2012 Imported Fire Ant Conference Proceedings Is Available on the Extension Web Site in .Pdf Format

Conference Proceedings

Nashville, TN April 16-18, 2012

Hosted by

Proceedings of the 2012 Imported Fire Ant Conference

Nashville, Tennessee, USA April 16-18, 2012

Conference hosted by:

Tennessee State University, College of Agriculture, Human, and Natural Sciences, 3500 John A. Merritt Blvd., Nashville, TN 37209

The University of Tennessee, Agricultural Extension Service, Plant and Pest Diagnostic Center, 5201 Marchant Drive, Nashville, TN 37211

Tennessee Department of Agriculture, Division of Regulatory Services - Plant Certification Section, Ellington Agricultural Center, P.O. Box 40627, Nashville, TN 37204

Organizing Committee

Tennessee State University Jason B. Oliver, Chair Alicia M. Bray, Co-Chair Nadeer N. Youssef, Co-Chair Nick J. Gawel Holly Hodges

University of Tennessee: Frank A. Hale

Tennessee Department of Agriculture: Walker G. Haun Steve D. Powell

Proceedings compiled by: Jason B. Oliver Tennessee State University, Nashville, TN

Proceedings Disclaimers These proceedings were compiled from author submissions of their presentations at the 2012 Imported Fire Ant Conference, held on April 16-18, 2012 at the Holiday Inn Express Downtown Nashville, Nashville, Tennessee. The 2012 annual conference was organized by Tennessee State University, College of Agriculture, Human, and Natural Sciences, Nashville, TN; The University of Tennessee, Agricultural Extension Service, Plant and Pest Diagnostic Center, 5201 Marchant Drive, Nashville, TN; and Tennessee Department of Agriculture, Division of Regulatory Services – Plant Certification Section, Ellington Agricultural Center, P.O. Box 40627 Nashville, TN. The opinions, conclusions, and recommendations are those of the participants and are advisory only. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by Tennessee State University, The University of Tennessee, or the Tennessee Department of Agriculture. The papers and abstracts published herein have been included as submitted and have not been peer reviewed. They have been collated and duplicated solely for the purpose to promote information exchange and may contain preliminary data not fully analyzed. For this reason, the authors should be consulted before referencing any of the information printed herein. This proceedings issue does not constitute a formal peer review publication. However, ideas expressed in this proceedings are the sole property of the author(s) and set precedence in that the author(s) must be given due credit for their ideas. A copy of the 2012 Imported Fire Ant Conference Proceedings is available on the eXtension web site in .pdf format. To access the 1984 – 2012 Proceedings use the following link: http://www.extension.org/pages/Proceedings_of_the_Imported_Fire_Ant_Conference

Table of Contents

Page

Proceedings Disclaimers 2

Table of Contents 3

Remarks from the Conference Chairman 4

Acknowledgements 5

Index to Submitted Presentation Abstracts / Manuscripts 6

Index to Submitted Poster Abstracts / Manuscripts 9

Submitted Abstracts / Manuscripts 11

List of Registrants 85

Conference Sponsors 90

Appendix 1: 2012 Program Agenda 93

Appendix 2: Conference Photos 105

Remarks from the Conference Chairman

Greeting from Tennessee to the imported fire ant and invasive ant participants,

I want to thank you for attending the 2012 Imported Fire Ant Conference held at the Holiday Inn Express Downtown, Nashville, Tennessee. I hope everyone had a great time in Music City Tennessee, and the conference provided valuable information and enlightenment on imported fire ants and other important invasive ants. The final tally for meeting attendance was 65. The conference began with a Monday evening reception that doubled as a poster viewing and corporate sponsor exhibit area in the Entertainment District reception room. The research conference began Tuesday morning with a welcome from the Tennessee Commissioner of Agriculture, Julius Johnson. Mr. Johnson provided compliments to our group for working to manage such a serious pest issue for citizens in Tennessee, the southern and western United States, and globally. Next, the Dean and Director of Research / Administrator of Extension from the Tennessee State University (TSU) College of Agriculture, Human, and Natural Sciences, Dr. Chandra Reddy, provided a welcome to conference participants from TSU and our co-hosting institutions. Dr. Reddy also conveyed a welcome from the President of TSU, Dr. Portia Shields, and Interim Provost and Executive Vice President for Academic Affairs, Dr. Millicent Lownes-Jackson. Dr. Reddy especially expressed the honor TSU had in co-hosting the Imported Fire Ant Conference this year because TSU was celebrating its Centennial Celebration during the same week as the Imported Fire Ant Conference, as well as TSU Ag Week. Following the opening Welcome Session, this year’s meeting featured a special session on Invasive Ants, which included Dr. Pat Zungoli (Clemson University) speaking on Asian needle ant, Dr. David Oi (USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology) speaking on invasive crazy ants, and Dr. Karen Vail (The University of Tennessee) speaking on other invasive ants. We were also fortunate to have three contributed talks this year on other invasive ants like little fire ants, European red ant, and crazy ants, which immediately followed the special Invasive Ant session. Due to the increased significance of other invasive ants besides imported fire ants, meeting participants decided during the business meeting to formally change the conference name to the “Imported Fire Ant and Invasive Ant Conference”. In addition to the three special session invasive ant presentations, this year’s conference had 17 oral papers and 9 posters on topics pertaining to invasive ants, ecology, genetics, behavior and physiology, management, biological control, fire ant identification, morphology, and extension. The conference concluded with the eXtension Imported Fire Ant Community of Practice workshop held Wednesday afternoon and Thursday morning. The 2012 conference would not have been possible without the dedication, commitment, and hard-work of our conference committee: Dr. Alicia Bray (co-chair), Nadeer Youssef (co-chair), Dr. Nick Gawel, Holly Hodges, Dr. Frank Hale, Walker Haun, and Steve Powell. I am especially appreciative to Alicia and Nadeer, who worked many hours on meeting planning, gift bags, registration, and program development. I hope to see all of you at next year’s conference in Virginia Beach. Please be on the lookout for emails from Dr. Peter Schultz (Virginia Tech, Hampton Roads Agricultural Research and Extension Center) with details on the 2013 Imported Fire Ant and Invasive Ant Conference.

