone affect other vetebratns. The particle size insects, including other ants? and timing of the application 2. It is an accept(~d practlCt~ are further designed to to treat at the edge of an The active ingredient in Pro- minimize the amount that infestation in order to limit as Drone could affect several might be eaten by non-target much reinfestation as other types of insects, species. To provide addi- possible and to prevent including mosquitoes and tional assurance that lab further spread. Kendall and flies, but because the bait has results accurately reflect Kerr Counties are the been formulated specifically field conditions, the USDA westernmost boundary of the in to attract the imported fire will gather wildlife samples fire ant infestation Texas. ant, other nontarget insects for observation before ahd should not be affected. Tests after treatment. 3. The County Commis- on this type of bait have sioners Court in both Kerr shown that 95% of the bait is 15. Does Pro-Drone have ahy and Kendall Counties picked up by the imported effect on rivers ot grourtd showed interest in past TDA fire ants within an hour in water? treatment programs to heavily infested areas. Its oily control the fire ant, and in nature, particle size, and low Pro-Drone will not be applied April they voted to participate application rate further to rivers, streams, or any in the Pro-drone treatment minimize the risk of uptake water supply even though to program after holdihg public by non-target insects. do so would have no effect on hearings in their counties. Although native ants were the water. Pro-Drone water observed in the Pro-Drone half-life is six days. Less than 4. The land use patterns in test plots in Georgia and 3% of the bait formulation both counties are precisely Texas after the treatment leaches into water. It is, of those for which Pro-Drone program was completed, course, never a good practice was approved. further research is needed to to apply any pesticide in a fully document the effect of way that it enters any water 18. How was the degree of fire Pro-Drone on native ants and supply. ant infestation in Kerr and Kendall Counties deter- other ground foraging mined? insects. Entomologists from 16. Why is n ' t Pro - D r 0 ne Texas Tech University will approved fot cropland? USDA and TDA conducted monitor the test plots in Kerr and Kendall Counties to The manufacturer has hot field surveys that involved document the effect of Pro- completed the residue actual counts of imported fire Drone on native ants and analysis on various crops as' ant mounds. Crews surveyed other insects. required by the EPA for parts of the counties by registation for use on ground transportation and by 14. Does Pro-Drone have ah cropland. The manufacturer foot and reached remote effect on other wildlife? anticipates completion of the areas by helicopter. analysis before September, 19. How can TDA assure me that In tests by the manufacturer 1983. my property will not be and reviewed by the treated? Ecological Effects Branch of 17. Why were Kendall County the EPA, Pro-Drone was fed and Kerr County the areas TDA is committed to a to Bobwhite Quail, Mallard designated for this treatment professional and efficient Ducks, Rainbow Trout, program? application program. The Bluegill Sunfish and Shrimp, following methods will be and the results of the test 1. Field surveys conducted used to insure that your indicate that Pro-Drone is by USDA and TDA docu- property will not be treated: essentially nontoxic to these mented a fire ant infestation nontarget organisms. averaging 30 fire ant niouhds 1. TDA will visit each site and 91

insure that the area to be 2. These test sites also have 8. respond to emergency omitted is properly marked had soil, water, vegetation, situations to protect on their map. insect and wildlife samples public health; and taken by the National 2. TDA will tie marker Environmental Monitoring C.treat isolated new infes- balloons easily visible from Laboratory of the USDA. tations. the air on the boundaries of The NEML will again take your land. samples after treatment. 23. Who can I call if I need more information about this treat- 3. ,TDA will instruct pilots as 3. In conjunction with TDA, ment program? to the location of land to be entomologists at Texas excluded from the treat- Tech University will For information in your ment area. monitor native ants and county, call the office of your other insects in test plots to County Judge (Kendall: 249- 4. TDA will have its own document the number of 2131; Kerr: 257-7093). inspector on site when native ants and other treatment occurs to moni- insects before and after For information about the tor application to insure treatment. Texas Department of that your land is not Agriculture contact: treated. 21. How is the treatment program funded? Ron White, Assistant Deputy 5. Application is only made Commissioner for Regula- when wind is less than 10 USDA is paying one-half the tory Activities, (512) 475- mph. cost of the entire treatment 6346. program; the other half is 6. All equipment is calibrated paid from TDA funds IMPORTED FIRE ANT PRO- to insure proper applica- allocated by the Legislature GRAM BASE OF OPERATIONS tion. exclusively for the purchase IN KERR COUNTY: and application of a 7. Should any problem arise registered pesticide to be (512) 896-2767 or should you have any used for treatment of the red questions, call the TDA imported fire ant. base of operations in Kerr County at (512) 896-2767 22. What other programs has or call Ron White, Asst. TDA initiated to control the Deputy Commissioner for imported fire ant? Regulatory Activities, at TDA's office in Austin 1.lncreased funding for (512) 475-6346. research to' develop an effective biological 20. What monitoring programs solution. are planned? 2. Development and distribu- 1. Fifty quarter-acre sites tion of updated informa- have been identified within tion to assist county or city the treatment area to governments to obtain evaluate pre-treatment various insecticides and ievels of the imported fire biological control sub-

James Bosworth and Robert K. Vander Meer

USDA/ARS, Gainesville, Florida

~I This paper presents the results of preliminary research only and will be published later through established channels with appropriate editing. The paper and data should not be referred to in literatlJre citations until they appear in press. Mention of a proprietary product does not constitute a recommendation by the United States Depart- ment of Agriculture nor does it imply registration under FIFRA as amended.

92 93

ABSTRACT

Colony Founding Minims: A New DoZ,mm,:n',s,:nV1:cta caste

James Bosworth and Robert K. Vander Meer, Ph.D.,

USDA/ARS,Gainesville, Florida

Colony founding minim (CFM) poison sacs contained only a single major alkaloid that was demonstrated to be different (gas chromatography and mass spectra) from any previously reported /7('!('IlO();:/;; venom alkaloid.

The two acknowledged female fire ant castes, workers and queens, are characterized by distinct venom alkaloid differences. CFM represent a third distinct venom alkaloid pattern. This data plus the behavioral constraints placed on CFMas opposed to mature colony workers led us to propose that CFMconstitute a third fire ant caste. Minim-sized workers from a 6 month old colony had a venom alkaloid pattern qualitatively identical to mature colony workers. We conclude that CFMare a transient caste with the specific function of aiding the queen in the vulnerable colony founding period. Further chemical, behavioral and morphometric studies are in progress. 94

Colony Founding Minims: A New SoZeno/)f;-':;: 1~1l7J'l:cIJlCaste James Bosworth and Robert K. Vander Meer

USDAjARS,Gainesville, Florida

After an alate queen drops to the ground following insemination, she must perform functions (such as grooming, nest excavation and maintenance as well as brood tending and brood feeding, etc.) which, in a mature colony are performed by the worker caste. After a nest has been established, the new queen, using food reserves accumulated prior to mating and also derived from flight muscle cell death and reabsorbtion, lays eggs and tends brood until they emerge as adults. These new workers also find themselves in an environment much different than workers in a mature colony. Instead of being in a colony with division of labor, these new workers, by innate mechanisms or via controls by the queen, must perform all functions. (They are therefore behaviorally different from mature colony workers.) Several years ago when analyzing hexane extracts of founding colony minim poison sacs, Dr. (Bob) Vander Meer noted a distinct differeRce in their alkaloid pattern compared to the typical worker pattern previously published in the literature. Based on distinct behavioral requirements and the distinct chemical difference in the poison sac contents, we PI9~??_~ that colony founding minims (CFM or NCM) represent a new transient fire ant caste. Last fall a set of experiments were undertaken to begin the investigation of this hypothesis. We are approaching this phenomena from three different points of view: 95

1. Morphological/morphometric 2. Behavioral/temporal 3. Chemical - both quantitatively and qualitatively We have initially concentrated on chemical aspects since pre1im- inary data was already available. We have analyzed for poison sac alkaloids, although we also are working on post-pharyngeal gland hydrocarbons and Dufour's gland patterns. I would like to present our current data comparing the poison sac venom components from colony founding and mature colony (MCM) minims. The experiments included recording of weights and head widths (HW) as well as extirpation and analysis of poison sac contents. Typically, three trials consisting of 3-5 ants/ trial/worker type were performed. Figure 1 presents graphically the total alkaloids/poison sac expressed as ~g/mm HWor ~g/mg body weight.

As the quantitative differences in venom between NCMand MCMwere con- siderab1e, an interim colony age group (designated OCM)consisting of ants from six month old (100-200 workers) immature colonies were also sampled. The graph suggests that as the colony matures the total amount of venom alkaloids increases, at least for minim sized workers. Gas chromatographic analysis of poison sac contents also revealed gua1itative differences in poison sac contents as well as quantitative differences. Figure 2 illustrates the GC profile of both MCM(above) and NCM(below) venom alkaloids. An internal standard, n-docosane, was used at a concentration of 0.5 ~g/poison sac. The column used was a

15-m DB-1 capillary column at 1500C isothermal. Kovacs Indices (KI) were determined for all major peaks in each sample (Table 1). Note that the predominate (94%) NCMpeak has the same KI as the double bonded 96

C13:l of the worker caste (MCM or Major). Also notable in the NCMpro-

file is the absence of the other predominate peaks (C13:0' C15:0 and

C15:l) of S. invieta workers. Wealso analyzed the alkaloids on a OB-225 capillary column

(Figure 3) which separates on the basis of polarity. There was a difference in retention time (Rt) of approximately 0.5 minutes between The KI values shown in Table 2 the NCM peak and the C13:l MCMpeak. reflect the differences between these peaks. Note that the saturated

(C13:0 and C15:0) and monounsaturated (C13:l and C15:l) alkaloids elute in reverse order on the OB-225 compared to OB-l.

Having established the qualitative differences between the MCM and

NCM peaks, we prepared a sample from the latter for GC/MS analysis

(chemical ionization, CI). The results using either methane or iso- butane as the reactive gas indicated that the compound had a molecular weight of 279 (Figure 4). The odd molecular weight indicated that the compound contained an odd number of nitrogens. The tenuous CI fragmen- tation pattern was similar to that of the familiar knownfire ant alkaloid.

The tentative structure of this compound (pending further spectral analysis) is shown in Figure 5. Based on the GC retention time characteristics of the colony founding minim alkaloid as well as the GC/MS data, the structure of the compound fits that of a C13 piperideine or a piperidine having the double bond shifted to another position in the side chain. Further spectral and chemical analyses are being carried out to determine the absolute structure of this major component. Conclusion

The two established female S. invieta castes, workers and queens, 97

are clearly distinguished by their venom components (Figure 6). Changes in mature colony worker size does not alter the qualitative aspects of the venom. The venom of colony founding minims is distinctly different from both female alate/queens and worker venom. We therefore propose that colony founding minims, which exhibit a distinct qualitative difference, represent a new, albeit, transient caste in Solenopsis invicta. FIGURE 1

COMPARISONOFTOTALALKALOIDVERSUSHEADWIDTHANDWEIGHT

U G 29fLKALOIDUS HEADWIDTH US WEIGHT /1111 HW- > ...... - T 0 15 T ~ A co L A 18 L /"G BODYWT-) K A L ...... 0 5 ...... I .:::::::: ...... D ...... S ...... 8 1 , I::f:l...~ ::::F:.... 0 1 2. 3 4 5 6 7 HC" OC" "C" FIG U R E 2

GASCHROMATOGRAPHTRACESOF MATURECOLONYMINIM (MCM) AND NEWCOLONY MINIM (NCM) VENOMALKALqIOS (OB-1) ,n

I I , MCM ,: ", I'I wi ~I 21 en !.

~ Ii I

st , I)L~ 5 10 15 20 25 30 MINUTES

1" ,I ,I :I 'I :i , , std NCM , , :I wi ~ eni zi 0, II a..~ en " Ii, w ., Ct: II ", I !I 'I

I I.t'l

I i~l L 5 10 15 20 25 MINUTES

99 100 FIGURE 3

GAS CHROMATOGRAPH TRACESOF MATURECOLONYMINUM (MCM) AND NEWCOLONYMINIM (NCM) VENOM ALKALO~OS (OB-225)

MCM w en z 0 0- en w a::

std "I! I---

III I 5 10 15 20 25 MINUTES

WI II std en z HCM 010- II , en W ex::

I I

I I 5 10 15 20 25 FIGURE 4

CHEMICALIONIZATION MASSSPECTRAOF NEWCOLONYMINIM ALKALOID

60 70 8(1 90 100 110 12~3 130 140 150 160 170 180 190 ...... a......

200 210 220 230 240. 250 260 270 280 290 300 310 320 330 FIGURE 5

POSSIBLE STRUCTUREOFNEWCOLONYMINIM ALKALOID

CH2(CH2)11CH3

PIPERIDEINE --' 0 N

CH2(CH2)2CH =C H ( C H 2 ) 7 C H ~

PIPERIDINE 103 FIGURE 6

GAS CHROMALOGRAPHTRACESOF FEMALEALATE AND MAJORWORKERVENOMALKALOIDS

n w CI) z I 0 I ALATE a.. CI) w Ii ;I a: I ,II' I I I' std d JU~Ul~ 10 15 20 25 30 5 MINUTES

II " M'A J 0 R "'I " W CI) :I z II ,I 0 I a.. ! wCI) a: I

iI I II , I ,I I I I std ! I _J JJ JI II I I I I 10 15 20 25 30 MINUTES 104

TABLE 1

KI VALUESOF VENOMALKALOIDS FROMTHREEs. INVICTA WORKERTYPES

COLONYFOUNDING MATURECOLONY MATURECOLONY

ALKALOID MINUM(%) MINUM(%) MAJOR()

C13: 1 2035 (59) 2035 (21)

C13:0 2065 (10) 2065 (23)

2035 (94)

C15: 1 2205 (27) 2205 (25)

C15:0 2210 (3) 2210 (16) ------__n------_.-

TOTAL ': 94 99 'j)

08-1 CAPILLARY COLUMN(15m) 150°C ISOTHERMAL 105

TABLE 2

KI VALUESOF VENOMALKALOIDS FROMTHREES. INVTCTA WORKERTYPES

COLONYFOUNDING MATURECOLONY MATURECOLONY

ALKALOID ~~1iB MINUM 0,) MAJOR('/,)

C13:0 2290 (8) 2290 (19)

C13: 1 2305 (52) 2305 (16)

2315 (1 00)

C15:0 2480 (5) 2480 (22)

C15: 1 2500 (35) 2500 (42) ------

TOTAL ~'s 100 LOO 99

OB-225 CAPILLARYCOLUMN(30m) 200°C ISOTHERMAL A Citrus Based Solvent as a RIFA Mound Orench

Presented at the Annual Imported Fire Ant. Conference Gainesville, Florida March 27 & ~8, 1984

NOT FOR PUBLICATION

D. Craig Sheppard Cynthia A. Gates

Department of Entomology University of Georgia Coastal Plain Experiment Station Tifton, GA 31793

106 107

A Citrus Based Solvent as a RIFA Mound Drench

In early 1983 a Jacksonville, Florida firm, All South Supply Co., approached us to test a fire ant mound drench material. Oirt

Squad@ was a citrus peel oil based solvent and they had reason to believe it would be effective in this application.

A limited preliminary test with 100, 33 and 17% material in water revealed that a 17% solution gave 100% control and the other rates (inexplicably) gave less favorable results. These mounds, and those in subsequent tests, were drenched with one gallon of dilution per square foot of mound area. Mound area for several mounddiameters was calculated, and subsequently determined from a table like this:

Diam Sq. ft. Diam ft. --o:T3" 1 31-33 6 14-19" 2 34-35 7 20-23 3 36-38 8 24-27 4 39-41 9 28-30 5 4(1-44 10

~ound size in pastures may vary considerably, and measuring volume of dn~nch for pi\ch mound is necessary to achieve a consistent ri1te of application within a treatment.

After our initial success two replicated tests were conducted with mounds in 1/10 A plots receiving like treatments. Active mounds in a 13' buffer ring around each l/JOA circular plots were also treated. The assumption was that possible movement of these mounds into the actual test plot would compensate for possible movement of test mounds out of'the plot. If only ~ounds in the

1/10A were treated movement of some surviving colonies could place them outside the plot and they would be counted as dead. 108

Results of the first replicated test are given in Table 1. Diazinon at 0.1% was used as a standard. Heavy unexplained

mortality in all four check plots limited the value of this test, but the Dirt Squad treatments were more effective than the standard

at 2 months. Percent control corrected with Abbott's formula for

8% Dirt Squad and 0.1% diazinon were negative values, since

reductions in these plots were actually less than in check plots.

Very little check mortality occurred in the seconrl replicated

test and only the corrected values are given in Table 2. Exceptionally good control was achieved with all three treatments

and may be due to extremely cold weather which followed treatment.

This cold weather may have impacted heavily on newly moved

(treated) colonies. Recently built shallow mounds would have been more susceptible to freezing. No winter kill was found in check plots in this test. In most of our mound drench tests 70-80% centrol is considered very good, and previously we had never achieved 100% control with any material.

A commerical citrus peel oil preparation (Dirt Squad) seems to be a very good RIFA drench material at rates as low as 8-10%.

Other tests (Appendix A) indicate that fresh citrus peels contain insecticidal factors and is not a matter of formulation. Limonene appears to be the major agent in the oil, but citral and 1inaloo1 are also active. Much work has been previously conducted in this area. Appropriate articles are cited in a manuscript to be published this spring in the Journal.of Agricu1tura'i Entomology entitled " Toxicity of Citrus Peel Liquids to the House Fly and Red

Imported Fire Ant". 109

Table 1. Percent Reduction of RIFA Mounds Dirt Squad 1/10A, 4 Reps Treated 4/12, 13/83

8% D.S. 17% D.S. 0.11, Dial. Check

1 mo. 4J% 29% 60% 48% 2 mo. 82% 100% 73% 87% 2 mo. -38% 100% -108% (Corrected)

Ti1blp ? P<'rcent Conty'o 1 (corrpcted) of RJ F/\ ~1()unds Dirt Squi1d, 11IDA, ~ Reps Tr(>~ted 12/12/83

20% D.S. 10% D. S. 0.1% Dial.

1 wk. 85 77 90 1 mo. 75 91 95 2 mo. 84 81 90 3 mo. 95 91 100 110

Appendix A

Tables from

TOXICITYOF CITRUS PEEL LIQUIDS TO THE HOUSE FLY AND RED IMPORTEDFIRE ANT

A manuscript submitted to The Journal of Agricultural Entomology

D. Craig Sheppard

Department of Entomology Univprsity of Georgia Coastal Plain Experiment Station Tifton, Georgia 31793 111

Mean number of ~pad Solen.Q£sis j~_v..iSJ:~for"gers (of 10) on 8.S 9 dicecl whole citrus peel in a covered 100 mmpetri dish. - ~--- -~ ~--~---

Time of Type of citrus tested Exposure f,rapefrui t Lime Lemon Orange Check a a a a 2.7a 5 min a.ob O.Ob O.Ob O.Ob 15 min 2.0 8.3a 0.0 b O.Oab O.Ob c 1 h 4.0 c S.7 4.7 c 10.0a O.Ob 2 h 9.0a 10.0a 9.3a 10.0a 0.0

Means in a row with different superscripts are significantly different (P<.OS).

Percentage of orange peel oil in a pl of acetone producing K010' values (from knockdown at 8h) in house flies in twoKD~O tnals. a~d K090 Trial K010 KOSO KOgO

1 9.54 (10.44-6.12)* 10.94 (12.54-9.67) 12.54 (20.15-11.43)

2 9.98 (10.57-9.17) 12.55 (13.22-11.99) 15.79 (17.59-14.73)

*Figures in parentheses indicate upper and lower 9S% confidence limits. Reinfestation Rates of Red Imported Fire Ants Following Toxic Bait Treatments

Clifford S. Lofgren and David F. Williams~

Insects Affecting Man and Animals Research Laboratory ARS -.USDA Gainesville, Florida 32604

A study was made of the reinfestation rates of the red imported fire ant (SoZenopsis invicta) on plots treated with toxic baits. Two parameters of ant density were used: active nests per acre and total numbers of worker ants per acre. The latter was based on estimates of mound size as shown in Table 1. Total worker ants per acre was obtained by multiplying nests per acre in each size category by a weighting factor, as shown in Table 1. All the data for five test sites are presented in Tables 2 and 3 and Figures 1 and 2.

The results show that along roadsides (Table 2). plots treated with

AmdrdID,as well as check plots. were more heavily infested 27 to 36 months after treatment than they were before treatment. This result was evident with both evaluation methods. No correlation in reinfestation rates attributable to the bait treatment was noted.