Jason B. Oliver, Chairman, 2012 Imported Fire Ant and Invasive Ant Conference

Acknowledgements

The organizers of the 2012 Imported Fire Ant and Invasive Ant Conference and participants would like to express their appreciation and gratitude to the sponsors for their generous support. Sponsor support was vital in making the conference a success and financially feasible. We are very grateful for their continued generosity. Sponsor ads can be found on page 90 of the proceeding.

Corporate Sponsors: Platinum

Bronze

We would also like to thank Kelli Galloway, Lauren Jensen, Kristin Palmer, and Wayne Coghiel of the Holiday Inn Express Downtown Nashville, Nashville, Tennessee for the excellent accommodations and service in meeting the needs of the conference participants and organizers. We also thank Dennis DeVoto from Conference Direct for his assistance with hotel selection, negotiations with hotel and meeting vendors, and for his invaluable advice on meeting planning. Special thanks go to Joseph Lampley (Tennessee State University, Entomology Laboratory Assistant, Otis L. Floyd Nursery Research Center, McMinnville, TN) for his artistic talent in designing the logo for this year’s conference program, t-shirts, and conference tote bag. Thanks also to former conference organizers and members, Paul Nester, Kelly Loftin, Kathy Flanders, Bastiaan ‘Bart’ Drees, and Neal Lee for their most helpful advice. I would also like to thank Scotts Miracle-Gro for supplying the note pads for the conference and everyone who provided door prizes including Nadeer Youssef (ant fishing flies and framed atlas moth), Alicia Bray (framed cross-stitched ant pictures), Charlotte Oliver (insect-related pottery mugs), Michael Owen (Advion bait), and Fire Ants book (donated by Texas A&M University Press). We also thank Carol Russell (USDA-APHIS) for her continued annual support of the conference with numerous door prize items from Hawaii.

Index to Submitted Presentation Abstracts / Manuscripts

NOTE: NS denotes Not Submitted. Speaker name in bold.

Page Submitted Abstracts / Manuscripts

Special Session on Invasive Ants

NS Asian needle ant. Patricia Zungoli (Clemson University, Entomology, Soils, and Plant Science, 230 Parkway Drive, Clemson, SC)

11 Invasive crazy ants. David Oi (USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology,100 S. W. 23rd Drive, Gainesville, FL)

NS Other invasive ants. Karen Vail (The University of Tennessee, Department of Entomology and Plant Pathology, 214 Ellington Plant Science Building, Knoxville, TN)

USDA Imported Fire Ant Program

13 Overview of the APHIS Imported Fire Ant Program – 2012. Anne-Marie Callcott (USDA, APHIS, PPQ-CPHST, Gulfport, MS), Charles Brown (USDA, APHIS, PPQ-EDP, Riverdale, MD), Katherine Hough (USDA, APHIS, PPQ-WR, Fort Collins, CO), and Ronald Weeks (USDA, APHIS, PPQ-ER, Raleigh, NC)

Other Exotic Invasive Ant Species

16 Little fire ants in Hawaii: Situation report, impacts, and ongoing research. Cas Vanderwoude,1 Michelle Montgomery (Hawaii Ant Lab, Hawaii Department of Agriculture, 16 E. Lanikaula St., Hilo, HI) and Neil Reimer (Hawaii Department of Agriculture, 1428 S. King St., Honolulu, HI)

17 Attempts to manage the invasive European red ant, Myrmica rubra. Eleanor Groden, Jennifer Lund, and Kerry Bernard (School of Biology & Ecology, University of Maine, Orono, ME)

18 Adding insult to injury in Texas grasslands: crazy ant extirpates imported fire ant and further reduces native ant and arthropod diversity. Edward G. LeBrun, John Abbott (Brackenridge Field Laboratory, Section of Integrative Biology, University of Texas at Austin, 2907 Lake Austin Blvd, Austin, TX) and Lawrence E. Gilbert (Section of Integrative Biology, University of Texas at Austin, 1 University Station #C0930, Austin, TX)

Ecology

NS Propagule pressure and colony social organization are associated with the success of fire ant invasion. Chin-Cheng Yang (Master Program for Plant Medicine, National Taiwan University), M. S. Ascunce, L. Z. Luo, C. J. Shih, R. N. Huang, and D. D. Shoemaker

19 Impact of red imported fire ant on Mexican rice borer in sugarcane and non-crop hosts. Matthew T. VanWeelden, Thomas E. Reagan, Blake E. Wilson, and Julien M. Beuzelin (Louisiana State University Agricultural Center, Baton Rouge, LA)

Behavior / Physiology

NS Ontogeny of the fire ant alarm pheromone. Robert Vander Meer and Man-Yeon Choi (USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL)

22 Introduction of a neurohormone in the fire ants. Man-Yeon Choi and Robert K. Vander Meer (USDA-ARS, Center of Medical, Agricultural, and Veterinary Entomology, Gainesville, FL)

24 Large-scale analysis of fire ant pheromones: A progress report. Robert Renthal (University of Texas at San Antonio, San Antonio, TX)

25 Sublethal dose insecticides affect fire-ant foraging and recruiting efficiency. Xing Ping Hu and Zhonglin Yuan (Department of Entomology and Plant Pathology Auburn University, Auburn AL)

NS Family ties and fluorescent dyes: searching for nepotism in Solenopsis invicta. Collin C. McMichael and Micky D. Eubanks (Texas A&M University, Department of Entomology, College Station, TX)

26 Influence of genetic variation on colony-level foraging behavior of the red imported fire ant. Alison A. Bockoven and Micky D. Eubanks (Texas A&M University, Department of Entomology, College Station, TX)

Fire Ant Management

NS Development of cold temperature techniques for certifying bulk soil for movement: Large scale trials. Karen Vail (University of Tennessee, Entomology & Plant Pathology, Knoxville, TN), Jennifer Chandler, Jeremy Shoop, Anne- Marie Callcott, Kevin Hoyt, and Richard Evans

27 “Fighting Texas’ fire ants: The team approach” 10 years and counting – renewing interest in the Lago Santa Fe Fire Ant Project. Paul R. Nester (Texas AgriLife Extension Service, Houston, TX) and Bastiaan “Bart” M. Drees (Department of Entomology, Texas A&M University, College Station, TX)

Biological Control

NS Colony impacts and host specificity of the red imported fire ant virus SINV-3. Sanford D. Porter, Steven M. Valles, and David H. Oi (USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL)

39 Death by 1000 cuts: Continuing to assemble communities of phorid fly parasitoids for the biological control of imported fire ants. Edward G. LeBrun, Robert M. Plowes (Brackenridge Field Laboratory, Section of Integrative Biology, University of Texas at Austin, 2907 Lake Austin Blvd, Austin, TX) and Lawrence E. Gilbert (Organizational Director, Section of Integrative Biology, University of Texas at Austin, 1 University Station #C0930, Austin, TX)

Other Topics

45 Impact of the invasion of the imported fire ant, does identification matter? S. Bradleigh Vinson (Department of Entomology, Texas A&M University, Collage Station TX)

Extension

46 Reinventing fire ant eXtension. Kathy Flanders (Department of Entomology and Plant Pathology, Auburn University, AL) and Bart Drees (Department of Entomology, Texas A&M University, College Station, TX)

Index to Submitted Poster Abstracts / Manuscripts

Page Submitted Abstracts / Manuscripts

Biological Control

47 Toxicity of alkaloids of red imported fire ant, Solenopsis invicta Buren against the green peach aphid, Myzus persicae (Sulzer). Tahir Rashid (Department of Agriculture, 1000 ASU Drive, #330, Alcorn State University, Alcorn State, MS), Paul J. McLeod (Department of Entomology, University of Arkansas, Fayetteville, AR), and Jian Chen (USDA-ARS, Biological Control of Pests Research Unit, Stoneville, MS)

51 Arkansas’ phorid distribution after 10 years of releases. Kelly Loftin, John Hopkins, Doug Petty, and Ricky Corder (University of Arkansas, Department of Entomology, Fayetteville, AR)

Ecology

NS The ant (Hymenoptera: Formicidae) fauna of the cedar glades and barrens of the Central Basin of Tennessee. JoVonn G. Hill (Mississippi Entomological Museum, Mississippi State, MS)

Fire Ant Management

52 Managing the red imported fire ant on a green roof in Friendswood, TX. Paul R. Nester, Anthony W. Camerino (Texas AgriLife Extension Service, Houston, TX), and Alejandro A. Calixto (Texas AgriLife Research, College Station, TX)

60 Trolling for red imported fire ant (Solenopsis invicta) control efficacy data in the Lone Star State's record-setting hottest, driest year in U. S. history! Paul R. Nester (Texas AgriLife Extension Service, Houston, TX), Bastiaan “Bart” M. Drees (Texas AgriLife Extension Service, College Station, TX), and Alejandro A. Calixto (Department of Entomology, Texas A&M University, College Station, TX)

Genetics

66 A novel use of microsatellite markers for distinguishing two imported fire ant species and their hybrid. David C. Cross (Mississippi State University, Biochemistry, Molecular Biology, Entomology and Plant Pathology), Rajesh B. Garlapati (Genesis Laboratories, Inc., Patna, India), Juliet D. Tang (Mississippi State University, Forest Products), Pummi Singh and Michael A. Caprio (Mississippi State University, Biochemistry, Molecular Biology, Entomology and Plant Pathology)

70 Cytochrome P450 gene expression profile in the red imported fire ant, Solenopsis invicta. Nannan Liu,*Feng Liu, and Ting Li (Auburn University, Department of Entomology and Plant Pathology, Auburn, AL) *Corresponding author

Other Exotic Invasive Ant Species

74 Specialized setae and spongiform lobes in Strumigenys and Pyramica ants (Hymenoptera: Formicidae). Joe A. MacGown and Richard L. Brown (Mississippi State University, Mississippi Entomological Museum, Box 9775, Mississippi State, MS)

80 Efficacy of hydramethylnon, indoxacarb, and methoprene baits against the little fire ant Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae). Susan K. Cabral, Arnold H. Hara, and Kris L. Aoki (University of Hawaii at Manoa, Komohana Research and Extension Center, 875 Komohana Street, Hilo, HI)

Morphology

NS Sensilla of the apex of the maxillary and labial palpi of Solenopsis invicta, Solenopsis richteri, and Nylanderia pubens (Hymenoptera: Formicidae). Gerald T. Baker and Richard L. Brown (Mississippi State University)

Raves & Rants About Invasive Crazy Ants

David H. Oi USDA-ARS, Center for Medical, Agricultural, & Veterinary Entomology, Gainesville, FL