Ant populations in both treated and untreated pasture plots (Table 3) decreased 27 to 36 months after Amdro@or EL-468 (N-12-all1ino-3-nitro-5-

(trifluoromethy1) pheny1]-2,2,3.3-tetraf1uoroproponamide) were applied to the treated plots. Thus, again, no difference in reinfestation rates attributable to the baits was detected.

112 113

Data on reinfestation rates in a large plot near Albany, Georgia treated by aircraft with Amdro, and a check plot, are shown in Figures

1 and 2. The Amdro was applied in April 1981 at the rate of 4 g AI per acre and a bulk application rate of 1 lb/acre. Ten ~-acre subplots were established in the treated and check areas for pre- and posttreatment evaluations. The latter were made after 6, 19, 44 and 116 weeks. The data were analyzed with a SAS program using general linear models. Nest

(mound) density decreased to 0 following the Amdro treatment (Figure 1) while the nest density in the check was static. After 20 to 40 weeks the subplots became heavily reinfested with small young colonies. The numbers of these colonies decreased dramatically after about 80 weeks and at 116 weeks posttreatment there was an average of 24 nests in the

Amdro subplots compared to 16 in the check subplots.

When the same data is viewed on the basis of total worker ant population (Figure 2), a different picture of the impact of the bait treatment is revealed. The population of total ants dropped dramatically as the pretreatment nests were eliminated. However, after 40 to 60 weeks when the nest counts rose dramatically, the total ant population was still very low. The reason for this is obvious when one recognizes that the reinfestation nests were small (categories 1,2 and 3 in Table 1) and thus each nest contained only a small fraction of the total ants in a mature colony nest. It is evident from the graph that the total ant population did not return to the levels in the check plot until about

100 weeks after the baits were applied.

In conclusion. areas treated with toxic baits already heavily infested with red imported fire ants (-20 mounds/acre) may be tempor- arily infested with large numbers of small young colonies; however, 114 these small colonies represent only a very small fraction of the total ant population prior to treatment. After 2 to 3 years, total nests and ants both return to a level consistent with the carrying capacity of the particular habitat involved. Thus, ~e can state that toxic fire ant baits significantly reduce overall worker ant populations for a period of up to two years even though data from individual nest counts gives the erroneous impression of large population increases with one year. 115

Table 1. Method of determining total RIFA worker populations in field plots.

Age of Estimated no. of Average no. of ants/colony Cate9.2I,t colony ants/colony weighting factor 1-2 Months < 100 100

2 3-5 Months 100-1,000 550

3 6-12 Months 1,000-10,000 5,500

4 13-24 Months 10,000-50,000 30,000

5 24-36 Months 50,000-150,000 100,000

~/ Total population (1 acre) = Sum of products obtained by multiplying total nests in each category by corresponding weighting factor. Table 2. A comparison of IFA PR9ulations pretreatment and 27 to 36 weeks posttreatment with toxic baits applied to roadsides.-

No. of Avg. no. of nests per acre Percent Avg. no. of ants per acre Percent Treatment plots Pretreatment After 27-36 months change Pretreatment After 27-36 months change

Homerville, GA (SR 122)

Amdro 4 54 65 +20 2,277 ,500 3,547,700 +56

Check 4 41 52 +27 1,616,000 3,151,400 +95

Baldwin, FL (Interstate 10)

Amdro 4 85 109 +28 3,989,500 8,760,400 +120 -' Check 4 116 122 +5 6,367,500 10,334,90Q +62 -' C"\

~ Maximumreduction in active nests following all treatments ranged 60-100%; bait applied by helicopter; application rate 6 to 9 9 of AI per acre at Baldwin, FL and 5 to 8 g AI per acre at Homerville, GA. Table 3. A comparison of IFA ~7pu1ations pretreatment and 27-36 months posttreatment with toxic baits applied to pastures.-

No. of Avg. no. of nests per acre Percent Avg. no. of ants per acre Percent Treatment plots Pretreatment After 27-36 months change Pretreatment After 27-36 months change

Jasper, FL

Andro 3 65 32 -51 4,565,000 2,362,800 -48

EL-468 , 3 60 37 -38 3,798,500 2,705,400 -29

Check 3 64 43 -33 4,472,300 3,331,000 -26

Earlton, FL

A'idro 5 48 26 -46 --, 2,452,700 2,012,500 -18 --' -.....: Check 1 54 26 -52 1,911,200 1,932,200 +1

~/ ;~aximumreduction in active nests following all treatments ranged 60-100%. Bait applied with ground equipment; application rate of Amdro4.3 g AI per acre, EL-468 8 to 16 g AI per acre. 118

140 , \ " \ I \ , \ 120 ,- \ I~ \ , \ ,e ", " \ 1"£ // \ \ 100 I~ / \\ II \\ I / \\ II \\ en II \' ~ 80 II \\ en II \ \ w z II \ \ -1 I , \\ ~ I , \\ 060 II \\ ~ II \\ I , \\ I , \\ II \\ 40 I , \\ II \\ I , \\ II \\ , I ' , II \ 20 II \ \ ! l Checkplot --.1 I - ,, ------\ \ 0 0 20 40 60 80 100 120 TIME AFTER BAIT WASAPPLIED (WKS) Figure I. Reinfestation rate in plot treated with Amdro based on nests. (One- half acre subplots) 119

22000

20000 I I I 18000 I I 0 I J 0 I 16000 I -)( I z I 0 '- I- 14000 ., / ~ 12000 ," I I 0- I / ,. CheCI

USDA/ARS Insects Affecting Man and Animals Research Laboratory P.O. Box 14565 Gainesville, Florida 32604

120 121

Duirnal and Seasonal Differences in Red Imported Fire Ant Roadside Abundance

Daniel P. Wojcik, R. J. Burges and C. M. Blanton

USDA/ARS Insects Affecting Man and Animals Research Laboratory P.O. Box 1~565 Gainesville, Florida 32604

Solenopsis invicta Buren, the red imported fire ant (RIFA), is a pugnacious, immensely successful invader of disturbed habitats throughout the southeastern United States. In many habitats where it occurs,

RIFA is a keystone species, that is, it may influence community structure.

Like its congener, S. geminata (F.) (Risch and Carrol 1982, 1983), RIFA dominates the ant fauna numerically and may affect other arthropod populations as well.

One reason RIFApopulations increase so rapidly may be their foraging success, which includes the ability to locate potential food resources, recruit foragers, and displace competitors. This study addresses this option by monitoring patterns of RIFA foraging at roadside baits throughout the day and year. Timing of foraging may determine or depend on 1) availability of prey and other resources; 2) activity patterns of dominate or aggressive ants or other "enemies"; and 3) temperature or rainfall. The last two alternatives are considered here.

The research was performed as part of a USDA study on long-term changes in Gainesville ant fauna as affected by urbanization and the increase in Rlf"J\ pOlJulations frOll1 1972 to the prf'sent.

------_._-_.._------Materials and Methods

Sampling was carried out at 9AM, 3PM, 9PM, and the following 3AMon

10 dates from July 1977 to July 1978. One honey-agar cube and one small 122 hamburger meatball were placed one to three feet apart on one inch by one inch aluminum sheet squares (Wojcik et al. 1975) and set out at each of 24 sites along Williston Road, Gainesville, Alachua County, Florida.

For each time and date sampled, the baits were located at the same 24 sites. Baits were collected after one hour, ants were preserved in alcohol, sorted to species, and counted.

Analysis of variance (ANOVA)was performed on the following response variables for RIFAand other dominant ant species: 1) number of occurrences, i.e., number of baits at which a species occurred;

2) percent of total occurrences of all ant species;

3) number of specimens collected;

4) percent of all specimens of all ant species. The model for ANOVAattributed variation to the following factors:

Time of Day Date

Type of Bait (meat or honey)

Time of Day * Date interaction

Time of Day * Type of Bait interaction

Date * Type of Bait interaction

This model accounted for 87% of the variation in the number of occurrences and 91% of the variation in the number of specimens of RIFA at the baits.

Pearson product-moment correlations were used to compare total

numbers and percents of occurrences and specimens of each species with

those values for all other species and with air and soil temperatures,

relative humidity, rainfall, and total radiation. 123

Computerized data analysis was performed using the Statistical

Analysis System at the Northeast Regional Data Center, University of

- Florida, Gainesville, Florida. Climatological data were provided by Dr. Frank Gardner, Department of Agronomy, University of Florida, Gainesville, Florida. Results and Discussion

The number of occurrences of RIFA at baits are given in Fig. 1.

The greatest values for RIFA foraging are usually for nighttime sampling

(9PM and 3AM). The lowest values for RIFA foraging are usually for

sampling at 3PMand during the winter months. RIFA occurs approximately

twice as often in the summer as in the winter. Fig. 2 summarizes the

four response variables analyzed for RIFA. The percent of RIFA occurrences at baits are given in Fig. 3. Relative to the other ant species sampled, RIFAforages: 1) more at night than in the daytime, 2) more in March

than any other individual species. This behavior may give RIFA a head

start on spring population buildup. The daily peak occurrences of three dominant species of native ants are given in Fig. 4. Peak foraging of

native fire ants (NFA) (s. gemimlta) and RIFA occurs in the warm summer months. Their occurrences at baits are not correlated because NFA

forages most in the daytime and RIFA most at night. Peak foraging of

Pheidole dentata Mayr and Prenolepis imparis (Say) occurs in the fall and

winter, respectively. They may be less likely than NFA to compete

directly with RIFA for food. The temperature data are summarized in Fig. 5. No correlation could be found with relative humidity, rainfall, total radiation, or soil temperature. Fig. 6 summarizes the significant correlations found in this study. 124

References Cited

Risch. S. J.. and C. R. Carroll. 1982. The Ecological Role of Ants in

Two Mexican Agroecosystems. Oecologia 55:114-9.

1983. Effect of a Keystone Predaceous Ant, .':"/"I/,'!';;/;; :1"/11/1/"(<1,

on Arthropods in a Tropical Agroecosyst~". Ecoloqy G3:1979-i)3. -

Wojcik. D. P.. W. A. Banks. and W. F. Buren. 1975. First Report of

'Pheidole moerens in Florida (Hymenoptera: Formicidae). Coop.

Econ. Insect Rep. 25:906. 9

8 *

7 -« 1W - -~ 6 0 * * 0 0 C8 cu ,,'

rn ""(" ;/k. ,>,'1-"- ~ 5 ;#~ c * ~f-a§ta;"8* U"1 ... ''&", $' "'~,. ~ 3 -« ~ <' ~ -« "<;""& ** ". ,{<> * E "'<; -2' ;:, z """..f 2 -« * *

0 AUG SEP

FIG. 1. DAILYANDANNUALPATTERNSIN RIFA OCCURRENCEATBAITS, JULY 1977-1978. SYMBOLS REPRESENT TIME OF DAY SAMPLED: 0 = 9AM,*= 3PM,'. = 9PM,*= 3AM. Line connects means for each sampling date. TIM E B.l.fA 1500 0900 2100 0300

X Number Occurrences 1.95 2.26 2..45 2.45 ~~~~~ ~a~aaaoc-~ .

x Number Specimens 185 352 376 448 ~ ~ -' N m

% Occurrences 3.6 8.9 11.2 12.4 5Zml9~~ \~

% Specimens 9.9 18.8 23.2 28.4 ~ ~~goOQOQ

FIG. 2. DIURNAL DIFFERENCES IN RIFA FORAGING AT BAITS. VALUES ARE MEANS COr1PAREDVIA DUNCAN'S MULTIPLE RANGE TEST, CALCULATED ON DATA FOR ALL BAITS (n=48) ON ALL SAMPLING DATES. VALUES UNDERLINED BY THE SAME LINE ARE NOT SIGNI~ICANTLY DIFFERENT (a=O.05). so * .

40 - "iij D * - t * co30 : I * Co) C . GI * * . * 0 -' . N * . '-J !Jco . .. 0 * * t o ' 0 * 0* 0 Q . 0 0 Q.L! * * * * * JUL I AUa I SEP I OCT HOV DEC JAH FEB MAR APR MAY .!UN JUL 1977I 1978 Date

FIG. 3. DAILY ANDANNUALPATTERNSIN PERCENTRIFAOCCURRENCEAJBAITS, JULY 1977-1978.

SYMBOLSREPRESENTTIMEOF DAYSAMPLED:a = 9AM,~= 3PM,. = 9AM,~= 3AM. 24 G " .,,..~ :\G...G., 20 :: ~ ~ ",.' .'.'. '"

:.. "" G - : G "" ..' 'cu 16 " - .Q !, , - '.: "G ./ co :, .- I/) ; ~ / Q) ; I I ',' ... ,.' u G: I I ,\... "'... G G... c ~ : ~ 12 ' . ... I \ """'''''''' I :J U U 0 /\ D I I I \\~ -l-D_-l1 . N D------D co - D\ I .~ I 0 \ I \ 1 ... ' Q) 8 I \ D I \ .' .Q D-rf \ I .\ i E ( ~ " ~ , . z \' .I 1 ..'.., \ ,. .,,.., 4 ~,. : .,,. .,,. .~aI _I 0

FIG. 4. DAILY PEAK OCCURRENCEOFNATIVE ANTS AT BAITS. G = Solenopsis geminata, D = Pheidole dentata, I = Prenolepis im~aris ..... '" "" '" '" "'''''''~''''''''' "'" "'''' '" '" '" ..... '" '" '" ""'", '" '" ".. "" '" ".. '" ".. '" ".. '" "" "" '" ".. Q) '" ".. '" ... * 1< .....'" '" '" .a 70 ".. .;""'" *,,,,..,-t< '" '" '" 1< '" * 1<1< ~"'''' tV "..'t t< "" ~ ... '" '" '" '" '" Q) '" "" 1< 1< Q. 60 '" '" '" "" 1<", E ".. '" '" "'~ Q) '" - 50 '" 1< 1< c: ".. '" 1< N tV ".. '" '" 1<* 1< <.D Q) 1< ~ 40 ... '" '"'" 1< '" ~ ",1< 30

20 AUG SEP

FIG. 5. WEEKLYMEANSOF MINIMAXAIR TEMPERATURES,GAINESVILLE,FL, 1977-1978. STARS INDICATE WEEKLYMINIMAX TEMPERATURESATFIVE FEET ABOVEGROUND. TRIANGLES SHOWMINIMAXTEMPERATURESONDAYSTHAT ANTS WERESAMPLED. Significant (ex: 0.05) correlations among occurrences of major ant species and climaticfactors. Positive correlation = +, negative correlation: -, and blank.: non-significant correlation.

* 8.g. P.d. P. i. Temp Rainfall

8olenopsis invicta - + - --' w 0 I Solenopsis eeminata - - + Pheidole dentata -

Prenolepis imparis -

*( 7 ~ rev i0 U s day s )

FIG. 6. SIGNIFICANT CORRELATIONS. A Technique for Marking Fire Ants

Daniel P. Wojcik, R. J. Burges and C. M. Blanton

USDAjARS Insects Affecting Man and Animals Research Laboratory P.O. Box 14565 Gainesville, Florida 32604

Markal@ball point paint markers were used to mark RIFAworkers. Dots of blue, red, white, yellow, orange, or green paint were placed on seven individual workers (one color per ant). Forty-two ants were marked in each of six queen-right colonies for a total of 252 marked

ants. The ants were grasped with forceps and the top of the gaster

touched into a drop of paint. The marked ants were returned to the ~olony while the paint was still wet. The colonies were checked daily for three days, then biweekly until less than one percent of the marked ants were recovered in two consecutive

examinations. The marks were slowly lost over a 100 day period with one mark lasting 119 days (Fig. 1.). No mortality could be attributed to

the paint. No attempt was made to mark newly-eclosed workers. Large

chips of paint were rarely found (three times). Marked dead ants were

found nine percent (23 out of 252 workers) of the time. No differences were noted in the longevity of anyone color.

~j This paper presents the results of preliminary research only and will be published later through established channels with appropriate editing. The paper and data should not be referred to in literature citations until they appear in press. Mention of a pr~prietary product does not constitute a recommendation by the United States Depart- ment of Agriculture nor does it imply registration under FIFRA as amended. 131 TOTANTS\ 45 44 43 ~ 41 40 39 38 37 t 36 t 35 t 34 t 33 32 31 30 29 28 27 26 25 t 24 t 23 t 22 t 21 t 20 19 18 17 W N 16 15 14 t 13 t 12 t 11 t 10 t 9 8 7 6 5 4 3 2 1 ~ 0 \ --t th_--t--- --th_--t-_h_t t t t t- t t -- -- -th_- -t t t t t t-- -- -t -- ---. f r , . 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 Icor Iff nO IIf ItS!

DAYELAPS

FIG. 1. SURVIORSHIPOF MARKSON RIFA WORKERSMADEWITH [email protected](A, B, C, D, E, F) REPRESENTSTHETOTAL SURVIVING MARKSOF ALL COLORSFORTHAT COLO~YONA GIVEN DAY. COUNTS WEREDISCONTINUEDWHENA COLONYHADLESS THAN 10% SURVIVING tlP.C«SIN 2 CONSECUTIVEEXAMINATIO~~S. "",""'11'.1 ",,'" II".I\MIJ

Hematologic and immunologic responses of Holstein calves to a fire ant toxicant *

Donald L. Evans, PhD; Karen L. Jacobsen, DVM, MS; Doris M. Miller, DVM, PhD

SUMMARY Pn'violls st,lIIlil's':' h;,v(' shown ),hat IIll'nt was ass('ss(.d hy hacille Cal- Ilolstein ('aUIl' an' s('nsitive immu- met.t.e-(;ucl'in vaccination and Holstein calves (;~ to [) months of nologically to the deleterious effects subsequent skin challenge with pur- age) were used to develop an animal of certain environmental toxicants. model sensitive to environmental ified protein derivative (PPD) of My- Only a few studies of toxic syn- cobacterium tuberculosis. toxicants. In the present study, the fire ant toxicant AMDRO was fed dromes in large animal species".7 have been conducted. Cattle exposed (113.5 g/day/caID to weanling cas- Materials and Methods trated calves (9 test and 9 controls) to polybrominated biphenyls (1'1313) developed changes in certain im- Animals-Eighteen Holstein bull for 7 weeks. As early as 14 days after mune response variables during calves between 3 and 5 months of age the start of the AMDRO feeding, treatment." These studies demon- and weighing between 75 and 87 kg were leukopenia was observed. Differen- purchased from 2 farms. Calves were ex- strated that the bovine species may tial counts revealed significant non- amined (KLJ) on the farms before pur- be an appropriate sensitive model for transient decreases in lymphocytes chase and were healthy, normally immunotoxicologic studies. and eosinophils. Eosinopenia was developed calves. All calves from a given Presently, AMDRO is widely used brm Wl're fed idl'ntical rations bl'fore observed from days 21 to 49 of as a lin' ant insed.icid(' in UH'south- l'ul'cha,.;p. To minimizp I'o,.;,.;ihlp non- AMnRO tn'atnwnt.. Variahility in eastl'rn and sOllthwl'stl'rll lJnitl'd t n'atmpnt dint'l'plll'(''';, calvp,.; W('J'(' a,.;- lll'matOtTit and hemoglohin valtu's States. Acconling- to the AMDl{O ,.;ignl'd to tl'catnwnt and contl'Ol groups, in treated and control calves pre- technical infimnation bulletin,H this ";0 that the 2 groups were nearly iden- cluded making a determination of compound is poorly absorbed by tical in ages and weights of the animals. trends due to toxicant exposure. mammals, and more than 95'/' is ex- Equal numbers of calves from each farm The AMDRO treatment did not were assigned to each group. Within these creted unchanged in the feces. Other produce significant decreases in than the technical infiwmation bul- constraints, the calves were randomly primary or secondary antibody allotted to the 2 groups. Calves were cas- letin, the scientific literature is de- responses to keyhole limpet hemo- trated 2 days after they were purchased void of publications regarding the and 45 days before entry into the exper- cyanin or to Brucella abortus vacci- effects of AMDRO in mammals. nation. It also did not produce iment. Deworming (1.1 g of morantel Large-scale applications of AMDRO tartrate"/caID, dusting for lice,b intra- suppression of cellular immunity, as are being proposed to attempt erad- nasal vaccination against infectious bo- determined by delayed-type hyper- ication of the imported fire ant (IFA), vine rhinotracheitis and parainfluenza sensitivity response to bacille Cal- type 3 viruses," and vaccination against mette-Guerin. Potential effects of but published reports regarding short- or long-term effects of any fire 7 species of clostridial organisms" were AMDRO on thermoregulatory mech- pprf(JI'ml'd at thp saml' time as castra- ant toxicant (including- AMnRO) on tion. Twicp daily, calvl's Wl'I'l'fl'd a 16(';' ani;;ms were indicat(~d by significant donH'stic animal lH'alth an' non- depressions of I'edal temp('rature (Tudp protein grain ration without addl'd existent. lmmunotoxicologic studies observed after 25, 32, and 39 days' antibiotics" 10.675 kg/caIn and ti'ee choice treatment. are clearly needed to determine pos- coastal Bermuda grass hay. sible biological effects of the IFA eradication campaign on the health AMDRO-A total of 113.5 g of of exposed animals. AMDRO' in a corn grit base; adminis- In the present short-term study, " N.'mall'l cattle wormer boluses, Pfizer Ine, weanling Holstein calves were Nl'w York. NY. Received for publication .June :10. I!lH:\. treated with the fin' ant toxicant " I{ahon fly and Iice dust. From Ihe Departments of Medica( MilTohiol. . IlIIt-PI.:1 Van'i,\\'. Bioceutic Laboratories. S( °I:Y IEvans. Jacohsenl. Larl:e Animal M..dimH' AMDf{O. Several clinical, hemato- .Ios,'ph. Mo, I.Jacob,,'ni and Patbolol(y (Millerl. (:011«1(1' or logic, and immunolog-ic variables ., llltrahac-7. Bl'echam Lahoml.ol'i.,s. Bristol. VeIPrinary Medicine. University or U"OI'I("" ""'1111. Athens. GA 30602. were measured in the treated and . Purina calf startina. Ralston Purina, St Louis. Supported by Veterinary Medical Experin1l'nl control calves. The in vivo effects of Mo; did chanl(t