The name “crazy ants” often refers to various species of formicine ants that are characterized by erratic running when disturbed. Some species can develop extremely large populations that can dominate landscapes. Two of these species, the yellow crazy ant and the Caribbean, or Rasberry [sic], crazy ant with their extremely large numbers and scurrying behavior are spectacular to watch until they start to crawl all over you. The yellow crazy ant, Anoplolepis gracilipes, formerly known as Anoplolepis longipes, is thought to be from Africa or tropical Asia. It thrives in moist tropical lowlands and has invaded many South Pacific islands. Successful eradication of this invasive ant has been reported from several sites in Australia. Another eradication project is being conducted under the auspices of the U.S. Fish and Wildlife Service (USFWS) on Johnston Atoll National Wildlife Refuge, which is located about 825 miles west-southwest of Honolulu, Hawaii. The refuge provides critical, rat-free habitat for central Pacific sea bird populations. While designated a wildlife refuge in 1926, the atoll was also a military installation since 1934, being used for submarine and aircraft refueling, nuclear weapons testing, and chemical weapons storage and disposal. By 2005, virtually all buildings were razed and military personnel removed. In 2010, a 90 acre yellow crazy ant infestation was found on one (Johnston Island) of the four islands that make up the atoll. By 2011 the contiguous infestation had increased to 120 acres and a 0.2 acre satellite infestation was also discovered. The USFWS has been sending teams of four or five volunteers to try to eradicate the ants. Because of the remote location logistical support is challenging, with teams and supplies being transported by ship (3 day one-way trip) approximately every six months, and communication to Honolulu is limited. Current attempts to control the yellow crazy ant include using ant bait consisting of canned cat food and dinotefuran, an EPA “reduced-risk insecticide”. In January of 2012 less than 1% of the sampling sites had yellow crazy ants. It was observed that nest initiation by the ground nesting red-tailed tropicbird had increased with the decline in yellow crazy ants. However in April, yellow crazy ants were found at 11% of the sampling locations, thus monitoring and treatment will continue. The Caribbean crazy ant, Nylanderia cf. pubens, and the Rasberry crazy ant, Nylanderia near pubens, have inundated landscapes in parts of Florida and Texas, respectively. They also have been found in Louisiana and Mississippi. Recent taxonomic and genetic studies indicate that Caribbean and Rasberry crazy ants are the same pest species. Monthly sampling of nests in Gainesville, Florida revealed that the presence of male alates was most prevalent from October to February. High numbers of female dealates and queens were easily observed in January to March, while brood was not consistently evident during that time period. Bait evaluations on laboratory colonies of the Caribbean crazy ant showed consistent colony death with gel bait containing imidacloprid (MaxForce® Quantum) diluted in sucrose solution, and granular baits containing hydramethylnon (MaxForce® Complete) and abamectin (Advance® Carpenter Ant Bait). Various bait formulations containing the insect growth regulators pyriproxyfen or (S)- methoprene caused average reductions in brood of over 80%. However reductions were not consistent across colonies and thus not significantly different from controls. In the search for biological control agents, RNA sequences from Caribbean crazy ants collected from various

regions of Florida were surveyed for pathogens. Three replicating sequences of viral origin were detected indicating potential virus infections in the crazy ants and the need for further characterization (Valles et al. 2012).

References Cited Valles, S. M., D. H. Oi, F. Yu, X.-X. Tan, and E. A. Buss. 2012. Metatranscriptomics and pyrosequencing facilitate discovery of potential viral natural enemies of the invasive Caribbean crazy ant, Nylanderia pubens. PloS ONE 7(2): e31828. doi:10.1371/journal.pone.0031828.

Disclaimer Mention of trade names or commercial products in this report is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Overview of the APHIS Imported Fire Ant Program – 2012

Anne-Marie Callcott,1 Charles Brown,2 Katherine Hough,3 Ronald Weeks4 1USDA, APHIS, PPQ-CPHST, Gulfport, MS 2USDA, APHIS, PPQ-EDP, Riverdale, MD 3USDA, APHIS, PPQ-WR, Fort Collins, CO 4USDA, APHIS, PPQ-ER, Raleigh, NC

The Federal Imported Fire Ant Quarantine was implemented in 1958 and is cited in the Code of Federal Regulations, Title 7, part 301.81 (7CFR301.81). The goal of the present day quarantine is to prevent the artificial spread of imported fire ants. Regulated items include nursery stock, grass sod, hay, soil and other items that can transport IFA. The current regulated area includes all or part of 14 states and Puerto Rico (AL, AR, CA, FL, GA, LA, MS, NC, NM, OK, SC, TN, TX, VA) and approximately 366 million acres. Numerous models indicate the potential range of the IFA is greater areas than those currently infested with IFA, primarily in the western states (OK, TX, NM, AZ and CA). Since the late 1980’s, the federal IFA quarantine program has been implemented by the states. States are responsible for inspecting nurseries, issuing compliance agreements, surveys, and conducting blitzes w/ USDA. Oversight of the program is by USDA-APHIS-PPQ and includes development of quarantine treatments, transfer of information to states and enforcement, including investigations and fines associated with violations. Oversight and management of federal IFA quarantine program is a team effort of PPQ led by the National Program Manager (PPQ-EDP), with team members from PPQ-ER, PPQ-WR and PPQ-CPHST. The PPQ-CPHST Lab in Gulfport MS is responsible for the development of methods and tools used in the IFA Quarantine for survey, detection, regulation and control. The group oversees the APHIS Phorid fly (Pseudacteon spp.) rearing and release program (biological control of IFA). Florida Department of Agriculture, Division of Plant Industries (FL-DPI) mass rears the phorid flies and state cooperators conduct releases. ARS-CMAVE, Gainesville, FL develops the rearing techniques and transfers them to FL-DPI. Recent accomplishments of the APHIS, PPQ IFA program included several new IFA quarantine treatments that have been developed and validated over the last few years with data from PPQ-CPHST, Tennessee State University and University of Arkansas. These include a new treatment for balled-and-burlapped nursery stock using a bifenthrin dip/immersion which is awaiting APHIS approval, and a new treatment for grass sod using bifenthrin also awaiting APHIS approval. In addition, APHIS has funded ARS to develop rapid an IFA identification field kit and trap, and a potential cold treatment for contaminated soils is under development with data from PPQ-CPHST and the University of Tennessee. To date, four species of phorid flies are in rearing and are being released. P. tricuspis and P. curvatus are established in more than 65% of the IFA quarantined area in the southern U.S. and releases of P. obtusus and P. cultellatus will continue through 2013. If no other phorids are available for rearing on red IFA, we will phase out the rearing and release program in 2013-2014. Special thanks to ARS- CMAVE (S. Porter), FL-DPI (G. Schneider) and all the state cooperators for making this program a success. So, where is the APHIS-PPQ IFA Program going and what is changing?

APHIS will be closing the Gulfport Facility (MS) in 2012. Existing CPHST staff and operations will be outsourced or relocated to other facilities. All other APHIS or PPQ staff that work at that facility will continue to work out of other locations in the local commuting area. The CPHST Analytical chemistry unit will transfer operational work to other units or will outsource the work. Environmental monitoring to support routine PPQ programs will be outsourced to other labs through cooperative agreements or contracts and managed by a CPHST scientist. Project work to support CPHST and PPQ will be relocated to CPHST Miami Lab and staff will be relocated to CPHST Miami or other CPHST or PPQ units. Most importantly for IFA interests, PPQ-CPHST will NO LONGER provide analytical support for IFA soil samples. The CPHST Imported Fire Ant unit will be outsourcing all methods development work through cooperative agreements and managed by a CPHST scientist. Anne-Marie Callcott will remain as CPHST IFA coordinator/contact and located in Biloxi MS. As soon as new contact information is available it will be distributed. Staff will be relocated to other CPHST or PPQ units.