., ,1('\.1 . Control E 6000 E 0 Treatment "- 10POO ~ a; '" 300 U '" E E 5000 I E "'- "" E 0 8000 ~I* If) 0 " If) CD '" ~0 200 >. 4000 co '" 0 ilo 6000 0 .s:::: 0 "E a. W :;: .ControlE . Control 100 :~ 3' , a Treatment 3000 ~ OT r I, , , , , I , i 0 Treatment -2 5 14 21 28 3542 49 OT , ,II, , , , , , Days Treatment -2 5 14 21 28 35 42 49 0 -r--rII,~ , , , , , 2 -2 5 14 21 28 3542 49 Days on Treatment 3 Days on Treatment Fig 1- Total WBCcounts determined in both control and AMoRO-treated calf groups. Signif- Fig 2-Absolute lymphocyte counts determined icant differences between the groups were at at various times during treatment with AMDRO. Fig 3-Effects of AMDRO treatment on absolute days 21. 28. 35. and 42. Data points with as- Significant differences in numbers of lympho- numbers of eosinophils. Differential counts were terisks indicate significant difference from con- cytes occurred after 14. 21. and 35 days' treat- done for 49 days during AMDRO treatment. On trols at P < 0.05. ment with AMDRO. Data points with asterisks days 21 through 49 significant differences were indicate significant difference from controls at P observed. Data points with asterisks indicate < 0.05. significant difference from controls at P < 0.05. tered orally with a commercial horse powder gun, was given to each of 9 calves once daily. Control calves were given corn Bruc('lIa ahortlls pa('cinatioll-Bru- TABLE 1-Skin test response to intradermal PPD grits" (without added AMDRo) admin- ('('//a ahortlls strain I!)'" vaccinp was in- inJ~~ion':" H____-- istered hy the same method. jPclpd suhcutarH'ously in thp cprvical (',011' Skin induration gl'llllp (nll'an nlln sill n.gion. Card tpsts, tuhe agglutination, - '---- . Jl('mato!(}~ic ('xa 11/ilIatioll-Peripheral ( ~onll'lll II;.!!? ' 1.% Rivanol prpcipitation, and complenlPnt TI'"al"d Hi.!! ' l.or, blood was collected

1024 Am J Vel Res. Vol 45, NO.5 U!J

34 . Control . Control 103 a Treatment 0 Treatment

~ 32 3 102 ~0 ..... 2 0 f= <1J E >- Q) "0 .... ~ 30 0 2 ::J 0 .D 101 > "6 .... c Q) Q) <{ 2° Q. u 28 I E ~ 100 . Control "0 0> ..L <1J 0 0 Treatment .:£ ....J U 0 oIi I, II , Q 26 " 25 39 at 7 14 21 28 35 42 7 1° Treatment 0 I, IIIII Days Post - Inoculation Days on 5 14 21 28 35 42 49 6 4 (KLH) Days on Treatment Fig 7-Effects of AMDRO treatment on body Fig 6---Responses of control and AMDRO-treated temperature. Treated and control animals were calves to KLH.Antibody responses (primary and monitored during 39 days treatment with AM- Fig 4-Hematocrit determinations of AMDRO- secondary) were determined during treatment ORO, and after approximately 3 weeks on AM- treated and control calves. Values were ob- with AMDRO. At primary sensitization, calves ORO, treated calves' temperatures were tained from 5 to 49 days of treatment. Signifi- had been treated for 7 days with.AMDRO. Pri- significantly lower than those of the controls. Data cant differences were observed at 0, 5, and 35 mary response at 14 days after inoculation is points with asterisks indicate significant differ- days' treatment. Data points with asterisks in- different from controls (p. 0.10). ence from controls at P < 0.05. dicate significant difference from controls at P . 0.05 was a trend for smaller hematocrit sponses were obtained. Peak primary values in the treated group than in anti-KLH responses for both AMDRO- the controls. Hemoglobin concentra- treated and control calves occurred tions manifested a similar trend (Fig 14 days after sensitization (Fig 6). II G); till' l1l'moglol>in valul's in tl1I' I'l'ak antibody response of the con- tn'atl'd gmup at 0, ;:,and :~;:,days wpn' trols at this time was greater than significantly Ipss than those of the Ihl' treated calf group (P <. 0,10), controls IP "'0 O.OG). Significant differences were not ob- 10 served at any other time (even after Serum proteins-Serum fibrino- secondary challenge), ~ gen concentrations were signifi- Serum anti-Brucella titers (com- 0> cantly IP < 0.051 different between plement fixation, Rivanol, card and ~ 9 contml and trpatpd groups only dur- tube agglutination) determined for 6 c: :D ing w('l'k Ii (1.111'p('riod of maxillllllll wp('ks aller inoculation with B ahor- 0 cllang('s in 1I('ll1alologic valll('s). Ills indicated that tn'atrnent titers g 8 'I'.'l'atn1l'nt val ups (mean - 4HH.HH were not significantly different from E .t- 59.3H g/dl) were significantly <1J . Control those of the control calves (regard- I larger than those of the controls less of the test) at any time during 0 Treatment (mean ~ ;{;38.8H ' I:3.8 g/dl) during the treatment period (data not 0 I, this timp (p. (I.OG), shown L 5 14 21 28 35 4249 5 Plasma protpin conl'l'ntrations, Days on Treatment IIIl'asllrpd each wepk during the Cell-lI/ediated immlillity-Table 1 treatment period, did not signifi- gives the total mean score results cantly differ between treated and (skin induration) and contains no Fig 5-Hemoglobin determinations made at control calves Imean = 7.05 g/L vs differences between the treated and various times during treatment with AMDRO. mean = 7.04 g/L, respectively I. Significant differences occurred after 0, 5, and control calf groups. 35 days' treatment with AMDRO. Data points with asterisks indicate significant difference from Serum 19-5era IgGl and IgG2 Physical examinations-All calves controls at P --::0.05. concentrations of both calf groups remained clinically healthy were not statistically significantly throughout the study. peri mental period, were sil,rnificantly different over the 7-week treatment Rectal temperatures at 25, 32, and different between treated and con- period, The mean -'- SI';Mf()!' thl' con- :39 days were significantly lower (p trols only during the 1st and the 5th trol calves for the period was 2,259.25 '- 0.05) in AMDRO-treated calves treatment weeks. At 0, G, and ;~G I 27 mg/dl, and that fiJl'thp tn'atPd than in control ('alves (Fig 7). days, Iwmatocrit valw's wl'n~ signif- calv('s, ~,:107AII I :m.H'1 mg/dl. icantly It,ss fill' tn'at.l'd c;a!VI'S t.II:11I N('('("()!ISV ('.rallliIIlIIIOIlS ~igllifi- for ('ontn.ls (I' 11.11:>; Fig ~). :\/lllhody jill'/lllIlio/l Both pri (";III t. gross It'siolls wI'n' 1101. pn's('1I1 'I'll rougllou I. till' ('x p(~ri nWII t, tllI'n' IIlary alld sl'colHbrv allti-KI.II 1'1'- 111 1.1"1'0111111'111.vs cOII!.rol calvI's. Mi-

May 1984 1025 136

croscopically, all animals had hyper- curred 14 days after inoculation. Such central physiologic defect. Signifi- plasia of the bronchiolar-associated similarities supported the conclu- cantly lower body temperatures at 25, lymphoid tissue and prominent lym- sion that the present control calves 32, and 39 days after treatment may phoid follicles in the spleen, lymph were from a representative popula- indicate a malfunction in thermore- nodes, thymus, and aggregated lym- tion. gulatory mechanisms involving the phatic nodules (Peyer's patches!. Hematologic data indicated that hypothalamus. Other investigators There was no premature degenera- AMDRO-treated calves had signifi- at our institution" have noted var- tion of the thymus in any animal. cant Ipukopenia. This occUlTPdfi)r 4 ious neurologic effects of AMDRO, Hepatic lesions were present in ~ conspcut.ivp wppks du ring the px!>pr- including convulsions in swine and AMDRO-tn'ated calves. Focal mi- inwnt.. From :kd t.hl'Ough()t.hwpeks, intolerance to heat stress in dogs. croabscesses, consisti ng of hepatic cell AM()I{O-t.n~at.pd calvps WPI'P Ipuko- Additional studies on the effects of necrosis with a neutrophilic infiltra- penic (Fig I L Th'p primary causp of AMDRO on the CNS, including his- tion, were present in these ~ calvps. this leukopenia at. ~l and :Hj days topathologic examinations, are war- was dup to lymphocytP dpcn'asps (Fig rant.pd. Because these changes were Discussion ~L Treated animals also were Iym- obsprved relatively soon (approx 3 weeks) after the start of the AMDRO The bovine species has been pre- phopenic as early as 14 days after viously shown to be an appropriate initiation of treatment. An extended treatment, the potential neurotox- sensitive animal for immunotoxicol- period of eosinopenia (for 5 weeks, icity of this chemical justifies fur- ogic studies. Kateley and Bazzellfi from day 21 to day 49 of treatment; ther investigation. examined many immune variables Fig 3) also was observed. Although In many southern and southwest- in cattle exposed to PBB, and evi- cortisone values were not deter- ern states AMDRO is used widely as dence of immunotoxicity was not ob- mined; these responses could have a fire ant toxicant. Approval of AM- served. Others2 demonstrated that been due to long-term endogenous DRO by the Federal government is dairy herds exposed to PUB(placed glucocorticoid release. However, conditional at the present time. Pre- in the feed) developed toxicosis. Ka- serum IgC concent.rations of treat- vious investigations on the immu- teleyet al4 and Moorehead et al" re- ment and contl'Ol groups were not notoxicology of AMDRO have not ported on PBBtoxicosis in heifers, but different indicating that other fac- been reported, even though domestic significant changes in hematologic tors than stress may be involved. animals may come in contact with values were not observed. Fever was Additional studies measuring he- AMDRO while grazing in pastures not present in PBB-treated Holsteins. matologic changes indicated that or in "feed bag accidents." This sit- Gross necropsy findings included hematocrit and hemoglobin values uation occurs when animals are large kidney, mucoid enteritis, and of treated calves were not signifi- housed in the vicinity of palatable secondary pneumonia." Although PUB cantly different from those of control toxic substances such as pesticidal and AMDRO are not chemically re- calves (except for 1 pretreatment and baits. Preliminary studies have lated, both compounds have now been 2 treatment points). However, a trend shown that the AMDRO-containing studied in the same model system toward smaller values for treated corn grit bait is palatable to cattle; and, as such, provide evidence for the calves was observed (Fig 4 and 5). and others" have demonstrated that biologic validity of this model sys- It seems that AMDRO may selec- the bait also is palatable for dogs and tem. In the present study, necropsy tively affect production of immuno- horses in small amounts. The dosage examinations revealed no major competent T and B cells, rather than of AMDRO used in the present study pathologic changes. However, there depress differentiation and func- may be larger than calves would en- were several hematologic and im- tional maturation at a peripheral site. counter in a similar time frame in munologic changes. Additional evidence. to support this field conditions (except in geo- Base-line values for an "immuno- idea was provided by the studies on graphic areas where grazing over competence" profile on Holstein cat- anti-KLH and B abortus responses, large areas could occur). However, tle have been determined by others. I and also by the delayed-type hyper- information provided in the present Hematologic values for controls ob- sensitivity results. Except for the study should point out the need for tained in the present study did not depression of KLHantibody titers at appropriate caution to avoid barn differ appreciably from those re- 14 days after inoculation, no differ- accidents, feed-mixing accidents (such ported by Kateley and Bazzell.I Con- ences were observed in any immune as occurred in Michigan with PBS), trol hematocrit values in the present response variable measured. Like- and chronic exposure of grazing an- study were between 27.8% and 33.8%. wise, anti-Brucella titers were not imals to AMDRO. This fire ant bait These were slightly different from the significantly less in the treatment which is absorbed percutaneously, 28.4% (min-max, 21.6% to 34.6%) group. If nonspecific endogenous im- undoubtedly also will be handled ex- value previously reported. I Like- munosuppressants (pYl'Ogens, glu- tensively by farmers and eventually wise, our hemoglobin values of 9.9 cocorticoids, prostaglandins) were by homeowners using the product to 11.9 gldl compare with published I active during AMDRO treatment, against fire ant garden pests. values, 8.4 to 13.1 g/d\. Also com- then B-cell andlor T-cell responses The lymphopenia-eosinopenia re- parable were a mean serum Ig (lgG 1 should have been concomitantly de- sponse observed after relatively short- and IgG2 combined) concentration of pressed. This did not occur. term treatment may produce dele- 2,259.25 mgldl (previously reported I Rectal temperature measure- " Dr:;. Caudle and Coulter, College of Veterinary 2,745 mg/dl) and the finding that ments perhaps supported the con- Medicine. University of Georgia: Personal commu- peak primary anti-KLH titers oc- cept of AMDRO producing a subtle nication. 1983.

1026 Am J Vet Res, Vol 45, NO.5 IJ/ terious consequences f()r a parasite- bl'Omi,,,,I.ed biplu'IIyl,colllamillal"d prol<.ill lion and liver biochemistry. I,'I//Jiron I/enllh cOllc,'nlral<' 10 dairy calLie. oj Alii V'" M..

*Reprinted by written permission of the authors and t:le editors of the American Journal of Veterinary ~esearch. References to this article should cite the original publication.

May1984 1027 lIi~111ir:hts of Hesearch with Pheromones anc~ :iehaviors

B.Michael Glancey, I SDA,ARi>,Imported Fire Ant Laboratory

Gainesville, ~lorida

1. Trailinc Studies

Dr. Vandert':eer'sr.roup has shown that the trail pheromone consists of both

-('arnesenes and homofarnesenes. Laborato:r;rsynthesis of the various isomers was

recently completed and we tested the comnounds usinr; our standard trailinr; biol.ssay

and these are the results (Glil1e). 'They look real ~ood, <'specially in lif,ht of

the fact that the ants will trail only those compounds that occur naturally.

2.Effect of the lluality of lir;ht upon the trailinr; response.

:..twas suggested to us that the quality of lir;ht ,.,illaffect the trailinr

response as much as the quanity of lic:ht. He therefore tested the effects of whi te, red, ;rellow, 'green, blue and infra-red upon the response of the ants trailing either to food or to their Queen. l'hevarious lir.hts ",ere suspended

about 11 inches over the trail, and the remaider of the lights turned off. ',fter we observed the response to the food, lhe mothe~ queen was ~laced in the arena

a~d the Rnts resr-mse noted. ~e results' ::;lide)show that the an"-s were more

sensi ti ve to '-,hequeen than to the foc ~, '1110that the in fra-re'l part of the

spectrum may indeed playa role in the trailinr; response.

3.Studies with multiple queen colonies.

Studies have been iniated to determine behavioral differences between

T'mltipIe <1ueen colonies collected from h senarate r;eor:raphical locations...

Texas, Mississippi, Georr;ia, and Florida.',-ehave found that multiple queen workers

Gueened with one of their own queens will kill newly ~ated introduced queens,

l'utHi] 1 accept more of therr own queens. ~~if;tcrs of thc;e .Torkers, ir r.ot queened (slide) '-Tillaccept a newly introduced queen, ',11[: !\'lviW~ accepteLt her,

138 139 are prone to accept more alien queens. Tf the colonies rejected the rirst queen thcy wi J1. re,ject ~,l1l):,ef1l1ent queens. It mir:ht be that the colenies are reactinr' to the Queen odor. Acceptence of newly mated queens mir:ht indicate an odor relationship between queens, since the queens all came from the same matinG flight and its possible that they might have even come from the same mound.

Or it ~ight indicate a disposition on the part of the workers to become more tolerant of additioanl queens. This is what ~ie;ht be hanpening with the Georgia colonies. 'i-Iesuspe.t that .palyp;ny has only reeently ber:un in Georgia, and that the workers have not built into the system a mechanism for acc!,=ptingmore queens whereas the older polYF'",ynous areas have done so.

:f.:~perm counts as determined automatically with a Coulter Counter.

':Iehave been able to automatically determine the inseminatiion rates of newly mated qaeens, physor;astric queen, laboratory reared queens, rmltiple queens from 4 geographical areas, nales, and virgins of the RIFA and counts of the black i~ported fire ant from t.1issississippi.'ihe followinr; slides show the results. Our counts give much higher rates than those reported by Ball and

Vinson.\lso, we discovered that we could ascertain the age of the queen up to 3yr by means of how quick the spermathecal contents dissolved in the solvent.

:e also disoovered that the "rope" :1Sseen in the seminal vesicles or sperma- theca is not necessarily an indicator of insemination. iome spermathecae which did not have the "/.ope" had counts of 2-3 million sperm. Finally, we found in both the red and black form, the males sometime have only one semiLal vesicle, lmt that one has enoup;h sperm to completely inseminate the female.

5. Polyethism

Our final report has to do with the division of labor or polythethis~. . iow

depending on who you believe, Vinson/Miranda or Wilson, ;'ouwill have a problem or describinG the division of labor. Vinson et al believe that age causes

defines the lal)ors while Hilson believes that si ze does. 11 ':e taken Vinson's 140

'\-lork and with these (slides )visual cues, put un the various labors he has found for each of the varous castes: brood tender, reserve, or forager.

He were interested in how the varous castes responded to a number of the pheromones and so we ran them through the various bioassays we use and these are the results.:e do have 1 separate castes and they do re::mond differently to the vari~us pheromones.

Our future work will focus on the neurochemical correlates of learning, olfactory conditioning and the effect of size on the response to the pheromones. Response of ants to synthetic homofarnesenes

No. ants responding in 1 minute

1000 pgl cm 100 pg/cm 10 pg/cm 1 pg/cm

...... ISOMER Colony 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 ......

E,Z 0 '-

...... Z,Z 0

"-- Z,E* 4 4 4 4 4 2 2' 3 1 0

...... E,E 4 4 4 4 4 0 0 4 0

'* Naturally occurring isomers 142

1 Effect of wavelength upon the ability of 4 ant colonies to trail Dufor's gland extract laid toward either a food supply or a queen.

No. of positive responses

2 DE IR Red Yellow Green Blue White Food Queenl Food Queen Queen i Food QueenlFood - Queen IFood -/- QueenI Food 4 4 i 4 4 I 4 4 4 4 4 4 I 4 4 I I I .1 4 4 4 4 4 4I 4 4 4 4 ! 4 4 I

I I .01 1 3 I 0 2 0 1 0 1 0 1 1 0

I I .001 0 0 I 0 0 I 0 O ! 0 0 0 0 I 0 0

lall lights 60 watts. IR = 6 watts.

2Dufour Equivalent SPEP2.; COU::;'='SOF 94iIEWLY }..1ATEDRIFA QUEmS AS DETERHnTED BY A COULTER COillITER

cou::r.:;s PT ;,:ULIONS ;TU'.~ER OF QUEENS PERCENTAGE

0-1 0 0

1-2 3 3.2 2-3 3 3.2

3-:" 2 2.2. 4-5 6 6.4 5-': 40 43 6-7 39 41

~ 7-= 1 1 w

X of 04 oueens = 5,669,683 !. 118,148

Range: l, l49, 300---7 ,228, ()36 SPEPJ-1COUNTSOF 9u COLONYREAREDRIFA I'IALES

Sperm count in mi} lions rumber of males Percentage

0-6 0 c

6-7 3 v'-'< ?

7-8 19 .28.4

8-) 38 L8.J

9-10 24 25.:

7 ~ 10-11 7 I '';

11-12 1 , - +0> ~ ~. 12-13 0 v

13-14 1 1.::'

~ 14-15 0 ..J

X of 94 males = 8, 761,637 ~ 36, ')95

Range: 6,155,018--13,299,525 SPERM COU;ITS OF FIELD COLLECTED P:trYSOGASTRIC RIFA QUEENS (UEK; # X SPERHcounT SE

1 551, 94 907

2 1,321,101 4,988 3 1,(89687 2,604

4 2,n9,345 7,428

5 2,470,140 3,297

6 3,J29,344 2,853

7 3,335,200 7,J21

8 4,')84,284 6,661

9 4,143,897 5 , (,69 .j:::. (J1 10 4,557,337 10 , 198

11 4,903,575 5,623

12 5,413,825 4,654

13 5,634,883 6,135

-, SPERM COUNTSOF LABORATORYREAREDQUEENSAND VIRGB ALATES (RIFA).

:;,ueen # Flif1:!1t date Age X count SE

1 7/7/78 5.25yr 5,067,090 6,789

2 7/9/79 4.25yr 5,207,450 7,029

3 6/12/80 3.33yr 5,::':45,703 4,440

4 5/7/81 2.42yr 5,217 ,794 9,']24

5 5/7/81 2.42yr 5,218,;38 7,281

6 5/21/82 1.12yr 6,:l-05,50 6,681

7 5/21/82 1.42yr 6,219,795 6,485

8 5/18/83 .42yr 5,101,074 6,485

9 5/18/83 42yr 5,577,768 6,118 +:> . 0"> 10-19 Virgins 2weeks 0 0

All Queensex~~ned 10/6/83. ~.2X1 SPEP!.1 COUNT OF 10 MULTIPLE 0,T..2KrS FROM VARIOUS GEOGRAPHICAL A..'REAS

=-aCKTJ:r -X em;:; ':.' SE

::-LORIDA 5,428,136 513,524

GEORGIA 5,C33101 48,968 mSSISSIPPI 6,OOO,les 71,01.0

'IEY.AS 5,186,337 590,616

~ ""'-J SPE&~ COlliiTS OF FIELD COLLECTED RIFA QUEENS FROM ~IDLTIPLE QUEB1 COLONI~S

QUEEH If X SPEHM COllin SE

'}EOHGIA

1 4,)09,122 5,450

2 4,933,526 5,280

3 5,140,188 5,019

4 5,429,526 6,750

5 5,731,941 5,)01

6 5,801,205 .j::> 4,805 00

7 5,957~117 7,933

8 5,)78,342 6 , 26 2

9 5,991,200 7,494

10 6,458,239 12,139 SPERH COUNTS OF FE:'] COLLECTED- RIFA QUEEHS FRO:-: :.ruLTIPLE QUEEN COLONIES C;UEEN# X SPEffi.J COUNT SE

~OLORIDA

1 2,691,560 7,913 2 4,cno, 717 4,603

3 4,428,260 5 ,998

4 5,059,157 5,477

5 5,235,840 7,959

6 5,469,399 9,165

7 5,473 ,770 2,042

~ 8 5,556,731 7,270 \.0

9 5,563,340 7,082

10 5,802,491 5,791 SPERH COUNTSOF FIELD COLLECTEDRIFA QUEENS FRm1 MULTIPIEQUZE;H COLONIBS

~..., ~UEEN # X SPERI1 COUNT ;::'z,

-nSSISSIPPI

- 4 , 190 ,511 6,:::59

2 5,254,172 6,036

3 5,952,529 5,724

4 6,129,307 5,140

5 6,229,584 6" ~,43 ()1 a I" 0 6,241,923 8,057

'7 6,422,912 5-,2.21

8 6,~30,505 lO,25l

9 6,527,')19 15,:138

10 6,624,310 5,557 - ?ERr.! COLEITS OF FIELD COLI2C::'::J RIFA Q.U~EHS FROM MULTIPL.-=:-;;7..EE::ICOLONIES

- ~~ -'lIBEl! # X 2?EP.:~ COUIIT '-'-

'_'EXAS

720,690 3,240

2 2,595,J69 5,453

3 3,85l,151 2..,;73 I. '-' 4,=72",40 4,462

5 5,267,636 ::;-' ,j-.\.) -;;1 Q -' -'U1 /' 0 5,26e,~-01 5,303

7 5,432.,629 6,;54 ,. U 5,:77 , ~42 L,018

0 / 6,0:0,352 6 , ~30

10 6,552,534 5, -=-91 SPERM COUNTSFOR 11 FIELD COLLECTEDQUEENSOF E. ~:ICHTERI(BIFA)

Queen # X sperm count SE 1 3S36,Q02 4,732

2 5 129 ,388 10,622

3 5218,666 8,629

4 5,230,338 13 501

5 5416181 6,058

6 5,423,265 8871

7 5602,880 8O10

8 5,636,312 6,402 U1 N 9 5698,044 8757

10 5 762 428 10,318

11 5?63,819 4 179 SPE?l.~ COlnTS OF 51 FIELD COLLEC?EDS. ?ICHTERI HALES(BIFA)

SDe~ count in millions Number of males Percentage

.-~, 0 0

5-6 2 3.9

I" ..., c.-: 4 7.[

7-8 3 5.9 U1 W 8-:; 29 56.9

9-10 13 25.5

~ of 51 males = 8,428,986 ~ 9,961

Range: ),429,793--9,732615 154

POL YETHISM The division of labor

Vinson/Miranda' Age determines labors.

Wilson 'Size determines labors. BROOD TENDER (NURSE)

1. Tend brood

2. Groom queen 3. Groom self

4. Receive food

5. Stridula te

-' U1 RESERVE 1. Move to nest periphery 2. Groom self

3. Serve for recruitment

4.Remain motionless

5. Help feed larvae

6. Help to forage -' U"1 0"\ 7.. Nest" construction

8. Nest defense FORAGER

1. Forage for food

2. Recruit reserves

3. Nest defense --' 4. Experience death U"1J 0.93mm or more

HEAD WIDTH

0.93-0.73 mm --' O.72mm or less coU"l 159

AGE - ITS INFLUENCEON THE PHEROMONAL RESPONSE

CASTE MEZN NO. SECONDSFOR BROODPICK-UP II DUNCANGROUP

BROODTENDER 8.8 125 A

RESERVE 33.2 125 B

FORAGER 52.2 125 C

RESPONSEOF VARIOUS CASTES TO FOOD. ANOVAWITHa. .05

CASTE MEAN TIME FOR FOODPICKUP SE II DUNCANGP

BROODTENDER 111.60 1.2 125 A

RESERVE 108.70 3.8 125 B

FORAGER 94.40 2.4 125 C

RESPONSE OF VARIOUS CASTES TO THE QUEEN RECOGNITION PHEROMONE IN All OLFACTOMETER CASTE MEAN NO. ANTS RESPONDING N DUNCAN GROUP

JROODTENDER 42.1 25 A

RESERVE 26.6 25 B

FORAGER 18.0 25 C

RESPONSE OF VARIOUS CASTES TO A SURROGATE QUEEN AND THE QUEEN RECOGNITION PHEROMONE

CASTE MEAN NO. ANTS RESPONDING N DUNCANGROUP

BROODTENDER 10.7 25 A

RESERVE 1.9 25 B

FORAGER 4-8 25 ::; 160

RESPONSEOF VARIOUS CASTES TO THE TRAIL PHEROMJNE

CASTE MEAN DISTANCE TRAILED{cm) N DUNCANGROUP

BROOD TENDER 6.20 500 A

RESERVE 6.55 500 B

FORAGER 6.64 500 B

RESPONSES OF VARIOUS CASTES TO RECRUITMENT PHEROMONE

CASTE MEAN NO. ANTS ON SPOT N DUNCANGROUP

BROOD TENDER 6.76 150 A

RESERVE 4.74 150 B

FORAGER 3.34 150 C ?ESPONSE BF VARIOUS CASS TO DUFOUR'S GLAND IN A Y--E (Weighted percent mean).

Dufour equivalent X SE X SE Y SE

:Brood Tenders Reserves Foragers

3 95.7 1.1 96.7 3.7 88.3 1.9

1 82.7 2.1 84.7 2.9 77.8 3.1

0.3 85.6 .86 4.6 69.3 3.0 75.0 --' 0'\--' C1 72.6 3.6 83.2 1.7 63.6 loG

0.03 68.9 1.5 58.4 1.2 59.2 3.6

0.01 51.8 1.'" 1 54. 2.5 56.7 1.7 Isolation and Identification of the Recruitment Pheromones of~ Solenopsis invicta and Solenopsis richteri Francisco M. Alvarez*, Robert K. Vander Meer and Clifford S. Lofgren

USDA/ARSInsects Affecting Man and Animals Research Laboratory Gainesville, Florida 32604

*University of Florida, Department of Entomology and Nematology

162 163

ABSTRACT

Isolation of the Recruitment pheromones of Soz'enopsis invicta and SoZenm'sis Y'~:chteri Francisco M. Alvarez, Ph.D. and Robert K. Vander Meer, Ph.D., and Cliff Lofgren, Ph.D., USDA/ARS,Gainesville, Florida

Z,E-~-Farnesene (6ng/WE), heptadecane (12ng/WE) and a third component of unknown structure designated "Cl" (0.075ng/WE) were isolated from

Soz'cnop;;isinvicta. The mixture was found to elicit 85% of Dufour's gland recruitment activity when tested using a recruitment spot bioassay.

The recruitment pheromone of Soz'enopsis Y'ichteY'i was composed of two compdhents found in Dufour's gland. Component "Cl" (the same as found in Soz'enopsis invicta) plus "HPLC-4" gave 85-90% of Soz'enopsis richteri recruitment activity (1:1, 4ng/WE). This represents the first isolation of a recruitment trail pheromone in ant species. 164

Isolation and Identification of the Recruitment Pheromones of Solenopsis invicta and Solenopsis pichtepi Francisco M. Alvarez*, Robert K. Vander Meer and Clifford S. Lofgren

USDA/ARSInsects Affecting Man and Animals Research Laboratory Gainesville, Florida 32604

*University of Florida, Department of Entomology and Nematology

For the past several years our laboratory has made a concerted effort to elucidate the chemica1 nature of the imported fire ant's trai1 pheromone system. Twomajor aspects of the trail pheromone have been investigated: a) trail orientation, and b) recruitment to the trail.

The concept of trail orientation and recruitment for Solcnopsis invicta was first reported in 1962 by Wilson (1). When a foraging worker dis- covers a food source it examines the material and if the object is too large to carry, the ant will lay a chemical trail back to the home nest. The chemical trail passes from the Dufour's gland through the sting to the ground. The Dufour's secretion acts as an attractant that draws workers over short distances to the trail and then orients them to the food source. Wilson also observed that the better the food source the greater the trail reinforcement and hence greater recruitment. Eluci- dation of trail orientation pheromone components for S. invicta were reported in 1981 by two laboratories: Vander Meer et al. of the United States Department of Agriculture (2), and Williams et al. of Texas A&M (3,4). The structure of an allofarnesene (Z,Z,Z-3,7,11-trimethyl-

2,4,6-10-dodecatetraene) was confirmed by William's group via synthesis and direct comparison with the natural product (3). Likewise, two a-farnesenes (ZE and E,E-3, 7,11-trimethyl-l,3,6,lO-dodecatetraene) and 165 two homofarnesenes (Z,E and E,E-3,4, 7,11-tetramethyl-l,3,6,10-dodecatetraene) were shown by Vander Meer's group to be part of the trail pheromone via synthesis and direct comparison with the natural substance (2,5). However, very little is known about the chemicals responsible for the recruitment activity elicited by Dufour's gland extracts. We would like to report the isolation and partial identification of the recruitment pheromones of S. invicta and S. richteri. Chemical isolation of the pheromone complex was monitored at every stage by a spot bioassay designed to measure recruitment. Test solutions of known concentration were placed equidistant (~7cm) from the ant cell.

Two of the 10 possible locations were used for a Dufour's gland extract standard and a hexane control. All positions were randomly assigned. Results were quantitatied by counting the number of ants recruited to a 1.5cm circle around the sample spot every five minutes for a total of 30 minutes. The sum of the six counts constituted the result. Each test was replicated on six different colonies. In addition to biological activity, the isolation scheme was guided by gas-liquid chromatographic pattern (DB-l fused silica capillary column 100° for 10 minutes then increased temperature to 250° at a rate of 25°/minute). This proved to be useful since we could get an idea of the complexity of the active mixture. The method of recruitment pheromone isolation for S. invicta is shown in Scheme 1. Whole ants were first rinsed with hexane to minimize contamination

of the pheromone with cuticular hydrocarbons. The ants were filtered

and homogenized with fresh hexane to generate the hexane extract after filtration. The insoluble residue was further extracted in methanol.

Recruitment bioassay of the hexane rinse, hexane extract and methanol extract showed that most of the pheromone activity was located in the 166

hexane extract. Thin-layer chromatography of the hexane extract on silica gel showed a complex mixture of substances of widely varying degree~ of polarity, therefore, the extract was chromatographed on a silica gel gravity column and eluted with hexane, chloroform and finally methanol to yield a hydrocarbon fraction, non-polar lipid fraction and a polar lipi~ fraction. Spot bioassay of the three fractions indicated all the activity to be in the hydrocarbon fraction. Gas-liquid chromatographic (GLC) analysis of the hydrocarbon fraction (capillary column,

DB-l) showed the majority of material to be five known saturated hydrocarbons: n-heptacosane, 3-methyl-heptacosane, 13-methylheptacosane, 3,9- dimethylheptacosane and l3,15-dimethylheptacosane. The best way to separate these saturated hydrocarbons from unsaturated hydrocarbons in the mixture is by the use of silver nitrate impregnated silica gel column chromatography. Hence, the total hydrocarbon fractionation was put through a silver nitrate impregnated silica gel gravity column, which genera~ed twenty fractions. The fractions were grouped into staturated hydrocarbons, hydrocarbons with one double bond (monoenes), hydrocarbons with two to four double bonds, polyenes and other polar compounds. Spot bioassay of the five groups showed the saturated hydrocarbons to have 40% of Dufour's activity with little or no activity in

the other fractions. However, spot tests of combinations of the five

groups showed that monoenes in the presence of di-tetraenes possess 90% of the recruitment pheromone activity. When combined with the saturated hydrocarbon fraction all of the activity originally found in the total hydrocarbon fraction was regenrated. At this point it was clear we had a multiple component system isolated in three fractions. The best way to deal with such a system is to hold two groups constant while puri-

fying the third group. Then tackle the second group holding the other 167 two constant and so on.... The saturated hydrocarbons were studied first.

GLCanalysis (DB-l capillary column) indicated the presence of the five heptacosanes described above as the major constituents of the total hydrocarbon fraction. And indeed the qggregation activity (40%) elicited by this fraction was due to high concentrations of these hydrocarbons

(53~g/WE). However, preparative GLCon OV-17 at 90° gave a single component with retention time of eleven minutes corresponding to n- heptadecane, which at nanogram levels in combination with the unsaturated hydrocarbon fractions increased the pheromone recruitment activity.

Preparative scale GLCof the monoene fraction yielded a minor component with a retention time of 10.5 minutes which was of major importance in generating the desired recruitment activity in the pres-

ence of n-heptadecane and the di-tetraene fraction. The compound of

unknown structure was designated as "Cl". Finally, analysis of the di- tetraene group on DB-l showed a relatively simple chromatogram composed

of three major peaks. Preparative GLCof the individual peaks followed

by spot bioassay yielded the major component, which corresponded to Z,E,a-farnesene, as part of the pheromone complex. In summary, n-heptadecane was isolated from the saturated hydrocarbon fraction, Z,E-a-

farnesene from the tetraene fraction, and "Cl" from the monoene fraction. n-Heptadecane and Z,E-a-farnesene are knownto be present in Dufour's

gland of S. invicta at levels of 12 and 6ng per gland, respectively.

GLCanalysis of S. invicta Dufour's glands have shown compound "Cl" to

be present at levels of 75pg/WE.

With respect to biological activity, Figure 1, represents the dose - response of Z,E-a-farnesene in the presence of one worker equivalent

of n-heptadecane and "Cl". The recruitment spot bioassay was used to 168 generate the data, and percent response is r~lative to one worker equivalent of S. invicta Dufour's gland. In the absence of Z,E-a-farnesene

(OWE)we see a little over 55% of Dufour's gland activity being due to just "Cl" and heptadecane. Not until 0.5WEwas applied did we observe an increase in activity. At one worker equivalent we obtained 87%of Dufour's gland activity, with a linear increase in recruitment activity upon further addition of Z,E-a-farnesene. Hence, at lWEof Z,E-a- farnesene we get a 30% increase in recruitment compared to just having

"Cl" and n-heptadecane. Similarly increase in activity with increase of pheromone concentration was observed with "Cl" (Figure 2). In this case each sample contains lWEof n-heptadecane and lWEof Z,E-a-farnesene, with the concentration of "Cl" varied. In the absence of "Cl" we observed about 45%recruitment activity due to just n-heptadecane and Z,E-a-farnesene.

However, at levels as low as O.lWE (7.5pg) of "Cl" we get significant increase in activity with 87%recruitment at lWE (75pg). This represents a 40% increase in recruitment activity when lWEof "Cl" is added to the n-heptadecane - Z,E-a-farnesene mixture. n-Heptadecane also increased the pheromone activity (Figure 3) but not to the same extent as "Cl" and farnesene. In the absence of heptadecane we see about 85%recruitment, due to just Z,E-a-farnesene and " " c1 . At O.lWEthere's a slight increase in activity and at lWEwe get 95% of Dufour's gland recruitment activity.

The second part of this presentation involves the isolation of the recruitment trail pheromone from SoZenopsis richteri. Very little is knownabout the trail pheromone of S. richteri. Preliminary character- ization of the main trail orientation pheromone component of S. richteri was reported in 1976 by Barlin and co-workers (6). We would like to 169

present today the first isolation of the recruitment trail pheromone from S. richteri. The isolation scheme (Scheme 2) is very similar to the one used for

S. invicta. Whole ants were homogenized in hexane and filtered, generating the hexane extract. The hexane insoluble material was extracted with methylene chloride followed by methanol giving methylene chloride and methanol extracts. Spot bioassay of these extracts showed most of the recruitment activity in the hexane extract. Purification of the hexane extract on a silver nitrate - silica gel column gave five fractions of which 90% of the biological activity subsided in the F-C (monoene) fraction, (Scheme 2). Purification of F-C on high performance liquid chromatography (HPLC)using a silver nitrate - silica gel column (7) yielded two active components of unknown structure and designated compound "HPLC-4" and compound "Cl", Scheme 2). Both "HPLC-4"and "Cl" are present in Dufour's gla~d at levels of 4ng per worker equivalent. In this Recruitment activity of "Clil is represented in Figure 4.

experiment pure "Clil was tested on S. richteri using the spot bioassay at increasing concentrations of the pheromone. Weobserved a linear dose - response relationship similar to that obtained with Z,E-a- Indeed this type of behav- farnesene and compound "Clil from S. invicta. ior is in accordance with Wilson's observation 22 years ago, pertaining to reinforcement of a trail to a food source (1). That is, the more

chemical the higher the recruitment activity. Also, it should be stated

at this point that "Cl" from S. invictais the same as "Clil from S. richteri.

Interestingly at 0.02WE (~75pg) of IIClll virtually no recruitment activity

takes place with S. richteri (Figure 4). On the other hand 75pg is very 170

active in recruitment of S. invicta as long as is in the presence of n- heptadecane and Z,E-a-farnesene.

The importance of component IIHPLC-4" from S. richteri is shown in Figure 5. The graph represents four samples tested on the spot bioassay with percent response being relative to lWEof S. richteri Dufour's gland. At lWE "Cl" yielded 60% of the recruitment activity and component

IIHPLC-4" 70% of the activity. However, when combined ("Cl" + "HPLC-4") the optimum activity of 85%was obtained.

In conclusion three components have been isolated from S. invicta that elicit 85-90% of recruitment activity. Likewise, two components isolated from S. richteri yielded over 85% of Dufour's activity. All the components are present in Dufour's gland and some are associated with trail orientation activity as well. For example, Z,E-a-farnesene is a major orientation trail pheromone for S. invicta while, the mixture of "C1" and "HPLC-4"represents the major orientation pheromone of S. richteri. Also, this represents the first isolation of a recruitment trail pheromone in an ant species. We are currently working on the structures of "C1" and "HPLC-4", which we hope to elucidate in the near future. 171

References Cited

(1) Wilson, E. O. 1962, Animal Behaviour 10:134-47. (2) Vander Meer, R. K., F. D. Williams, and C. S. Lofgren. 1981, Tetrahedron lett. :1651-4.