Where can you get IFA soil samples analyzed for pesticide residue?

There is no federal requirement to analyze routine soil samples annually at nurseries under compliance agreements, however it is encouraged. Historically, these analyses were conducted either by the USDA, APHIS, PPQ-CPHST lab in Gulfport MS or by a local state pesticide lab. In late 2011, a letter was sent to all PPQ-SPHDs in impacted states to share with their SPROs. States may use their state pesticide lab or a neighboring state lab. States may enter into an agreement with USDA-AMS-National Science Lab in Gastonia, NC to conduct the analyses for them. AMS NSL-Gastonia has provided cost estimates for the IFA samples for FY12 (ca. $125/sample for a single pesticide analysis; ca. $38/sample for bulk density determination). However, states MUST negotiate with AMS directly. CPHST staff is available to discuss/provide analytical methods to state labs, and states may contact CPHST for contact information for AMS NSL. State inspectors will need to notify all growers in the state about this change since every year there are +20 samples submitted independently to the CPHST Gulfport Lab by nurseries (not through their state inspector).

What are we working on? • Environmental Assessment (EA): getting EA into the Federal Register • Once EA is final o Add new quarantine treatments for B&B and grass sod to PPQ Treatment Manual and IFA Program Manual o Update Program Aid “IFA 2007: Quarantine Treatments for Nursery Stock and Other Regulated Articles” o Update IFA Program Manual . Adding new quarantine treatments . Adding section on “policing the quarantine” • PPQ staff will be working to update/modify all existing publications (including online sites) regarding submission of IFA soil samples • Continuing development of regulatory treatments o Grass sod: bait + contact insecticide treatments o Field grown stock: replacement for infield bait+chlorpyrifos treatment o Combined infield and B&B treatment for long term stored B&B

o Continue/validate cold treatments of small containers of bulk soils o Continuing releases of P. obtusus and P. cultellatus

Website of interest: http://www.aphis.usda.gov/plant_health/plant_pest_info/fireants/index.shtml

Little Fire Ants in Hawaii: Situation Report, Impacts, and Ongoing Research

Cas Vanderwoude,1 Michelle Montgomery,1 and Neil Reimer2 1Hawaii Ant Lab, Hawaii Department of Agriculture, 16 E. Lanikaula St., Hilo, Hawaii 96720 2Hawaii Department of Agriculture, 1428 S. King St., Honolulu, Hawaii 96814

Little Fire Ant (LFA) is a small, stinging, arboreal tramp species which form polygynous polydomous colonies. It’s honeydew collecting behavior and protection of homopteran insects causes outbreaks of these pest insects and loss of agricultural production. Their stinging behavior has negatively affected harvesting and also been implicated in keratopathy of pets and livestock causing blindness. LFA was first reported on Hawaii island in 1999 and has since spread to Kauai and Maui. The Hawaii Department of Agriculture’s efforts using hydramethylnon and pyriproxifen ground applications combined with indoxacarb paste baits applied to vegetation resulted in the eradication of LFA from Maui within 1 year. Ongoing research studying palatability and efficacy of other baits was described. Granular baits with hydramethynon were more palatable than baits with methoprene and pyriproxyfen. Paste baits containing indoxacarb, imidicloprid, dinotefuran or clothianidin were all equally effective in reducing ant populations. The development and use of a paste bait applicator to reach and stick to vegetation which uses compressed air and a spray gun was described.

Attempts to Manage the Invasive European red ant, Myrmica rubra

Eleanor Groden, Jennifer Lund, and Kerry Bernard School of Biology & Ecology, University of Maine, Orono, ME 04469

The non-native European red ant, Myrmica rubra, is an invasive, stinging ant that has become established in many communities along the coast of northeastern United States and Maritime Canadian provinces. This northern temperate ant is native to Europe and Western Asia and though first reported in the U.S. in 1908, pestiferous populations have developed in the past 15-20 years. Colonies of these polygynous, polydomas ants establish nests in moist soils or organic matter, under or within a variety of natural and artificial substrates including stones, down woody debris and potted plants, in a variety of deciduous habitats. Because of the high density of nests in infested areas (ave. 1.4/m2) and the cryptic nature of nests, treatment of individual nests has limited utility for managing infestations. Broadcast granular ant baits have reduced populations somewhat, but populations rebound quickly. Although drawn to a variety of bait stations designed for either liquid or granular baits, labeled baits indicated that effective dispersal from the bait station is limited to ca. a 4 m radius around the bait station. Food (bait) preferences of foraging workers shifts from sugars post diapause to protein in mid summer, and transfer of all food types (labeled sugar, protein and oil) were equally detectable in brood and queens throughout the season. In 2011 we initiated a site-specific management strategy for M. rubra at a number of sites in Acadia National Park on Mount Desert, Maine. Infestations were mapped throughout the sites on a 10 m X 10 m grid. Treatment plans were developed based on the use of the site, its vegetation cover, and the intensity of the infestation both within the designated area and in the surrounding landscapes. Treatment plans usually consisted of an initial broadcast of baits (indoxacarb (Advion®), spinosad (Greenlight fire ant bait), and fipronil (Maxforce® FC Fire Ant Bait)) throughout the site. Persistent hotspots were identified with follow up sampling and were retreated with broadcast baits and/or bait stations were deployed with liquid (imidacloprid (Maxforce® Quantum)) or gel (fipronil (Maxforce® FC Ant Killer Bait Gel)) baits. Of six intensively managed sites in 2011, by mid September, one small, isolated infestation appeared to possibly be eradicated. However, remnant populations persisted within all other sites, particularly in densely vegetated and wet areas. Sequential mapping of the ant populations throughout the season at the managed sites suggest that small remnant colonies are persisting within the treated areas and resurgence of infestations is not primarily due to emigration from surrounding infested areas.