(3) Williams, H. J., M. R. Strand, and S. B. Vinson. 1981, Tetrahedron 37:2763-7.

(4) Willams, H. J., M. R. Strand, and S. B. Vinson. 1981,

Experientia 37:1159-60.

(5) Alvarez, F. M., and R. K. Vander Meer. 1983. Synthesis of Homo- farnesenes: Trail Pheromone Components of the Red Imported Fire Ant. Presented at the American Chemical Society Annual Meeting,

August 28 - September 2, Washington, D.C. (6) Barlin, M. R., M. S. Blum, and J. M. Brand. 1976,

J. Insect Physiol. 22:839-44.

(7) Heath, R. R. and P. E. Sonnet. 1980, J. Liquid Chromatogr. 3:1129-1135. Figure 1.

138-L% RESPONSEUS

" 115 R E 109 S

P ...... J 0 N H 85 S E 78

S5 L I1111"'.""''''''''''''''''''''''''''''''f' aoo 0.01 0.10 0.10 1.00 2.00 10.0 100

WORKEREQUIVALENT Figure 2.

138 ~RESPOHSE US C-1

" 113

" -' J 0 W 79 I 62

I "" 45 '"".".-"-'.. '- .. .~ f. 0.0 0.1 0.8 1.0' 8.0 8.0 , ~ 81

MORKEREQUIVALENT 174

Figure 3.

lee % RESPONSE US

" 90 R 80 P 0 H 79 S E 68

se...... 0.00 (101 0.10 0.10 \00 2 .00

WORKEREQUIVALENT Figure 4. RECRUITMENT ACTIVITY OF .C 1- IN- a.RICHTERI 7.0 88

-- 8.01 /' t85 :D ... m - (I) ID ." 0 5.0 50 0 II: Z --' A. -...,J CD (J'1 m 4.0 -I A ..16

3.0 3

0.02 0.06 0.2 0.5 1.0 .4.5 10

WORKER EQUIVALENT Fi g ure S. R E C R U I T MEN T ACT I V I T Y 0 F .. HPLC - 4 .

AND "C1- IN S.RICHTERI

100

III. 80 z 0 -' ! Q. Q) 80 .118 II:

tl 70

- - ..

Scheme 1 .

S. inviat 1) Hexane Ext. 2) Filtration

Hexane Ants 1) Hexane Ext. 2) Filtration

Hexane Ext. Inso1ub1es

1) MeOHExt. 2) Fil tration

Sil ica gel Col. Chrom. MeOH Inso1ub1es

Hexane MeOH CHC13 I Hydrocarbon Lipids Polar Lipids

I Silver nitrate Col. Chrom.

Hexane 5% Ether 10% Ether 120% Ether 50% Ether

Monoenes Di-tetraenes Po1yenes Polar Sat. Hydrocarbons (F-13) (F-3) (F-9) Prep. GLC Prep. GLC Prep. GLC

C17:0 "C1 " Z,E-a-farnesene 178

Scheme 2.

S. Y'1:ChtCP1:

1) . Hexane Ext. 2) Filtration

Hexane Insoluble

1) CH2C12 Ext. 2) Filtration I Insolubles CH2C12

1) MeOHExt. Sil ica gel 2) Filtration Col. Chrom.

MeOH Inso1ubles

Hexane MeOH

150% Ch2C12 ICH2C12 I Hydrocarbon Lipids Lipids Po1ar Lipids Silver nitrate Col. Chrom.

Hexane 5% Ether 10% Ether 20% Ether I 50%Ether

Sat. Hydrocarbons Oi-tetraenes Polyenes Polar Compds.

Monoenes I I F!B F-C F-D

AgN03-HPLC I HPLC-4 "c1 " .FORAGING STRATEGIES OF NATIVE ANTS IN FIRE ANT INFESTED AREAS OF CENTRAL TEXAS

/ Sherman A. Phillips, Jr., David M. Claborn, and Dave Wester

Texas Tech University Lubbock, Texas

ABSTRACT

Bait transects consisting of oil, honey, and meat were run in

oak, juniper, cypress, and improved pasture habitats of central

Texas. Of the thirteen different ant species attracted to the baits,

only four species dominated. The most prevalent ants were Solenopsis

invicta Buren, Monomorium minimum (Buckley), Iridomyrmex pruinosum

(Roger), and Pheidole spp. While S. invicta forages during the coolest

period of the twenty-four hour interval, I. pruinosum and ~. minimum

forage when temperatures are highest. Although ~. !:!1victa-isgenerally

a much better forager and recruiter than the other species, I. pruinosum

is a better competitor in the juniper habitat as evidenced by its

ability to competitively exclude S. invicta at the bait stations.

INTRODUCTION

The western spread of the red imported fire ant, Solenopsis

i!2.:':"_~c ta Buren, has been occurring unimpeded throughout Texas.

Although no predators or parasitoids have been found, some researchers believe that other ant species at the western leading edge may, in

fact, help delay or slow this migration into western Texas.

179 180

Therefore, this study was undertaken in the summer of 1983 to determi-ne

those species most likely to compete either directly or indirectly with the red imported fire ant (RIFA) for food sources.

MATERIALS AND METHODS

Four bait transects were run for 24 hour intervals in fire

ant infested Bandera County, Texas. These transects were through oak,

juniper, cypress, and improved pasture habitats. Each transect con-

sisted of baits placed on 16 ounce cup lids approximately 3 meters

apart. Twenty each of honey, soybean oil, and dog food containing meat were placed on the plastic lids for a total of 60 bait stations

in each transect. Half the baits were left in the field continuously

(stationary); whereas half remained in the field for 30 minute inter-

vals (transitory). Every 3 hours, plastic cups were snapped onto the

lids, thus capturing all the ants at each bait station. Both ground

and air temperatures were recorded. A new cup lid containing the same bait type was then placed in the same spot for the stationary baits.

Ants were then killed with alcohol for identification and tabulation.

Since this procedure was performed at 3 hour intervals for 24 hours,

a total of 240 stationary and 240 transitory gamples were taken from

each transect, making a total of 1,920 samples from the four transects.

For purposes of this talk and ease of presentation and display, data

from the three bait types have been combined. 181

RESULTS

Looking at fire ant infested Bandera County, we find that thirteen species of ants were taken from the baits. The most commonly encountered species include~. invicta, Monomorium minimum (Buckley),

Iridomyrmexpruinosum (Roger), and Pheidole spp. Also encountered, but in fewer numbers, were~_~~atoga~t~_~ _~_~iu~_cul~ Emery, C. punctu- l_a_t_a Erne ry, ~_

Paratrechina~rue~~ (Wheeler), f<2-~_myrmaspp., Atta te~~na (Buckley),

Pachychondyla ha!"pax Fabricius, and Pogonomyrmex barbatus (F. Smith).

The imported fire ant is found in greater numbers than the other species in three of the four habitats. As seen from Figure 1, ~. invicta dominates the oak habitat, followed in turn by ~. minimum,

PI1l'idole spp., and 1. PEtl_i~n!)~c;_u_T1l'However, in the juniper hahitat

(I<'igure 2) we see l1w t I .PY...t~ip!)~~tl.!l]dominates,followed in turn by

S. _i_n_~!.s_t--,~,!'E_~~~_ol-_~ spp. and M. E1_~~!mu_I1]. Again we find that in cypress habitats (Figure 3) ~. jnvicta dominates almost to the exclusion of the other species. Finally, in pasture habitats (Figure 4), the two most dominant species are ~. _~nvicta and I. pruinosum.

The numbers of ants foraging for each species at 3 hour-intervals over a 24 hour period were also determined for the different hahitats. If we look once again at the oak hahitat (Figure 5), we find tlt;ll 1H';tk fOLlging forM. m_icTl_i~~m occurs approxim;ltely1000 hours; wher-eas peak foraging for ~11_ej~9J~-spp. and ~. in_yi~ta occurs at 2200 hours. We might also note that neither M.--- minimum nor Pheidole spp. are able to compete with~. invicta with respect to foraging numbers. 182

- 250" - OAK :z: - .....0 - (HAS/SPECIES) ex:I- J - 0:::> - - V>I.J.J - CD - -0 50 - -...... I.J.JV> - -. I.J.JU - -V>a.. - -. V> - . I- - - ex::z: 40 - ex:...J - -. 0;::) - ...... - - :> - .....0 - -. :z: 30 - LI...... - -. 0 - - 0:: - .- CDI.J.J - :E: - .- ;::) - :z: - :z: . ex: 20 - - :E:I.J.J - - .- - .- 10" - - - -. - -. .-- -. I II Si Mm P'h Ip A B B B

FIGURE1. DOMINANTANTSPECIESDETECTEDATBAIT TRMJSECTSIN OAKHABITAT,BANDERACOUNTY,TEXAS. JULY4, 1983. ... 183

JUNIPER (HAS/ S PECIES) 14"

z .0.... ex:I-- - La.J0::::> - co'" - -0 5 - La.J'" - .... u - Cl..La.J - '"- - I-- - I ex:z 4 - ...J - 0.... - I >-<> - c'..c. - . >-< 3 - I.J.. - a - 0::: - coLa.J - :::E: - :::> - -- z - - La.Jc::(z: 2 - -. :::E: - -. - -. 1-1 - -. - -. - .- Si Mm Ph Ip A AA S

FIGURE2. DOMINANTANTSPECIESDETECTEDATBAIT TRANSECTSIN JUNIPER HABITAT. BANDERA COUNTY. TEXAS . JULY 7. 1983. 184

60 CYPRESS (HAS/SPECIES) :z: 0 l-- ::-e:: c:: 50 I.&J . V) a:I 0 ...... V) -I.&J U I.&J Q.. V) 40 ...... I-V) ~ -I

10 ..- Si Mm Ph Ip A B B B FIGURE3. DOMINANTANTSPECIES DETECTEDAT BAIT TRANSECTS IN CYPRESS HABITAT, 8A~DERA COUNTY, TEXAS. JULY 10, 1983. 185

- PASTURE 604 - z: - (HAS/SPECIES) 0 - I-..... - 0::::>ex: - Lr.J - en 50 - co - -0 - (/') - .....Lr.J - u0...Lr.J - -en 40 - enI- - ex:z - :::>ex:-I - ...... 0 - :>...... - 0 30 - :z:: - ...... - u.. - 0 - 0::: - Lr.Jco - :::> 20 - II z - ex:z - ::E:Lr.J - - I 10" - - I - I --- .- . I Si Mm Ph Ip A B B C FIGURE4. DOMINANTANTSPECIESDETECTEDATBAIT TRANSECTSIN IMPROVEDPASTUREHABITAT.BAtJDERACOUNTY.TEXAS. JULY13. 1983. 186

OAK 40 (SPECIES x TIME) ~-~ ... - ~ -:. ~ - ...~ -:. - ~ -:. 35 ~ - ~ -:. ~ -:. ~ -:. z: ~ - 0 ..... ~ -:. l- -~ -:.- e( - - :> - - Q: 30 - - LLI - -- V') - - CO 0 -: ' """''''' ~ -V') = ~ LLI ~ ..... - U LLI Q.. 25 -V') . ~ ' V') ~ -"e: ~ I- ~ ,U= -" ~ #~ c:: ~ ~ ~ . " ' ...J '. ", :. = ~ e( ## =:) ~,\ -: ' 0 ": ~ ..... t.8 .- :> r... (I)- ...... ttt : 0 tttt t. -: Z , ...... --,.-- LI- ~ : 0 ~, , Q: 11;.. - -:.-- : - LLI ::J CO , \ %: 15 =:) e. -:.: z -. " -:. : z '=" , \ : e( LLI e' ' \ : ::e:: . ,. -:.: ---:.:-- ~ . -- C., '. ---- 10 .I \ -- .,. " i .',. 5 I . . ; t. ." ,. " ' . . ." ,. . ',',.,." ,.

7 13 16 19 22 1 4 7 TIME NIGHT DAY I(

FIGURE5. NUMBEROF ANTSPERSPECIESFORAGINGAiTHREEHOUR INTERVALSIN OAKHABITAT. BANDERACOUNTY.TEXAS. JULY 4. 1983. 187

However, looking at juniper habitat (Figure 6) once again, we see that ~.invicta forages when night temperatures are low, approximately

0100 hours, as does Pheidole spp.; whereas peak foraging for~. mi~imum and I. pruinosum are 1600 and 1900 hours, respectively. Here we see

that I. pruinosum dominates foraging-activity to the exclusion of the other species, including~. invicta. Only ~. invicta was found

foraging to any extent in the cypress habitat (Figure 7). As in other cases, peak foraging occurs in the cool of night between 2200 and

0400 hours. Finally we see the same pattern developing in the improved pasture habitat (Figure 8). The peak foraging of ~. invicta occurs between 2200 and 0400 hours, while peak foraging of I. E£~inosum and M. minimum occurs when daytime temperatures are high, corresponding

to the lowest foraging level by ~. invicta.

By plotting species and duration with respect to transitory versus

stationary baits, we can effectively nullify the effects of temperature

as relates to time. By plotting in this manner, we can identify those

species which are better foragers and recruiters and therefore deter-

mine which species are most likely to competitively exclude other

species from the baits. From Figure 9 we see that S. invicta finds

the food source much more quickly than ~. ~ini~_~~ and Pheidole spp.

in the oak habitat. However, it would appear that M. minimum and

Pheidole spp. are not competitively excluded from the baits since

their numbers increase over time, corresponding to a decrease in

number of S. invicta. In the juniper habitat (Figure 10), in which

I. pruinosumdominates,~. invicta numbers decline at the baits 188

JUNIPER (SPECIES x TIME) 50

z 0 ..... l- 30 e::( :> 0:: LU tn co 0 -tn LU ..... 25 u 0..LU -tn I-tn Z e::( ...J 20 e::(::I C :>- ..... c z: ..... u... 0 15 0:: LU co :E: ::I Z :z: e::( LU:E: 10

5 ", " " . , . , 7 10 13 16 19 22 1 4 7 TIME DAY NIG HT I I' I I FIGURE 6. NUMBEROFANTS PER SPECIES FORAGINGAT THREEHOUR INTERVALS IN JUNIPER HABITAT, BANDERACOUNTY,TEXAS. JULY 7.1983. 189

120 z 0- e:(l- CYPRESS :> 0::: I.LJ (SPECIES x TIME) In CO 100 ,. 0- ..."$. In ...... I.LJ ~ .. U- ... .. I.LJ ...... c...... In ... -.. - ~ .. InI- .~ ~ z: 80 ...... e:( CJ~ ~ I -:- ~ .. e:( :::::> 0 ~ -'" ~ - ~~ t::~ ~ :> ~ # ~ .. c- ~ ~ ... .. z: ...... #... ## 'A. ~ ..~ .- 60 ~ # -" ... .. LI.. ... ## ~ .. 0 ~ #... .. 0::: I.LJ cc .. :IE: "... '" f. '.''.f ~~ .. :::::> '" f" ~ .... z: ...... z: 40 ..'~ ~ .~ e:( -" ,.~ ~ I.LJ :E:

7 10 1613 19 22 1 4 7 TIME DAY NIGHT II

FIGURE 7. NUMBEROFANTSPERSPECIESFORAGINGATTHREEHOUR INTERVALSIN CYPRESSHABITAT,BANDERACOUNTY,TEXAS. JULY 10. 1983. 190

PASTURE (SPECIES x TIME)

~ 90 - #.. ~ z: ~ = #.. ~.. .. 0 .... ~., -:'.#~...' .'. .. ~ -~s. ~ ..." -:" #.. ~..,' ~ .. ::> .~ . "" -. .'~ . .. ex: .~"- .. . -.'- . I.J.J 75 . ~... . - . -, ~ V') -" (J- .. a::! : ~ . " ..: ~ 0 .-" ~. ..."",:,,= _: .: -V') = : I.J.J . ~ . " 1::-. .: .... = : ,,= : c...> . ~ . ... -' . I.J.J = : v,'III" -= .... 0.. . . . " -. ... V') - .. - 60 ~ = .. (/) =" ~ ' . .. .. ~ .'- . ' - .. z: - . ... ~ .- . - ~ , -= .:.. I . , - . ... ~ =" ~ = .. - .. c - ft , .... " .. . . >- . . , ...... ~ -' ... ~.. c 45 ~ ~ ' . z . ~ : ",,,,,,': ~ .... ~ .. - , . ~ : u.. ~ . 0 ~ . . ex: Ed~.. ~ -! " I.J.J ::J~ .... - ~ \ a::! ~ '" : ~ \ ~ . .. % 30 o~ - \ 1:: ' : $ z: ' ..- ~ , I.J.J :J~ : $ \ ::: ' ':.... -~ , Q. - ~. ..~ , ...:' ~ 15 I \ .f ~4f. \. .' 41". "'.:::'11' '" .' " -":'111 " ~ ..,.-' -.::",'..."- '"'" 7 10 13 16 19 22 1 4 7 TIME DAY ]( NIGHT ~

FIGURE 8. NUMBEROFANTS PER SPECIES FORAGINGAT THREE HOURINTERVALS IN IMPROVEDPASTUREHABITAT, BANDERA COUNTY, TEXAS. JULY 13. 1983. 191

35 OAK (SPECIES x DURATION) '" '" 30 '" #', z '"" ....0 l- e( #', .n > '" Q- 0:: #', .. U.J '" / A V) a::I 25 #'i'J, . 0 "'/(\ '" '" 1~ V) U.J ",V .... #', U U.J #'" Q.. #'" V) '" '" V) '" I- 20 '" ~ #" -J #', e( '" :::;) 0 "" .... '" > .... #',. 0 z 15 .... u.. 0 0:: U.J a::I ::E: :::;) Z 10 Z « I.J.J ::E:

5 t,A.11I\"\I1IU~.-,-'-'-'-'- ..-.-.-.-.-.PHEIOOLE

TRANSITORY STATIONARY

FIGURE 9. COMPARISONOF NUMBEROF ANTS PER SPECIES DETECTEDAT TRANSITORYVERSUS STATIONARYBAITS IN OAK HABITAT. BANDERACOUNTY.TEXAS. JULY 4. 1983. 192

30 JUNIPER z 0 l-- (SPECIES x DURATION) e( >- c::: I.LJ (/) a:l 0 25 (/)- -I.LJ U I.LJ ~ ~~ -(/) ~~ I-(/) Z e( 25 ~~ ...J ~~ e( :;) ~~ -c >-- ~ ~~ c tb~"I~ ..,.- 15 u.. .~o~~ . 0 ~~~~"I c::: I.LJ ,.~~ a:l :E: :;) ~"I Z Z ~~ e( 10 I.LJ PHEIDOLE . ~ 4~~

S. invicta J 5 1n ...... -.-.-.-.-.-.-.-.-.-.-.-..M. minimum

TRANSITORY STATIONARY

FIGURE10. COMPARISONOF NU~1BEROFANTS PER SPECIES DETECTEDAT TRANSITORYVERSUS STATIONARYBAITS IN JUNIPER HABITAT, BANDERACOUNTY,TEXAS. JULY 7, 1983. 193

through time; whereas~. minimum, Pheidole spp., and~. pruinosum numbers increase. These data would again indicate competitive ex- elusion or competitive displacement at the baits. In the cypress habitat (Figure 11), as would be expected, S. invicta numbers increase unimpeded at the baits with time because of recruitment and the lack of competition by other species for the baits. Finally, in improved pasture habitats (Figure 12), ~. invicta competitively excludes I. pruinosum at the baits. Therefore, the low level foraging at 1300 hours (Figure 8) by S. invicta is not related to maximum foraging activity of I. pruinosum and M. minimum at that same time.

SUMMARY

1) Rarely is more than one species found at a bait.

2) The first species to find the bait generally dominates the bait.

3) Of thirteen species encountered at baits, only three have the

potential for competitive exclusion.

4) Of the four most common species, a temporal partitioning of

resources appears to occur.

5) While~. pruinosum and ~. minimum forage at mid-day, ~. invicta

and Pheidole spp. forage at night.

6) Of the four habitats,S. j_~~icta dominates all except juniper, in

which case I.pruinosum dominates

7) S. invicta is the first to find baits and recruits more rapidly to

those baits.

8) By comparing number of individuals with the amount of time spent 194

CYPRESS (SPECIES x DURATION)

100 z 0 l-- .." e( .." > .." c::: ~ UJ .." V') .." cc .." 0 80 .." - ~.. V') ..'" UJ .." - .." U UJ .. .." Q.. .JtlT:.." -V') CJ~ .." I-V') -\.~ " Z ,~.:!J...~ " .." e( "' .." .." ...J 60 e( ~ .." :::::I L"-" " -0 It -J .." > ~ " .-0 ~O .." z ~ " - y .." L.&.. ."V .." 0 I'!.:.O .." 40 ..., .." c::: .." UJ .." cc .." ~ ..ff; :::::I .." Z .." ..ff; Z .." e( ..ff; UJ .." ~ .. 20

TRANSITORY STATIONARY

FIGURE11. COMPARISONOFNUMBEROF ANTS PER SPECIES DETECTEDAT TRANSITORYVERSUSSTATIONARYBAITS IN CYPRESSHABITAT, BANDERACOUNTY,TEXAS. JULY 10, 1983. 195

PASTURE (SPECIES x DURATION)

100 z 0 ..... I- ~~~ ~ ~~~ ::>- e::: ~~~ LJ.I ~~~ V') ~~~ CO 80 ~~~ 0 ...... ~~~ V') ~~~ LJ.I ..... ~~~~ U ,,-v ~~ LJ.I Co. -". (,' ~~~~~ V') ...... (t.' ~~~ t/') ~~~ I- 60 , Z c:,. ~~~~ ~ ~~~ --I ~~~ ~ ~~~ ::::> ~~~ 0 ..... ~~~ > ~~~ ..... ~~~ 0 ~~~ z 40 ..... ~~~~ L.L.. 0 e::: w CO :5..,.. I. pruinosum z 20 .,..,..,..,..,..,..,..,..,..,..,..,..,..,..,..,..,..,..,..,~ z ~ LJ.I ::E:

TRANSITORY STATIONARY

FIGURE12. COMPARISONOFNUMBEROFANTSPERSPECIESDETECTEDAT TRANSITORYVERSUSSTATI~NARYBAITSIN IMPROVEDPASTURE HABITAT. BANDERACOU~HY)TEXAS. JULY 13. 1983. 196

foraging, ~. Jnvicti! accounts for the overwhelming majority

of foraging which occurs.

CONCLUSION

Although certain species of ants may have a delaying effect on the western migration of the red imported fire ant, it is more likely that the red imported fire ant is having a detrimental or negative impact on the native ant fauna of central Texas. An Update on Ro 13-5223 (l.a:acTM Fire Ant Insecticide)

G. L. Benson, J. T. Bridges, R. W. Bagley, R. H. Stanton and J. H. Wojciak MAAGAgrochenicals, Hl.RSciences, Inc., Vero Beach, Florida

Introduction

Ro 13-5223, ethyl [2-(p-phenoxyphenoxy)ethyl]carbamate, proposed coomon name fenoxycarb, was discovered and is being developed by Dr. R. Maag Ltd., Dielsdorf, Switzerland, and MAAGAgrochenicals, HLRSciences, Inc., for use in insect control. Fenoxycarb exhibits strong juvenile hormone activity which induces ovicidal effects, inhibits metamorphosis to the adult stage and interferes with the molting of early instar larvae in certain insect species. The susceptibility and response of a particular insect may be quite specific.

Chemical and Physical Properties

Molecular weight 301.3 Form white, crystalline solid (pure a.i.) CXior odorless Melting point 53 - 54° C (pure a.i.) Solubility (organic) >250 g/l solvent--acetone, chloroform, ethylacetate, etc. Solubility (water) 6 ppn (2CfC) Vapor pressure 1.3 x 10-7 Torr = 1.7 X 10-7 mbar Log. P (Octonol /water) 4.28 Stability stable under normal conditions

Toxicology

Fenoxycarb has demonstrated very low order of toxicity. The oral IDso is greater than 16,800 rrg/kg, the dermal IDso is f"eater than 2000 rrg/kg and the inhalation l.Cs0 is greater than 480 rrg/m (rat).

No eye irritation or allergenic potential has been observed in the guinea pig. Eye irritation in the rabbit is minimal, according to the Draize test.

The Ames test has proved negative.

In wildlife studies fenoxycarb has proved to be non-toxic to birds while demonstrating moderate toxicity to aquatic organisms.

Evaluation of the environmental fate of fenoxycarb indicates low environmental mobility, no bioaccumulation tendency in fish, little potential for environmental persistance and fairly rapid dissipation in soil and plants. This, coupled with low use rates and low toxicity, indicates little environmental hazard.

197 198

--Fire Ants

Fenoxycarb has been tested for several years by the USDA Imported Fire Ant Lab, Gulfport, Mississippi, and was found to be very active in controlling fire ants (Solenopsis invicta) at dosages of 4 to 6 grams ai/acre. An Experimental Use Permit was granted in the Spring of 1983 for testing fenoxycarb 1% bait (IffiIC") on 5000 acres in seven southern states.

Working with USDA Imported Fire Ant Station at Gulfport, Mississippi, trials were established on airport properties at Longview, Texas; Shreveport, Louisiana; and Albany, Georgia. Plots were approximately 150 acres at each location for the expanded corn cob grit (EXX;) and the pregel defatted corn (PGD) formulations of IffiIC. A fourth trial was established at the Glynco Jetport, Brunswick, GA, in cooperation with the USDA Imported Fire Ant Lab, Gainesville, FL.

Applications were made by a USDA pilot using a Cessna Ag-Truck equiPPed with a Transland sllln-line spreader (MO 20241) with.a Ram-air system in the hopper to prevent bridging. Speed was 120 mph at 50' altitude and a 50' working swath. Treatments began in Texas on May 3 and were canpleted in Georgia on May 14, 1983.

Evaluations were made 6 and 12 weeks following treatment using the Lofgren Scale (Lofgren and Williams).

Table I presents values that are an average fran three locations. IffiIC at 6 weeks after treatments (WAT) resulted in less colony mortality than AMDRO; however, it should be noted that population index values show a large reduction in the number of ants. By 12 WAT the colony mortality and. population index values for IffiIC had improved considerably and control could e considered canparable to the commercial standard. (* colony mortality value on the En; were 74% as canpared to 87% for the PGD and 86% for AMDRO).

Values obtained at Brunswick, GA (Table 2) indicate 26 to 27% colony mortali ty 6 WAT and by 12 WAT 89 to 94% colony mortality was obtained. The population index was greatly reduced at both evaluation dates. The En; and PGD carriers resulted in very similar control.

IffiIC was evaluatedusing a tractor mounted Herd Model GT 77A applicator set to deliver 1.0 lb of the En; or PGD formulation. Locationsselectedwere newly planted non-bearing citrus groves in Indian River County, FL. Table 4 indicates again a ratherslow initialcolonymortalityby 6 WAT; however, at 16 WAT80 to 88% colony mortality was obtained. The IJ9Pula tion index was greatly reduced at both evaluation dates. The dramatic increase in values in the untreated plots give an indication of the favorable habitat provided for fire ants in the citrus growing areas.

Results of single mound treatments with 1 to 3 tablespoons of IffiIC spread around the mound (Table 4) indicate I.ffiIG to be very active. Control of the individual mounds was faster canpared to the aerial application with canplete control achieved in four weeks.

MAAG Agrochemicals has submitted a petition for registration to the EPA for fire ant control withI.ffiIC. Approval is exPected by Spring, 1984. fenoxycarb

...... \.0 ~ WILDLIFE HAZARD (fenoxycarb)

Birds LD50 (mg!kg) LC50 (ppm)

Mallard Duck >3,000 > 20 ,000 Bob White Quail >7JOOO 25,317

Aquatic Organisms 96 hr LC50 ppm Rainbow Trout 1.6 N Carp 10.2 a Bluegill 2.9 Daphnia 0.4 (48 hr EC50) 201

Table 1.

SUMMARY OF SPRIl'{; 1983 FIElD '!RIALS

WIW IFA BAIT 'IDXICANI'S 1

Im}X>rted Fire Ant Station. USDA, APHIS, PIQ Gulf}X>rt, Mississippi"

Application Rate X% change in per acre X%Colony Mortality2 }X>pulation2 Trea1ment Bulk (lbs) AI (gms) 6 WAT 12 WAT 6 WAT 12 WAT Ro 13-5223 1.0% IGI)3 1.0 4.5 41 87 -92 -99

Ro 13-5223 1.0% 1.3 5.8 33 74 -88 -96 .EXX;3

AMDRO 0.88% 1.0 4.0 93 86 ..:...98 -97 ffiD

Untreated ------28 28 -27 ..-33

Footnotes:

1 - unpublished data, Collins, Homer et.al USDA APHIS Gulfport MS 2 - Averages based on results obtained at Longview, Texas; Shreveport, Louisiana; and Albany, Georgia 3 - ffiD = pregel defatted corn carrier .EXX; = expande

Table 2.

SUMMARY OF SPRINJ 1983 IMIDRTED FIRE ANI' FlEW 'IRIALS

WIlli Ro 13-5223 - GLYNCD JEIroRT, B~UNSWICK, GA

USDA, ARS, GAINESVIlLE, FL1

Application Rate :x% change in per acre x% Colony Mortality2 population 2 Treatment Bulk (lbs) AI (gms) -6 WAT 12WAT -6 WAT 12WAT

Ro 13-5223 1% 0.98 4.4 27 94 -89 -99.5 ffiD3

Ro 13-5223 1% 0.97 4.4 26 89 -88 -98 EXXi3

untreated ------11 22 17 -29

1 - Unpublished data Banks, W.A., D. Williams et al USDA, ARS, Gainesville, FL

2 - Values based on Lofgren scale (Lofgren & Williams 1982. J.Econ.Ent. 75:798-803)

3 - ffiD = pregel defatted corn carrier BOG = expanded corn cob carrier 203 Table 3.

SJMMARYOF SPRIm 1983 IMroRTEDFIREANTFIELD'!RIAlS

WIm Ro 13-5223 - OONBEARINJCITRUS'"Fl.l

Application Rate - XX change in per acre XX Colony Mortality 2 population 2

Treatment Bulk (Ibs) AI (gms) -6 WAT 16 WAT 6 WAT 16 WAT Ste I (Prange Grove) . Ro 13-5223 1% 1.0 4.5 27 88 -92 -99 FGJ)3

Ro 13-5223 1% 0.9 4.0 24 80 -88 -94 KG3 untreated ------+60 +49 +61 +50

Site II (Penney Grove)

Ro 13-5223 1% 0.92 4.1 52 84 -89 -98 FGD

Untreated ------+33 +99 + 4 +284

1 - Material applied~sing' tractor mounted Herd Model GT77A applicator

2 - Values based on Lofgren scale (Lofgren & Williams 1982. J .Econ.Ent. 75:798-803)

3 - PGD = prege1 defatted corn carrier KG = expanded corn cob carrier 204

Table 4.

.. IMroRTEDFIRE ANI' SINJLE ~ 'ffiEA'IMENTSWIlli Ro 13-5223

ARKANSAS, 1983

- .. 2 X<,tchange in X% Colony Mortality population2 1 Application rate Trea 1ment tablespoons per mound 6 WAT 12 WAT-- G WAT 12 WAT

Ro 13-5223 1% 1 33 75 -66 -84 ffiD Ro 13-5223 1% 3 33 100 -76 -100 FGD

AMDRO 3 22 44 -62 -59 FGD untreated --- 13 13 -16 -16

1 - Nine replicates~ per trea1ment. Values based on Lofgren scale (Lofgren & Williams 1982. J.Econ.Ent. 75:798-803)

2 - FGD - pregel defatted corn carrier Research Report From Clemson University

Accudose Aerosol Injection Method of Fire Ant Control with Dursban **

For InformationContact: CESSCO, INC. 1109 Central Ave. Charlotte, N.C. 28209 704.375.9824

205 206

CHLORPYRIFOS AEROSOL MOUND INJECTIONS FOR THE CONTROL OF THE RED.lMPORTED FIRE. ANTI, 2

Paul M. Horton3, J. B. Kissam3 S. B. Hays3 and G. W.Query4 Department of Entomology, Fisheriesand Wildlife Clemson University,Clemson, SC 29631 (Accepted for publication Apr. 28, 1982)

ABSTRACT

Chlorpyrifos aerosols injected into active S,,/enopsis invicta Buren mounds provided 100% control within 7 days. The treatments of 0.5% and 1.0% chlorpyrifos aerosols at rates of 0.17 and 0.44 g AI/mound, respectively, prevented reoccupation of the mounds for 35 days post treatment. The g AI/mound rates used represent a 75.90% reduction in the currently labeled chlorpyrifos drenches. .. .

Key Words: Insecticides, So/enopsis invicta Buren, mound treatments

INTRODUCTION

An aerosol injection device containing 0.7% pyrethrins was shown to be effective in controlling red imported fire ant (So/enopsis invicta Buren) colonies by individual mound treatments during field studies conducted in 1979-1980 (Hays, unpublished data). Based on these data, the device was registered with the U.S. Environmental Protection Agency and is currently being marketed. , Although the 0.7% pyrethrin aerosols give acceptable levels of control immediately following mound treittment, the mounds are often subject to rehabitation because of apparent lack of residual action. For this reason, additional field studies were implemented during the summer of 1981 to determine efficacy of chlorpyrifos aerosols when applied via the same injection system. Previous work conducted by Morrill(1977) showed chlorpyrifos provided good control when applied as a drench. Chlorpyrifos (Dursban) is now labeled for use as a mound drench at rates of 1.6-4.25 g AI/3.75 1 water/mound.

I Published by permission of the Director, South Carolina Agricultural Experi- ment Station, Technical Contribution No. 2023. 2Mention of a commercial or proprietary product does not constitute an endorsement by Clemson University or the Cooperative Extension Service. 3Department of Entomology, Fisheries and Wildlife, Clemson University, Clemson, SC 29631.' ". 4Cessco, Inc., P. O. Box 18452, Charlotte, NC 28218. 207

HORTON t:t ilL: ChIorpyri(os Aerosol (or Fire Ant Control 479 MATERIALS AND METHODS

Two consecutive field tests were conducted on the premises of the Wateree Correctional Institute, Boykin, Sumter County, SC. The tests were established in grazed mixed.grais pastures. Test sites included soil types ranging from sandy-clay to sandy-loam. In addition to the grazing of catde, the study area was subject to tractor- drawn fertilization and bushhog type mowing. No pesticides had been used in these pastures for severalyears prior to this study. The pastures were infested with red imported fire ant (IFA) colonies at a rate of approximately 181 active mounds/ha (73 mounds/ac).IFA mounds present averaged 30-60 em in diameter and 15-30 em -inheight. Larger mounds were evident along ditchc=s and fence rows. Before beginning the tests, a number of aerosol formulations were screened for effective dosage rates. These formulations included single and combination aerosols containing 0.5,0.7, and 1.0% pyrethrin, 0.5 and 1.0% chlorpyrifos, 0.5 and 1.0% . ~chlorvO&,0.5% permethrin, and 0.5% resmethrin. The screening process consisted of injecting individual mounds with the test aerosols. Test mounds were later excavated at intervals ranging from several minutes to 168 houn (7 days) post treatment to determine the efficacy of the materials used. The screening tests clearly showed that the pyrethrin and chlorpyrifos formulations caused rapid mortality to all IFA forms then present in the mound. Following the dosage determinations and based upon the 7 day results, the fust test was begun on July 28, 1981. Morrill(1977) indicated "Ihat mortality estimates taken at 7 days were reliable and that colonies did not 'recover' after several weeks or months". Our tesls were set up to compare 0.5 and 1.0%chlorpyrifos aerosols, alone and in combination with 0.025% pyrethrins, against the previously registered 0.7% pyrethrin aerosol. Table I presents a summary of all field treatments used in this test. Replicated circular plots 15.24 n in radius (0.07 ha) were selected within the test area which contained a r:linimum of five active mounds.. All active and inactive mounds within the plots were mapped prior to treatment using a modified plane table procedure (Brown et at. 1980). Treatments were assigned using a random numbers table. Each treatment and controls were replicated four times. Two sets of control plots were used. In one set, all mounds were injected with an aerosol consisting of the carrier only (15% methylene chloride plus oil). The second set of control plots received no treatment. The aerosol formulations were prepared by Cessco, Inc., Charlotte, NC 28295. The 0.7% pyrethrin aerosol is marketed under the tradename of Acc:u- dose, which is a. trademark of Cessco. All aerosol formulations used in this test were packaged in 1.14 Kg aluminum aerosol cannisten with dip-tube type valvesof the Accudose design.A 74 cm long x 8 mm diameter Fiberglas probe affixed to a standard aerosol pushbutton type cap is attached to the cannister. TIle probe has a 3 mm diameter hollow core throughout its length. At the tip of the probe four 0.06 mm diameter 9rifices are present for release of the aerosol once the cap is depressed. All active mounds within each plot were treated. TIle treated mounds in 208

480 1. GeOJBia EntomoL Sac. Vol. 17, No.4 Table 1. - Summary of field treatments used for control of individual imported fire ant colonies by aerosOl-injected~ticides. 1981.

"",All EquiY 1'818"" Treatment colony AI...--

0.7% pyrethrins 0.24 12.0 0.5% chlorpyrifos + 0.17 8.5 0.025% pyrethrins +0.01 +0.5 0.5% chlorpyrifos 0.17 8.5 1.0%chlorpyrifos 0.44 21.0 1.0%chlorpyrifos + 0.44 22.0 0.025% pyrethrins +0.01 +0.5

aBased on an infestation level of 50 active colonies per acre. the outer 4.24 m were used as barrier treatments to reduce the effects of new colonies. Previous work by Hays (unpublished data) demonstrated this size barrier zone to be effective for testing individual mound treatments. Prior to each plot treatment the cannister was weighed and the weight recorded. A timed application was made to each active mound based on size. Mounds " IS cm diameter received a 5 sec application; 15-30 em diameter a 10 see application; and greater than 30 cm diameter a 15 see application. The following procedures were used to treat each mound. The tip of the probe was held immediately above the mound's center and the aerosol cap was depressed. As soon as the aerosol began to be ejected from the probe orifices, a stopwatch was started and the probe was slowly inserted into the mound. After inserting the probe as deep as possible, it was slowly withdrawn and reinserted in another area of the mound. At least four or more probings were made in each mound> 15 cm to insure thorough coverage. Only I. probe was required in the smaller mounds. From the start to the end of the treatment the formulation was released to prevent soil particles from clogging the probe orifices. The elapsed time was counted aloud to insure proper timing. The total treatment time for each mound was recorded on the map for that plot. After all mounds within a plot were treated the aerosol cannister was reweighed and the final weight recorded. Control determinations were made at 7 and 35 days post-treatment. Each mound within the center subplot of 11 m radius (0.04 ha) was re-located using the plane table map made at the time of treatment. The mounds were individually examined and rated as either active or inactive: The 7-day post- treatment exam was conducted by beating on the ground ~mediately around the mound. When surviving ants demonstrating aggressive behavior on or immediately adjacent to the mound surface were observed, the mound was recorded as being "active". The 35-day post-treatment exam was conducted by excavating the mound to determine its activity level. A second test was begun on September 3, 1981. This test was conducted in the same fashion as the previous test with the following exceptions: (1) only one treatment, 1%chlorpyrifos (0.44 gm AI/mound) was compared to a 209

HORTON .1 A:. ChlorpyriCoa o4.erOlOI for Fire Ant Control 481 no-treatment control, (2) both active and inactive mounds were treated initially, (3) all new active mounds found in treatment plots during the 7.day examination were treated, (4) the last control determination was made 30 instead of 35 days post-treatment. The purpose of initially treating both active and inactive mounds, and treating new mounds found after 7 days was to reduce the effects of new mound establishment. The appearance of new mounds in the vicinity of treated mounds has consistently been a problem. It increases the difficulty in assessingthe control levelsattained with any individual mound treatment. No method has been demonstrated that effectively differentiates between new mounds built by survivors of a treated mound and a new colony. Morrill (1977). Hays (unpublished data) and others have discussedthis problem. RESULTS AND DISCUSSION

When the treatments were injected into each active mound, typical aggressive behavior by the ants WI.,observed. The ants attempted to climb up the probe but the cooling effect of the released aerosol apparently prevented them from doing so. This is an important side benefit of this application method, since it does provide some protection for the applicator. Within one minute after treatment by pyrethrins and 2-5 minutes by chlorpyrifos, ants on the surface were either dead or dying, and had lost their ability to sting.

Table 2. - Control of individual red imported fire ant cglonies within test plotsa by aerosol-injected insecticides. July, 1981.