Adding Insult to Injury in Texas Grasslands: Crazy Ant Extirpates Imported Fire Ant and Further Reduces Native Ant and Arthropod Diversity

Edward G. LeBrun,1 John Abbott,1 and Lawrence E. Gilbert2 1 Brackenridge Field Laboratory, Section of Integrative Biology, University of Texas at Austin, 2907 Lake Austin Blvd, Austin, TX 78703. 2 Section of Integrative Biology, University of Texas at Austin, 1 University Station #C0930, Austin, TX 78712

Biological invasions reduce biological diversity by causing local extinction of native species, and by homogenizing local biotas, replacing regionally distinct species assemblages with cosmopolitan taxa. Over the past 100 years, the Southeastern United States and Texas have been subject to invasion by a series of exotic, ecologically dominant ant species with each subsequent invader replacing its predecessor. Central Texas is in the early stages of invasion by an ecologically dominant exotic ant species novel to the region: Nylanderia nr. pubens. Using a combination of baits and pitfall traps, we sampled two areas of disturbed Gulf coastal prairie invaded by N. nr. pubens to evaluate the degree to which this invader impacts the diversity and abundance of co-occurring ants and other arthropods. In invaded parts of these habitats, N. nr. pubens rapidly attains densities up to 2 orders of magnitude greater than the combined numerical abundance of all other ants in the system. Overall ant biomass also increases in invaded habitat. As N. nr. pubens spreads, the current ecologically dominant invader, the imported fire ant (Solenopsis invicta) is entirely eliminated from areas with high densities of N. nr. pubens. Compared to imported fire ant dominated habitat, non-ant arthropod species richness and abundance are greatly reduced by the invasion and these impacts vary some with trophic category. Further, N. nr. pubens significantly reduces both the abundance and species richness of the remainder of the ant assemblage and does so in a non-random manner. Regionally distributed species are strongly negatively impacted but globally distributed species are largely unimpacted. The result is a net biogeographic homogenization of the local ant assemblage, a process that may be general to systems where an invasive ant species enters habitat previously colonized by tramp taxa. S. invicta impacts wildlife and the structure of arthropod assemblages and is currently nearly ubiquitous in non-forested habitats regionally. Its displacement by N. nr. pubens has critical implications for the natural systems of this region. The nature of the changes that ensue will depend upon key differences between the biology of this ant and that of imported fire ants.

Impact of Red Imported Fire Ant on Mexican Rice Borer in Sugarcane and Non-crop Hosts

Matthew T. VanWeelden, Thomas E. Reagan, Blake E. Wilson, and Julien M. Beuzelin Louisiana State University Agricultural Center, Baton Rouge, LA 70803

Abstract A study was conducted in the summer and fall of 2011 to assess the effect of predation by the red imported fire ant, Solenopsis invicta (Buren), on Mexican rice borer, Eoreuma loftini (Dyar), infestations in sugarcane (Saccharum spp.) and johnsongrass, Sorghum halepense. Sugarcane and energycane plots at the Texas A&M AgriLife Center in Beaumont, TX were either treated with insecticidal baits to suppress ants or left untreated. A 50% increase in E. loftini injury expressed as a percentage of bored internodes was observed where ants were suppressed. For non-crop hosts, plots of johnsongrass adjacent to rice fields in Texas were left untreated or treated with insecticidal baits to suppress ants. E. loftini injury across all treated plots increased over 2-fold when compared to untreated plots. This data suggests that S. invicta can decrease both pest build-up in sugarcane and plant injury in non-crop hosts.

Introduction Over the past few decades, the Mexican rice borer, Eoreuma loftini (Dyar), has become a significant pest of rice and sugarcane in the southern region of Texas, surpassing previously established stem borers in terms of economic importance (Johnson and van Leerdam 1981). In 2008, E. loftini was discovered in pheromone traps in Calcasieu Parish, Louisiana (Hummel et al. 2010), the same year the species was predicted to enter the state (Reay-Jones et al. 2007). Though management tactics including insecticide applications (Johnson 1985; Meagher et al. 1994; Wilson et al. 2011), irrigation (Reay-Jones et al. 2005), and cultivar resistance (Reay- Jones et al. 2003) have shown some success in suppressing E. loftini in sugarcane, the pest is expected to continue its advance eastward through Louisiana (Reay-Jones et al. 2007). Attempts to use biological control for the suppression of E. loftini have had little success. In contrast to the sugarcane borer, Diatraea saccharalis (F.), parasitic species have been unable to inflict any significant form of control against E. loftini (Meagher et al. 1998). The parasitic braconid species, Cotesia flavipes (Cameron), has been attributed to controlling D. saccharalis populations in the Lower Rio Grande Valley of Texas, but has shown little evidence in suppressing E. loftini (Meagher et al. 1998). In addition to suppression by parasitic species, generalist predators such as the red imported fire ant, Solenopsis invicta, have exhibited successful control of D. saccharalis (Negm and Hensley 1967, 1969; Beuzelin et al. 2009), however little is known as to whether these same effects occur against E. loftini. Our objective of this study was to assess the impact of predation by S. invicta on E. loftini in both sugarcane and a non-crop host.

Materials and Methods The first experiment was initiated in the summer of 2011 at the Texas A&M AgriLife Center at Beaumont, TX in plots of sugarcane by establishing ant suppressed and unsuppressed control areas. Ant populations were suppressed using a granule bait formulation of hydramethylnon and S-methoprene (Extinguish Plus, Zoecon Professional Products, Schaumburg, IL) which was applied to the rows and bases of the plants at a rate of 1.5 lb/a.