Man8Ctift " colonies reduct"li' A-.... T"'_c colonies/plot 7 cAys 36''''

0.7% pyrethrins 5.50 82.5 b 45.8b 0.5% chlorpyrifos + 9.30 96.8 a 90.6 a 0.025% pyrethrins 0.5% chlorpyrifos 5.80 10().Oa 100.0 a 1.0%chlorpyrifos 5.80 100.0 a 100.0 a 1.0%chlorpyrifos + 10.00 100.0 a 100.0 a 0.025% pyrethrins Carrier only 6.80 12.1 c 46.6b No treatment 8.00 11.0 c 12.8 c

aplOI size = 0.04 ha. bOnly active mounds within treatment plots were treated. cCompletely randomized design. Tests consisted of four replications for each treatment. . dMeans followed by the same letter are not significantly different at the 1% level based on Duncan's new multiple range test. 210

482 J. Georai&Entomol. Soc. Vol. 17. No.4

Table 3. - Control of individual red imported fire ant coloniesbwithin test plotsa by aerosol-injected chlorpyrifos. September, 1981.

MIen " reductloa Nthle ooIonI88 Awe,. TrNtmentC ooloniftlplot 7 days 30days No treatment 6.8 13.1 a 6.3 a 1%chlorpyrifos 7.0 100.0 b 92.3b aPiot size =0.04 ha. bActive and inactive mounds within test plots initially treated. All new active mounds found at 7 days post.treatment were treated. cCompletely randomized design. Tests consisted of four replications for each treatment. dMeans followed by the same letter are not significantly different at the 1% level based on Duncan's new multiple range test.

Table 4. - Developmentof new active moundsof red imported fire ant colonieswithintest plotsawhenexi~tingactivemoundsareinjected withaerosolinsecticides.July,1981.

New MalInda f'o8Itr88tm8nt A-. TrNtmentC oolonieslplot 7 daysd 3Sd8ysd

0.7% pyrethrins 5.5 2.3 4.3 0.5% chlorpyrifos + 9.3 1.5 5.0 0.025% pyrethrins 0.5% chlorpyrifos 5.8 4.5 6.5 1.0%chIorpyrifos 5.8 1.5 4.0 1.0%chIorpyrifos + 10.0 2.0 5.5 0.025% pyrethrins Carrier only 6.8 1.5 2.5 No treatment 8.0 1.3 1.5 aplot size = 0.04 ha. bOnly active mounds within treatment plots were treated. cCompletely randomized design. Tests consisted of four replicates for each treatment. dYaIues shown had no significant differences at the 1%level based on Dun. can's new multiple range test. 211

HORTON et td. : ChIorpyrifOi AerOlOl for Fire Ant Control 483 Treatments resulted in probe holes, but did not physically destroy the mound ~tructure. Examinations made during the preliminary screening tests had shown mortality of all adults present within the mound at the time of treatment to occur within 1-5 minutes. This wu even observed in some mounds built to a depth of 0.8 m. IFA cadavers were still present in all treated mounds excavated during the 30/35 day post-treatment exam. Tables 2 and 3 summarize the results of the treatments in both tests. It can be seen from these tables that the treatments containing chIorpyrifos provided significantly greater control than the 0.7% pyrethrins. This control remained significant 30-35 days post-treatment. Randomly selected mounds treate4 during the first test were still inactive when examined 65 days post- treatment. It was recognized that substantial numbers of IFA workers would be foraging away for the mounds during treatment. The possibility existed that these "survivors" of a treated mound would not reenter their original mound and would instead build a new mound within the treatment plots. Some of the new mounds found during the 7 and 30/3S day post-treatment exams could have resulted due to the activity of these survivors. In these tests a careful examination and comparison of the plot maps made at the time of each treatment and during the 7 and 30-3S post-treatment counts allowed some discrimination to be made between mounds built by "new" IFA colonies and "treatment survivors". If significant differences were observed in new mound development between the treated. plots VI. the no-treatment control plots, it could be at- sumed that the new mound development was primarily due to these survivors. As shown in Tables 4 and S, however, there were no statistically significant differences in the development of new active mounds between treatments or

Table S. - Developmentof new active moundsof red imported fire ant colonieswithin test plotsa when existingactivebmoundswerein- jectedwithaerosolchlorpyrifos.September,1981.

D-.-.-.New Mounds A..... T- ooloni81/plot 7 D8ysd 30 Dayad No treatment. 6.8 1.8 O.S 1%chlorpyrifos 7.0 1.3 0.5

"'ot size=0.04 hectares. bActive and inactive mounds within test plots initially treated. All new active mounds found 7 days post-treatment were treated. cCompietely randomized design. Tests consisted of four replicates for each treatment. dValues shown had no significant differences at the J% level based on Dun- can's-new multiple range test. 212

484 J. Geoqia Entomol. Sac. VoL 17. No.4 Table 6. - Comparison of insecticide rates and methods for individualmound treatment of red imported fue ant colonies.

MedIad crt Rate lit l.-cticid8 . Applic8tlon . Ail-lid

0.7% pyrethrins aerosol Soil injection 0.24 .. 0.17 0.5% chlorpyrifos aerosol .. 1.0%chlorpyrifos aerosol 0.44 0.5% chlorpyrifos ec Drench 3.83 .. 4.25 6.7% chlorpyrifos ec .. 44.4% chlorpyrifos ec 1.60 .. 50% carbaryl wp 6.81 .. 25% diazinon ec 7.46

treatments and controls within the plots. A comparison of the results shown on Tables 4 and 5 indicate, however, that the procedures fonowed in the second test were. effective in reducing new mound development. Initially treating all active and inactive mounds and treating new mounds developing within the 7 days foUowing treatment did reduce the number of active mounds found after 30 days. These data and other studies (Hays, unpublished data) indicates that this practice does reduce total activity. Based on these data and our observations in the field, the 1%chlorpyrifos aerosol injection provided the best control in these tests. No benefit appeared to be gained by the addition of the 0.025% pyrethrins and. in fact, the 0.5% chlorpyrifos plus pyrethrins gaveslightly poore! control. The aerosol injection method is quick and efficient. A cursory comparison of current market costs for the drench treatments indicated that the 1% chlorpyrifos aerosol would be competitive. In addition the data presented on Table 6 indicates that the chlorpyrifos aerosol treatments. applied according to the above procedures, results in a substantial decrease of the amount of active ingredient placed in the soil asopposed to the currently labeled drench treatments. The g AI/mound rates shown for the chlorpyrifos aerosol repre. sents a 75-90% reduction in the currently labeled chlorpyrifos drench rates.

UTERATIlRE CITED

Brown, R. E., J. C. Nickerson,and C. H. Gaddis, Jr. 1980. Circular method of sampling and mapping open.field plots. Florida Entomologist. 63: 257.8. Morrill, W. L 1977. Red imported fire ant control with diazinon and chlor. pyrifos drenches. J. Georgia Entomol. Soc. 12(2): 96-9.

J. Georgia Entomol. Soc. 17(4), Oct., 1982,478-484 ** Reprinted with written permission of the senior author and the editor of the Georgia Entomological Society. APPENDIXA

AUG2 6 1383

FIRE ANT PESTICIDE FACT SHEET , This is a summary of those products (pesticides) currently registered for Fire Ant Control, including approved. Experimental Use Permits (EUP's), and those pending products under review. This fact sheet identifies formulation types, dosage rates, sites of application, application methods and related points of interest for each of the pesticides listed.

Editor I S note: For information regarding the current registration status of imported fire ant pesticides~ contact:

Paul N. Parsons Special Review Branch (TS-767C) U. S. Envi~onmental Protection Agency 401 "W; St. SW Washington, D.C. 20460 703-557-7400

213 214

REGISTERED PESTICIDES

1. Chemical - Amdro PM-IS

Company - American Cyanamid EPA Reg. No's. 241-260 and 241-261 (0.88% formulation) Date Registered - 8/20/80 & 8/17/81 respectively

Description of Application

Site - Pasture and Range Grass, Lawns,Turf and Non-Agricultural Lands Tolerance - 0.05 ppm (pasture, rangeland grass and grass hay) Dosage Rate - 6.0 grams a.i. per acre (5 level tablespoons per individual mound) Method - Bait broadcast (ground or air application) & mound to mound treatment. Retreatment 4 to 5 months after first treatment if necessary Is use for quarantine program? No Restriction - No restriction on user group.

Points of Interest - Added control of harvester ants and big-headed ants to existing label. Increased storage stability of the product up to three months after opening.

2. Chemical - Baygon PM-12 Company - Boyle Midway Inc. Reg. No. - 475-173 (2% formulation) Date Registered - 6/10/77 Formulation - 2% Containerized Bait

Description of Application

Site - Around the Home (lawns, yards, etc.) Method of Application - Place ant trap near or on mounds Is use for quarantine program? - No

3. Chemical - Bendiocarb (Ficam W) PM-12

Company - BFC Chemicals Inc. Reg. No. 45639-1 (76% WP formulation) Date of Application - 3-25-81 Date Registered -8-11-81 Site - Mound drench Dosage ~te - 0.4 oz./8 gallons water Method - sprinkler can Is use for quarantine program treatment? No Restriction - For use by pest control operators only

Company - BFC Chemicals, Inc. Reg. No. - 45639-3 (1% Dust) Date Registered - 6/14/82 Site - Mound Drench Method - sprinkler can (slurry treatment) Is Use for Quarantine Program? No 215

Restriction - No restriction on user group.

4. Chemical - Carbaryl

Company - Amchem Products Inc. Special Local Need (SLN) No. = SC-790030 (50% WP Formulation) Date Issued by State - 8/13/79

Description of Application

Site - Lawns Dosage Rate - 1.0 - 1.5 pounds active ingredient (lbs. a.i.) per 100 gallons of water. 1 quart of dilution per 6 inches of mound diameter

Method - ~ound drench. Repeat if mound activity resumes Is use for quarantine program? No

5. Chemical - Ch1orpyrifos

Company - Chevron Co. Reg. No. 239-2423 (Amendrnent)(5.3% EC formulation) Date of Application - 3/29/79 Date Registered - 6/24/80 Description of Application

Site - Ant1nounds Dosage Rate - 2.5 ounces product per gallon of water Method - Sprinkler can treatment Is use for quarantine program? No

Company - Dow Chemical Co.

Reg. No. 464-343, 464-360 (22.4% and 40.8% EC formulations respectively) Date of Application - 11/5/79 Date Registered - 11/17/80

Description of Application

Site - Potting plants

Dosage Rate - 8 ounces per product per 100 gallons of water Method - Dip and drench treatments Is use for quarantine programs? Yes

Points of Interest - USDA has certified effective control with this product.

Company - Dow Chemical Co.

Reg. No. 464-553, (5% granular formulation) Date Registered - 12/12/79 Description of Application

Site - Potting media, nursery bench 216

Dosage Rate - 1.0 lb. product per cubic yard of potting soil Method - Mix into potting media granular 5% Is use for quarantine programs? Yes

Points of Interest - USDA has certified effective control with this product; however, Dow has withdrawn this product from the market due to phytotoxicity problems.

Company - Best Products, Inc. Reg. No. 20954-46 (6.7% EC formulation) Date Registered - 8/25/78

Description of Application

Site - Fire ant mounds Dosage Rate - 2 ounces product per gallon of water Method - Sprinkler can treatment Is use for quarantine program? No

Company - Dow Chemical Co.

Reg. No's. 464-343, 464-360 (22.4% and 40.8% EC formulation respectively) Date of Application - 1-17-80 Date Registered - 1/22/81 Site - Mound drench Dosage Rate - 1 fl. oz./2 gallons water Method - sprinkler can treatment Is use for quarantine programs? No

Company - Cessco, Inc.

EPA Reg. No. - 6959-67 (1% spray formulation) Appl. date - 11/12/81 Site - Mounds - homeowner Method - pressurized~ound injection tube Is for quarantine? No Dosage Rate - 1% - dependent upon mound size as to time tube is left in mound. Date Registered - 3/26/82

5. Chemical - Diazinon PM-IS

Company - Ciba-Geigy Corp. and others Reg. No. (25% EC) 100-456 Date of Application - 10/31/77 Date Registered - 12/18/79

Description of Application

Site - Lawns and recreation areas Dosage Rate - 0.016 lbs. a.i. per gallon of water; 1 or more gallon per . mound. Method -/~~ drench Restrict~ - do not apply to feed/food producing areas Is use for quarantine program? No 217

Points of Interest - Claim is only for "aids in control of fire ants." Product directed towards homeowner market.

Company - Thompson-Hayward Chemical Co. and others

Reg. No. (48% EC) 148-1130 Date Registered - 8/11/81

Description of Application

Same as above

Company - Hi-Yield Chemical Company and others Reg. No. - 34911-23 Granules (5% and 14%) Date Registered - 7/2/81

Description of Application

Site - Lawns and Recreation areas Method - Mound Application Dosage Rate - 0.0125 lbs. a.i./mound Is use for quarantine program? No Company - Pennwalt Corporation Special Local Need (SLN) - TX-920015 (23% Flowable Micro-encapsulated) Date Issued by State - 4/2/82

Description of Application

Site - Lawns and Recreation areas Dosage Rate - 1 fluid ounce/gallon water Method - Mound drench (sprinkler can) Is use for quarantine program? No

6. Chemical - Methyl Bromide PM-16

Company - Velsicol Chemical Co. Reg. No. - 876-257 (99.9% pressurized gas) Date Issued - 09/17/69

Description of Application

Site - Around horne, Non-cropland Dosage Rate - 1 ounce of product per 10 square feet mound area Method - Soil fumigation Is use for quarantine program? No

Points of Interest - According to Company this product has not been marketed for IFA control in the last four years.

7. Chemical - Orthene PM-16

Company - Chevron Chemical Co. EPA Reg. No. 239-2436 (15.6% EC formulation) Date Issued - 1-21-81 218

Description of Application

Site - Around the Home

Dosage Rate - 1/2 fl. oz./l gal. H2O Method - Apply on mound and treat a four (4) foot diameter circle around the mound with sprinkling can. - Treat new mounds as they appear Is use for quarantine program? No

8. Chemical - Pyrethrin I & II PM-17

Company - Cessco, Inc. Reg. No. - 6959-58 Date of Application - 10/18/79 Date Registered - 7/2/80

Description of Application

Site - Pastures, golf course, woodlots, building lots, feed lots, buildings, parks, playgrounds Dosage Rate - Spray as necessary Method - (Pressurized spray) spray directly on ants Is use for quarantine program? No Restrictions - Not for household use

9. Chemical - Imidan N-(mercaptomethyl) phthalimide PM-15 S-(O,O-dimethyl phoshordithioate)

Company - Zoecon Corp.

EPA Reg. No. 20954-14-(50% WP in premeasured water soluble packets) Date Registered - 7/8/82 Sites - Residential, Institutional, Commercial and Recreational Areas Method of Application - individual ~~und treatment (drench) Dosage - 1 packet (3.8 gms a.i./gal. water/mound) Is use for quarantine program? No Restrictions - for sale and use only in the state of Texas

10. Chemical - Rotenone PM-16

Company - Pennick Corp. EPA Reg. No. 432-677 Date Registered - 7/20/82

Description of Application

Site - Gardens, lawns; fields, agricultural land, golf courses, recreational areas, camp grounds and other similar areas. Method - Drenching the Ant Mouna Is Use for Quarantine? No

11. Chemical - Heptachlor PM-IS

Company - Do-It-Yourself-Pest-Control Inc. EPA Reg. No. 13283-4 (5% granular formulation) Date Registered - 10/21/74 219

Description of Application

Site - Buried telephone cable closures Method of Application - Place 4 ounces (premeasured packet) into buried telephone cable closure) Is Use for Quarantine Program? No Restriction - Packaged for the Telephone Industry

12. Chemical - Methylchloroform (l,l,l, Trichloroethane) PM-16

Company - Trichem Industries, Inc. EPA Reg. No. - 40708-2 (94% liquid formulation) Date Registered - 8/2/82

Description of Application

Sites - Urban/Rural/Agricultural & Non-Agricultural lands Dosage - 2 ounces product/ant hill Method of Application - Individual mound treatment - Pour 'chemical directly on ~op of mound. Is Use for Quarantine? No.

Company - Malter International Corp. EPA Reg. No. 1266-190 Date Registered - 1/3/83 Sites - Urban/Rural/Agricultural & Non-Agricultural Dosage - 2 ounces/ant hill - Method of Application - Individual mound treatment - Pour chemical directly on top of mound.

Company - Southern Chemical EPA Reg. No. 4000-94 Date Registered - January 10, 1983

Description of Application

Sites - Urban/Rural/Agricultural/Non-agricultural Dosage - 2 ounces/products/ant hill Method - Individual Mound Treatment

Company - Orlik Inc. EPA Reg. No. 47006-4 Date Registered - 12/3/82 Description of Application

Sites - Urban/Rural/and Non-agricultural lands Dosage - 2 ozs./ant hill Method - Individual Mound treatment

Company - Southern Chemicals Products Co. EPA Reg. No. 4000-94 Date Registered - 1-10-83 220

Description of Application

Sites - Urban/Rural/and Non-agricultural lands Dosage - 2 ozs./ant hill Method - Individual Mound treatment

Company - Apollo Industries, Inc. EPA Reg. No. 11623-26 Date Registered - 3-25-83

Description of Application

Sites - Urban/Rural/and Non-agricultural lands Dosage - 2 ozs./ant hill Method - Individual Mound treatment

13. Chemical - MV-678 (Pro-drone) Insect Growth Regulator PM-17 Company - Stauffer Chemical Co. EUP File SYmbol - 476-2211 (1.2% bait formulation) Date Registered - 2/18/83

Description of Application

Sites - Pastures, rangegrass, lawns, turfs and non-crop areas Dosage Rate - 4.8 gm/ai/acre (O.8~ lbs product/acre) Method - Aerial and ground application Tolerance - An exemption from tolerance on pasture and rangegrass was granted. "Is Use For Quarantine Program? No Points of Interest - Make two applications per year. 221

APPROVED EXPERIMENTAL USE PERMITS

1. Chemical - 1-(8~ethoxy-4,8-dimethylnonyl)-4(1-methylethyl benzene) PM-l 7

(MV-678) - Insect Growth Regulator (IGR)'{}) j' i'f r/UJ.U,C»{,:- Company - USDA-Stauffer EUP No. - 42634-EUP-2 (2.4% bait formulation) Date of Application - 3/2/79 Date EUP Issued - 5/5/79 Date EUP Extended to - 9/30/83

Description of Application

Site - Non-agricultural land Method - aerial application Is use for quarantine program? No Restriction - No grazing of animals Doage Rate - 0.0025 lbs. a./i. per acre or 0.00025 ounces a.i. per mound

Points of Interest - Existing stocks could be used until'9/30/82 under cooperation of USDA, State and local government officials.

Company - same as above

EUP No. 42634-EUP-2 USDA requested an extension and additional use of MV-678 to be used in Brazoria County Texas. Date Extended - 4/1/82 to 9/30/83

2. Chemical - Ethyl [Z-(p-phenoxyphenoxy)ethyl] Carbamate (RO 13-5223) PM-17

\, 0l1./t:... 'I Company - MAAG Agrochemicals EPA EUP No. - 35977-EUP-2 (1% formulation) Date of Application - October 12, 1982 Date Issued - March I, 1983 to March I, 1984

Description of Application

Site - Non-crop areas lawns and turfs (single mound treatment) Method - 1 to 3 level tablespoons per mound by uniformly distributing material 3 to 4 ft. around the base of the mound. Should be uniformly distributed over the infested area. Application can be made with ground or air equipment. -'Dosage - 1.0 to 1.25 Ib./A Is use for Quarantine? No Restriction - Maximum 75 lbs. active ingredient over 5000 acres in Alabama, Arkansas, Georgia, FJ.orida, Louisiana, Mississippi, Texas. " II 3. MK-936 (Avermectin B1) /I f f I fLl'/1

Company - Merck, Sharp & Dohme EUP No. - 618-EUP-IO (0.011% and 0.0055% formulations) Date of Application 11/25/81 222

Date Issued - May 3, 1982 Date EUP Extended to - March 23, 1984

Description of Application

Site - Non-Cropland Dosage Rate - 25 mg-50 mg a.i. per/acre Method - Broadcast (ground and air) Is Use for Quarantine? No Restriction - Maximum 178.13 grams a.i. over 4750 acres in Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina and Texas. Do not graze treated fields.

"Pending" Registration Actions and EUP's for Fire Ant Control

1. Chemical - Amdro PM-IS

a. Company - American Cyanamid EPA Reg. No. 241-260 (Amendment) (PP-2F2627) Date of Application - 1/7/82 & 12/1/81

Description of Applications

Site - Cropland (including pineapple and sugarcane) for control of IFA, harvester ants-and big-headed ants. Tolerance - 0.05 ppm for all rac's. Dosage Rate - 6.0 gros/ai/acre for Imported Fire Ants and Harvester Ants/lO.O gros/ai/acre for Big Headed Ants Method - Bait Broadcast (ground and/or air) and mound to mound treatment. Limitations - All Food Crops - Do not treat within seven days of Harvest. Pineapple - do not apply more than two broadcast applications per crop during any twelve month period. Sugarcane - Do not apply more than three broadcast applications per crop. Is Use for Quarantine? No

Status - Company has submitted outstanding long term chronic studies. Review completion expected by end of September 1983. b. Company - American Cyanamid EPA Reg. No. 2211-260 (Amendment) Date of Application - 6/24/83

Description of Application

Site - For use in non-bearing fruit and nut crops, in nurseries (field planting only). Tolerance - WF Dosage Rate - 6.0 gms/a.i./acre (5 level tablespoons per individual mound). Method - Broadcast (ground or air application) and mound to mound. Is use for Quarantine? No 223

Status - Application under review. Review completion expected by end of September 1983.

2. Chemical - Disodium Octaborate Tetrahydrate PM-16

Company - R. Value, Inc. EUP #44313-EUP-R Appl. date - 9/11/81 Site - mounds on unspecified sites Method - Broadcast application and individual mound treatment. Is use for quarantine? No Dosage Rate - 4 lbs. a.i./A (broadcast):1/4 cup product (mound) Formulation - 5% bait Status - Objection letter sent 12/18/81; no response received to date;

3. Chemical - Resmethrin PM-17

Company - Cessco File Symbol - 6959-AR, 6959-LO Date of Application - 10/21/79

Description of Application

Site - Buildings, playground, pastures, parks, feed and woodlots. Dosage Rate - Spray as necessary Method - (Pressurized spray) spray direct~y on~. Is use for quarantine program? No Restrictions - Not for household use. Status - Preliminary acceptance letters issued 6/28/82

4. Chemical - Methylenebis (thiocyanate) PM-31

Company - Vineland Chemical Company EPA Registration No. 2853-43 Date of Amendment Application - November 2, 1981 Formulation - 10% Liquid

Description of Application

Site - Non-agricultural land Method - Individual ffioundtreatment(Drench) Dosage - 2 oz. product/gal. water/35 sq./ft. In use for Quarantine? No Restrictions - Not for Homeowner Use as labeled. Status - Additional testing requested of applicant by Fire Ant Laboratory (USDA) Gainesville, Florida

5. Chemical - Carbaryl (Sevin) PM-12

Company - Union Carbide EPA Registration Nos. - 264-314, 316, 333, 334, 335 (WP, Sprays) Date of Amendment Application - August 6, 1979 224

Description of Application

Site - Crop & Non-crop areas (Homeowner and/or Commercial Use) Method - Sprinkler ca~/moUria_:~renchonly Dosage - 13-27 gms/a.L/acre Is use for Quarantine? No Status - Preliminary acceptance letter has been sent to company. Awaiting revised draft labeling before acceptance.

6. Chemical - Diazinon (Microencapsulated) PM-IS

Company - Pennwalt Corporation EPA File Symbol - 4S81-GLR Date of Application - October 29, 1982 Formulation - 23% Flowable Micro-encapsulated Description of Application

Site - Rangeland, pastures, lawns and recreation areas Dosage Rate - 1 fluid ounce/gallon of water Method - ~noun~ drench (sprinkler can) Is use for Quarantine program? No Status - Objection letter sent June 16, 1983. Additional bird and fish toxicity studies requested.

7. Chemical - Tetramethrin, Pydrin, Petroleum distillate PM-17

Company - C & J Chemical Compan¥ EPA File Symbol - 40849-RG Product Name - Enforcer Inject-A-Kill Fire Ant Killer Date of Application - September 15, 1982 Formulation - Aerosol spray

Description of Application

Site - Playgrounds, yards, pastures, parkst farms and golf courses Method - Individual mound treatment with mound penetrating rod. Is use for Quarantin~? No Status - Objection letter sent December 9, 1982. Additional acute toxicity data requested for this new formulation plus petition for tolerance for use on pastures and rangeland. 8. Chemical - Sodium bicarbonate, sodium silicat~, ammonium PM-IS chloride

Company - Naturally Green Inc. EPA File Symbol - 48374-R Product Name - Naturall~ Green Fire Ant Control Date of Application - May 27, 1982 Formulation - Two Product Pour-On Solution

Description of Application

Site - Mound$ on unspecified sites Method - Individual mound treatment. Apply solution "A" followed by solution "B" as pour-on or spray. 225

Dosage - unspecified Limitations - Product does not kill ants but claims the "gel" formed from the combination of solutions A & B interrupts colony life cycle causing rapid decline. Is use for Quarantine? No Status - Objection letter sent December 31, 1982. Additional testing requested of applicant through Fire Ant Laboratory (USDA), Gainesville, Florida.

9. Chemical - Dursban & DDVP (dichlorovos) PM-1S

Company - NCH Corporation EPA Reg. No. - 1769-233 Product Name - Dichloron Date of Amendment - September 29, 1982 Formulation - 3% Dursban & 2.8% DDVP concentrate

Description of Application

Site. mounds on unspecified sites. Method - Turn over mound with shovel and then apply full spray. Dosage - 7.5 ounces/product per 2 gallons water Is use for Quarantine? No Restriction - For PCO (serviceperson) Use Only Status - Objection letter sent July 25, 1983. Amendment action subject PR Notice 83-4. Application under review. lO.Chemical- Fenoxycarb PM-17

Company -MAAG Agrochemicals EPA File SYmbol - 3S977-L (technical)and 3S977-U (end-use) Product Name - LOGIC Date of Application - August 5, 1983 Formulation - 1% bait

Description of Application

Site: Lawns, turf, rangegrass, pasture, and nonagricultural land. Method: Unifornly distribute 1-3 tablespoons b~it 3-4 feet around base of moundi apply with ground equipment at 1 to 1.5 Ib/acrei apply aerially at Ib/acre. Tolerance - An exemption from tolerance on grass and grass hay has been requested.

Is Use for Quarantine? No Status: Application under review Point of Interest - Fenoxycarbis claimed to be an insect growth regulator. APPENDIXB

PARTICIPANTS IN THE 1984 FIRE ANT CONFERENCE

226 227

Mr. Richard L. Barzin Mr. Charles F. Adams Wickhen Products, Inc. American Cyanamid Company Big Pond Road One Cyanamid Plaza Huguenot, NY 12746 Wayne, NJ 07470.

, Dr. G. Lee Benson Dr. S. A. Alfieri, Jr. MAAG Agrochemicals, HLR Sciences Division of Plant Industry P.O. Box X P. O. Box 1269 Vero Beach, FL 32961-3023 Gainesville, FL 32602 Mr. Mike Bishop Mr. Ahmed D. Ali USDA!APHIS!PPQ!NMRAL Louisiana State University P.O. Box 3209 Dept. of Entomology, 402 Life Sc. Building Gulfport, MS 39503 Baton Rouge, LA 70803 Mr. Fred Blum Dr. Francisco Alvarez SCM Corp. USDA!ARS P.O. Box 389 P.O. Box 14565 Jacksonville, FL 32217 Gainesville, FL 32604 Mr. John C. Boltin Mr. C. R. Andress Stauffer Chemical Company Stauffer Chemical Company 2009 Orient 1:toad P.O. Box D?l Tampa, FL 33619 Houston, TX 77251 Dr. James Bosworth Mr. John C. Anzalone USDA!ARS Cline-Buckner, Inc. P.O. Box 14565 100 Calhoun St., Box 610 Gainesville, FL 32604 Independence, LA 70443 Dr. John M. Bowen Mr. Pedro Araoz College of Vet. Medicine MoBay Chemical Company P. O. Box 1508 The University of Georgia Athens, GA 30602 Vero Beach, FL 32961-1508 Dr.J. T. Bridges Dr. David Ball ,. Maag Agrochemicals Texas A & M University P.O. Box X Entomology Department Vero Beach, FL 32960 College Station, TX 77843-2475 ,,:,.','r:". '," Mr. Ralph E. Brown Mr. W. A. Banks Division of Plant Industry USDA!ARS Florida Department of Ag. & Con. Services P.O. Box 3269 P.O. Box 1269 Gulfport, MS 39503 ,:~Gainesvil1e, FL 32602 ,. ~"~!>..r,!,,; j' - Mr. Charles H. Bare Mr. Ed Butler USDA!APHIS!PPQ Overland Marketing Corporation 6505 Belcrest Rd., Federal Bldg. P.O. Box 5523 Hyattsville, MD 20782 Alexandria, LA 71301 228

Mr. David W. Bryon Dr. Carlos Cruz Dept. of Ent., Fisheries & Wildlife Ag. Experiment Station, U.P.R. .1 Clemson University Box 506 Clemson, SC 29630 Isabela, PR 00662 Ms. Marsha Cameron I Ms. Joe DeNicola Ward Archer and Assoc. Box 30012 University of Florida McCarty Hall - Ent. Dept. Memphis, TN 38130 Gainesville, FL 32611 I

Mr. T. Don Canerday Dr. Bastiaan M. Dress University of Georgia Texas Ag. Ext. Service Entomology Division I 200 Barrow Hall Texas A & M University 301 N. Main Street Athens, GA 30602 Bryan, TX 77803 I Mr. Ca~los Cardenas Dr. Richard Dybas SCM Organic Chemical Division P. O. Box 389 Merck & Company, Ag. Research Hil1sborough Road Jacksonville, FL 32201 I Three Bridges, NJ 08887 Mr. Avis u. Cherry USDA!ARS Dr. J. E. Eger P.O. Box 14565 Ag. Products Dept. I Dow Ch~mica1 Company Gainesville, FL 32604 P.O. Box 22300 Tampa, FL 33630 Dr. Richard C. Chromecek, President .1 Wickhen Products, Inc. Mr. Eddie W. Elder Big Pond Road USDA/APHIS/PPQ Huguenot, NY 12746 4080 Woodcock Dr. I Mr. Randall H. Clark Jacksonville, FL 32207 MAAG Agrochemicals, Inc. #174 Vestavia Hills Mr. Robert E. Eplee ..' USDA/APHIS/PPQ, Whiteville Methods Dev. CenterI Northport, AI. 35476 P. O. Box 279 .- Whiteville, NC 28472 Mr. Roy P. Clark " '" . EPA Region #4 I Ms. Dorothy Erlanger 345 Courtland Street, NE American Cyanamid Company Atlanta, GA 30369 ." One Cyanamid Plaza' I Mr. Homer Collins Wayne, NJ 07470 USDA/APHIS/PPQ ,;. (->".J.:.<, ,...:~.<:,,;.~.. Mr. Rob Esworthy Methods Development Environmental Protection Agency Gulfport, MS 39503 I 401 M. Street, S.W. Washington, DC 20640 Dwight B. Coulter, DVM Dept. of Physiology & Pharmacology Dr. Donald L. Evans I College of Vet. Medicine University of Georgia University of Georgia College of Vet. Medicine Athens, GA 30602 Athens, GA 30602 II 2-29

Mr. Homer E. Fairchild Mr. Richard Gaskalla USDA/APHIS/PPQ Division of Plant Industry Federal Center Building P. O. Box 1269 Hyattsville, MD ,20782 Gainesville, FL ,32602

Mr. W. T. Ferer Ms. Sherry P. Gause Merck & Company, Inc. Florida Farm Bureau P.O. Box 2000 "Florida Agriculture" Rahway, NJ 07065 P.O. Box 730 Gainesville, FL 32602 Mr. Tom Fernander Rt. 1, Gengl Newman Road Dr. B. M. Glancey Rockoale, TN 37153 USDA/ARS P.O. Box 14565 Mr. Richard G. Fifield Gainesville, FL 32606 AL Farm Bureau Federation Montgomery, AL 36198 Mr. W. Kenneth Glenn Clemson University Mr. Scott C. Fleetwood Rm. 210, Barre Hall American Cyanamid Clemson, SC 29631 1917 Penbrook Road Arlington, TX 76015 Leocadio r.ondim 78700 Caeeres, Mato Grosso, Brazil Mr. Tom Fleishel Caixa Postal 191 Dow Chemical 20 Perimeter Center, E., Suite 2005 Mr. Ge~rge Gosen, Jr. Atlanta, GA 30346 Merck and Company, Inc. P.O. Box 2000 Dr. Sam S. Fluker Rahway, NJ 07065 FL Cooperative Extension Service Building 803, Room 4, University of FL Mr. Monroe Hall Gainesville, FL 32611 Illinois Cereal Mills, Inc. 616 S. Jefferson Street Mr. Joseph H. Ford , Paris, IL 61944 USDA/APHIS/PPQ ,- 3505 25th Ave; P.O. Box 3209 Mr. Don Harlan Gulfport, MS 39503 Mid-South Agriculture Research ".': ,,1323 S. Avalon Dr. Oscar F. Francke Texas Tech University W.,/~:mph~s,,AR 72301 "'..:;',~~:,,; """, ',,' ' Department of Biological Science Mr. 'Don Harris Lubbock, TX 79409 :Division of Plant Industry P. O. Box 1269 _,.,Ms.Suzanne Fraser Gainesville, FL 32602 Division of Plant Industry . P.O. Box 1269 Mr. John Hodges Gainesville, FL 32602 Rt. 1, Box 20-A Tifton, GA 31794 Mr. John C. French, Head Ext. Pest Management 201 Ext. Hall Auburn Univ., AL 36849 230 .

Mr. F. J. Howard, Jr. . 212 Barre Hall Mr. Al Martin 'j Clemson Univ., SC 29631 N.C. Dept. of Agriculture Plant Industry Division - Plant Protection Mr. J. Brewer Huber, Sales Rep. Raleigh, NC 27611 American Cyanamid Company Pesticide Dept. Mr. Larry McKinney 5026 Lofty Pine Circle Griffin Corp. P.O. Box 1847 Jacksonville, FL 32210 j Valdosta, GA 31601 Dr. H. B. Jackson Mr. John Milio Clemson University Room 210, Barre Hall University of Florida I Clemson, SC 29631 Department of Entomology Gainesville, FL 32604 Mr. Norman A. Jacobson j Johnson Wax Mr. Michael Mispase1 1525 Howe Street Dept. Physiology & Pharmacology Racine, WI 53403 Univ. of Georgia Athens, GA 30602 I Dr. Donald Jouvenaz USDA!ARS Mr. Greg Mitchell ..- P.O. Box 14565 USDA!ARS P.O. Box 14565 I Gainesville, FL 32604 Gainesville, FL 32606 Dr. J. L. Knapp Mr. G. R. Moore !FAS , I 700 Experiment Station Rd. Stauffer Chemical Company Lake Alfred, FL 33855 Nyala Farm Road Westport, CT 06881 I Dr. Neil A. Lapp Merck & Company Mr. L. Ross Morgan 7208 Madiera Ct. Rm. 212, ~arre Hall Raleigh, NC 27609 Clemson University I Clemson, SC 29630 Ms. Lisa A. Lemke - Col. Moufied A. Moussa -Dept. of Ent., Fisheries & Wildlife DOD Research Liaison Officer I Clemson University Clemson, SC 29630 USDA ~ P.O. Box 14565 Gainesville, FL 32604 Dr. Richard Lipsey I Kenco Chemical & Manufacturing Corp. Mr. Ken Muzyk 10 West Adams Street/p.O. Box 6246 American Cyanamid Company Jacksonville, FL 32236 408 Larrie Ellen Way ,Brandon, FL 33511 I Dr. Clifford S. Lofgren -. USDA/ARS Dr. J.C. Nickerson P. O. Box 14565 Florida Department of Agriculture & Con. servll Gainesville, FL 32604 Division of Plant Industry P.O. Box 1269 Gainesville, FL 32602 I 231

Mr. Jack A. Norton Mr. Don E. Rawlins Merck and Company, Inc. American Farm Bureau Fed. Hillsborough Road Three Bridges, NJ 08887 225 Tougy Avenue Park Ridge, IL 60068 Dr. Martin Obin USDA/ARS Dr. T. E. "P,eagan . P.O. Box 14565 LA State University Gainesville, FL 32604 Department of Entomology 402 Life Sc. Building Mr. John B. O'Neil Baton Rouge, LA 70803

American Cyanamid Company, Dr. James A. Reinert 2997 Gant Place Ft. Lauderdale REC Marietta, GA 30062 3205 S.W. College Avenue Dr. JohnM. Owens University of Florida Ft. Lauderdale, FL 33314 SC Johnson' & Sons, Inc. 1525 Howe Street Racine, WI 53403 Dr. Jose M. Rodriguez P. O. Box 10163 Dr. Dennis Paustenbach Sanqurce, PR 00908

Stauffer Chemical Company Mr. David T. Rowlands Nyala Farm Road Westport, CT 06881 American Cyanamid Company 3197 Ferns Glen Drive Tallahassee, FL 32308 Mr. Sherman A. Phillips, Jr. Texas Tech University Dept. of Ent., Box 4169 Mr. T. A. Scarborough Union Carbide Lubbock,TX 79409 P.O. Box 2558 Mr. Ken Plumley Wayside, MS 38780 USDA/ARS '1600 S.W. 23rd Drive Mr. Max Sconyers Gainesville, FL 32601 MoBay.Chemical Company P.O. Box 1508 Dr. Sanford D. Portor Vero ,Beach, FL 329611508, Dept. of Biological Science Dr. D. L. Shankland '; FL State University ;(Tallahassee, FL 32304 University of Florida

" Entomology & Nematology Gainesville, FL 32611 i:,", Mr. Grady W. Query :Cessco Company, Inc. i',1109 Central Avenue Dr. Craig Sheppard University of Georgia " Charlotte, NC 28205 L: , "" ' Coastal Plains Exp. Station .,Mr. O. Grady Query Tifton, GA 3l79~ Cessco Company, Inc. 1109 Central Avenue Mr. Frederick Singleton Charlotte, NC 28205 1725 N. Main St., Suite 105 Summerville, SC 29483 232

Mr. Jim Smith Mr. David VanderHooven USDA/APHIS/PPQ The Andersons Cob Division P. O. Box 3209 P. O. Box 119 Gulfport, MS 39503-1209 Maumee, OH 43537

Mr. Richard H. Stanton Dr. Robert K. VanderMeer Maag Agrochemicals USDA/ARS P. O. Box X P. O. Box 14565 Vero Beach, FL 32960 Gainesville, FL 32604

Mr. Robert Stevens Dr. S. B. Vinson American Cyanamid Company Texas A & M tTniversity P. O. Box 2577 Department of Entomology Nacogdoches, TX 75961 COliege Station, TX 77843-2475

Dr. Jerry L. Stimac Dr. F. VondeMuh11 University of Florida MAAG Agrochemicals, Inc. McCarty Hall, Entomology Department P.O. Box X Gainesville, FL 32611 Vero Beach, FL 32961

Mr. Charles A. Strong Mr. J. Noel Wagner USDA! ARS Stauffer Chemical Company P.O. Box 14565 P. O. Box 17207 Gainesville, FL 32604 Raleigh, NC 27619 Mr. Jack Swarthout Ms. Theodora Wang Illinois Cereal Mills, Inc. American Cyanamid Company 616 S. Jefferson St. P. O. Box 400 ' Paris, IL 61944 Princeton, NJ 08540

Mr. John W. Taylor, Jr. Mr. D. C. Weeks USDA Forest Service Clemson University 1720 Peachtree Street, NW Plant Pest Regulatory Service Atlanta, GA 30367 Clemson, SC 29631

.- Mr. W. Kent Taylor Mr. Donald E. Weidhass Nor-Am Chemical Company USDA!CR , .' Rt. 6, Box 194 P. O. Box 14565 Tifton, GA 31794 Gainesville, FL 32604

Mr. James Trager Ms. Diane Weigle University of Florida " USDA!ARS McCarty Hall -Ent. Department P.O. Box 14565 Gainesville, FL 32611 Gainesville, FL 32604

".. Mr. Mark R. Trostle ~ Mr. George O. White Texas Department of Agriculture Texas Farm Bureau P. O. Box 12847 Rt. 1 Austin, TX 78711 Harwood, TX 78632 233.

Dr. David F. Williams USDA/ARS P. O. Box 14565 Gainesville, FL .32604

Dr. Karen G. Wilson NC Dept. of Agriculture P.O. Box 27647 Raleigh, NC 27611

Dr. Daniel P. Wojcik USDA! ARS P. O. Box 14565 Gainesville, FL 32604

Miss Lois Wood University of Florida McCarty Hall-Ent. Dept. Gainesville, FL 32611

Mr. John Wyokoff American Cyanamid Company One Cyanamid Plaza Wayne, NJ 07470