Fields were divided into four main plots and assigned as either ant suppressed or unsuppressed. Plots were divided into four subplots measuring 12 ft in length across three adjacent rows. Pitfall trapping was used to estimate ant densities in each subplot. At the end of the season, bored internodes and emergence holes from E. loftini were recorded on 10 randomly selected stalks from each subplot using destructive sampling. The percentage of bored internodes and number of emergence holes were compared between treatments using a generalized linear model analysis from Proc Glimmix (SAS Institute 2008). A second experiment was conducted in 2011 to assess the impact of predation by the S. invicta on E. loftini injury to a non-crop host. Two rice farms in eastern Texas were surveyed for this experiment (Chambers County, 29°51'13.02"N, 94°32'12.85"W; and Jefferson County, 30° 3'50.10"N, 94°16'53.23"W). Areas adjacent to rice fields at each farm were surveyed for weedy, non-crop hosts of E. loftini. Three transects containing the non-crop host johnsongrass, Sorghum halepense, were located along the margins of each field. Each transect measured approximately 140 ft in length and was divided into 2, 50 ft subplots, separated by a 40 ft buffer zone. Each subplot was assigned as either ant suppressed or unsuppressed. An application of ant bait was applied to subplots designated as ant suppressed. Pitfall traps were inserted into each subplot and monitored biweekly during the season to quantify densities of S. invicta. In mid-October and early November, 1 m2 samples of weeds around each trap were cut and collected to quantify borer injury. Samples were examined for borer injury. Borer injury and density were compared with the level of ant suppression using a general linear model analysis from Proc GLIMMIX (SAS Institute 2008).

Results and Discussion In sugarcane, damage to plants by E. loftini was approximately 50% greater across all ant-suppressed areas. The percentage of bored internodes averaged at 8.8% in unsuppressed plots, increasing to 13.8% in plots where ants were suppressed with insecticidal baits. Though a numerical trend was present, statistical analysis did not detect differences (F = 1.48; df = 1,1.88; P = 0.284). Differences in the number of emergence holes per stalk were detected in association with ant suppression (F = 2.43; df = 1,25.25; P = 0.023). The mean number of emergence holes per stalk across all unsuppressed areas was 0.16 and increased to 0.36 in areas where ants were suppressed. In johnsongrass, injury by E. loftini was higher between levels of ant suppression, but was not shown to be significantly different (F = 3.84; df = 1,30.65; P = 0.059). In unsuppressed plots, an average of 3.62 tillers per m2 had bored internodes, with the number of bored tillers increasing to 7.38 per m2. Leaf sheath injury was found on an average of 0.32 and 1.02 tillers per m2 in unsuppressed and suppressed plots, respectively. Although differences in overall injury (bored internodes and leaf sheath damage) were borderline on a statistical standpoint (0.1 > P > 0.05), numerical trends in borer injury associated with ant suppression should be noted. Results from this experiment suggest that predation of E. loftini by S. invicta has the potential to reduce pest build-up in sugarcane and plant injury to non-crop hosts.

References Cited Beuzelin, J. M., T. E. Reagan, W. Akbar, H. J. Cormier, J. W. Flanagan, and D. C. Blouin. 2009. Impact of Hurricane Rita storm surge on sugarcane borer (Lepidoptera: Crambidae) management in Louisiana. J. Econ. Entomol. 102: 1054–1061. Hummel, N. A., T. Hardy, T. E. Reagan, D. Pollet, C. Carlton, M. J. Stout, J. M. Beuzelin,

W. Akbar, and W. White. 2010. Monitoring and first discovery of the Mexican rice borer Eoreuma loftini (Lepidoptera: Crambidae) in Louisiana. Fla. Entomol. 93: 123–124. Johnson, K. J. R. 1985. Seasonal occurrence and insecticidal suppression of Eoreuma loftini (Lepidoptera: Pyralidae) in sugarcane. J. Econ. Entomol. 78: 960–966. Johnson, K. J. R., and M. B. van Leerdam. 1981. Range extension of Acigona loftini into the Lower Rio Grande Valley of Texas. Sugar y Azucar 76: 34. Meagher, R. L., Jr., J. W. Smith, Jr., H. W. Browning, and R. R. Saldaña. 1998. Sugarcane stemborers and their parasites in Southern Texas. Environ. Entomol. 27: 759–766. Meagher, R. L., Jr., J. W. Smith, and K. J. R. Johnson. 1994. Insecticidal management of Eoreuma loftini (Lepidoptera: Pyralidae) on Texas sugarcane: a critical review. J. Econ. Entomol. 87: 1332–1344. Negm, A. A., and S. D. Hensley. 1967. The relationship of arthropod predators to crop damage inflicted by the sugarcane borer. J. Econ. Entomol. 60: 1503–1506. Negm, A. A., and S. D. Hensley. 1969. Evaluation of certain biological control agents of the sugarcane borer in Louisiana. J. Econ. Entomol. 62: 1008–1013. Reay-Jones, F. P. F., A. T. Showler, T. E. Reagan, B. L. Legendre, M. O. Way, and E. B. Moser. 2005. Integrated tactics for managing the Mexican rice borer (Lepidoptera: Crambidae) in sugarcane. Environ. Entomol. 34: 1558–1565. Reay-Jones, F. P. F., M. O. Way, M. Sétamou, B. L. Legendre, and T. E. Reagan. 2003. Resistance to the Mexican rice borer (Lepidoptera: Crambidae) among Louisiana and Texas sugarcane cultivars. J. Econ. Entomol. 96: 1929–1934. Reay-Jones, F. P. F., L. T. Wilson, M. O. Way, T. E. Reagan, and C. E. Carlton. 2007. Movement of Mexican rice borer (Lepidoptera: Crambidae) through the Texas rice belt. J. Econ. Entomol. 100: 54–60. Wilson, B.E., J.M. Beuzelin, T.E. Reagan, and A.T. Showler. 2011. Aerial insecticidal control of the Mexican rice borer in sugarcane, 2010. Arthropod Management Tests 36: F95

Introduction of a Neurohormone in the Fire Ants

Man-Yeon Choi and Robert K. Vander Meer Center of Medical, Agricultural and Veterinary Entomology, ARS-USDA 1600 SW 23rd Drive, Gainesville, FL 32608

Abstract Neuropeptides are the largest group of insect hormones, and are synthesized in the central and peripheral nervous systems. One neuropeptide family is the PBAN/pyrokinin family defined by a common FXPRLamide or similar amino acid fragment at the C-terminal end. Over the past years we have extended knowledge of the PBAN/pyrokinin family of peptides to ants, focusing mainly on the fire ant, Solenopsis invicta. The fire ant is a well studied and good model as social insect. We introduce here our research to date on the molecular structure, diversity, and characterization of PBAN and additional peptides as well as determine the potential function and application of these peptides for fire ant control.

Introduction Insect neurohormones function as intercellular communicators that regulate a variety of physiological and behavioral events during development and reproduction. The Pheromone Biosynthesis Activating Neuropeptide (PBAN)/pyrokinin family is a major class of insect neuropeptide hormones that is synthesized in central nervous system (CNS) including subesophageal ganglion (SG), and released into the hemolymph. These peptides have been conserved throughout evolution, and are expected to be present in all insects. PBAN/Pyrokinin family peptides regulate multiple physiological functions including: pheromone biosynthesis, melanization, embryonic diapause, and insect development. Social insects depend on sophisticated pheromonal communication to maintain colony cohesiveness and sociality. The fire ant is probably the most studied ant species in the world and a great deal has been published on the identification and characterization of fire ant pheromone systems, e.g. a variety of queen produced primer pheromones, and queen and worker releaser pheromones. Virtually nothing is known about how pheromone production and release is regulated. Recently, we demonstrated the presence of PBAN/pyrokinin immunoreactive neurons in the fire ant, and identified and characterized PBAN and additional neuropeptides. We have mapped the fire ant PBAN gene structure and determined the tissue expression level in the central nervous system of the ant. Here we report on the molecular structure and diversity of fire ant PBAN/pyrokinin peptides and potential application using insect neuropeptides.

Materials and Methods Insect: Field collected monogyne fire ant colonies (Solenopsis invicta) were maintained in the lab using standard procedures. Fire ant brain-subesophageal (Br-SG) was dissected from male and female sexual forms, as well as sterile workers, and used to isolate mRNA and synthesis cDNA. Whole-mount immunocytochemistry: Fire ant Br–SG and ventral nerve cord were dissected in a cold saline and fixed for 3 h in 10% formalinphosphate-buffered saline (PBS: 100 mM NaH2PO4, 150 mM NaCl, 3 mM KCl, pH 7.4). The tissues were incubated overnight in PBS containing 2% triton X-100 (PBS-T). Subsequently, tissues were incubated for 4–6 h in PBAN

anti-serum (diluted 1:2000), goat anti-rabbit anti-serum-peroxidase (Sigma) (diluted 1:2000), and horseradish peroxidase-conjugated rabbit anti-peroxidase (Sigma) (diluted 1:400) in PBS-T after washing with PBS-T overnight with slow agitation at room temperature between each incubation step. After the last incubation, the tissues were washed with PBS and then incubated in 50 mM Tris–HCl buffer (pH 7.6) for 10 min. Visualization of the immunoreactivity was performed with diaminobenzidine and H2O2 (Sigma) for about 3 min. After satisfactory color development was achieved, the tissues were transferred to PBS, then dehydrated placed in a graded series 40–80% glycerol. Tissues were examined under a Nikon Eclipse E800 microscope equipped with a digital camera. Reverse Transcriptase (RT) –PCR: Total RNA was isolated from head, thorax, and abdomen of female alates using the PureLink™ RNA Kit (Invitrogen). The total RNA (50ng) was used to amplify a 501-bp DNA fragment of Soi-PBAN with a Soi-PBAN specific primer set (5’-AGGAATTCCGTTAATCGTGC-3’ and 5’-GTTGCGT AATTGACGTCCG-3’). A 100-bp fragment of the fire ant 18S rRNA gene was also amplified for a positive control using primers (5’-CCCGTAATCGGAATGAG TACACTTT-3’ and 5’-ACGCTATT GGAGCTGGAATTACC-3’). The one-step RT-PCR was performed as follows: 1 cycle at 50°C for 30min, 40 cycles at 95°C for 30s, 50°C for 30s, and 72°C for 1 min using RT/Taq mix polymerase (Invitrogen).

Results and Discussion 1. Three groups of cell clusters found in the SG corresponded to the mandibular, maxillary and labial neurons as in Lepidoptera. PBAN-like immunoreactive neurons in the VNC were found in thoracic (two pairs) and abdominal ganglia (three pairs). 2. Identified fire ant PBAN cDNA, encoding 176 amino acids including PBAN and three additional neuropeptides. 3. The structure and pre-prohormone positions of the fire ant PBAN gene are more similar to Lepidoptera PBAN than the PBAN found in other insect groups. 4. Difference between immunocytochemistry and PBAN gene expression by RT-PCR suggest that a FXPRL peptide is being dominantly expressed in the abdomen that is derived from a gene other than the Soi-PBAN gene.

To clarify specific physiological modes of action of PBAN/pyrokinin peptides in the fire ant the identification and characterization of a specific receptor(s) for these peptides is required. The study includes measurement of binding activity of PBAN and other peptides to the PBAN receptor expressed in an in vitro system, and determination of the receptor location, e.g. tissue, gland, or organ, which would suggest a potential target site and role for PBAN/pyrokinin peptides. Our study here lays the groundwork necessary for successfully determination of specific functions, and may lead to novel control invasive ants.

Large-Scale Analysis of Fire Ant Pheromones: A Progress Report

Robert Renthal University of Texas at San Antonio, San Antonio, TX 78249

Ants may use short-range chemical signaling for nestmate recognition and task coordination. We previously identified basiconic sensilla on the distal antennal segment of red imported fire ants as likely sites for contact chemoreceptors (Renthal et al. 2003), and a similar conclusion was reached in studies of carpenter ants (Ozaki et al. 2005). We are interested in identifying the nature of short-range chemical signals that are displayed on fire ant cuticle and received by antennal contact. Nestmate recognition by carpenter ants involves a mixture of 18 alkane components displayed on the cuticle (Ozaki et al. 2005). By contrast, the 5 alkanes on Solenopsis invicta cuticle may be too sparse for a signaling system. Thus, we have begun an analysis of the fire ant's polar cuticular lipids (fatty acids and esters) to test their involvement in nestmate recognition and other signaling. We developed an HPLC method involving four-solvent gradient elution from a C-18 reversed phase column with evaporative light-scattering detection to isolate more than 20 different components in the polar fraction of fire ant cuticular lipids (see figure). To select candidate components for behavioral testing, we assume that compounds important in contact chemoreception will bind strongly to antennal olfactory binding proteins. Using proteomics analysis, we identified two chemosensory proteins and three odorant-binding proteins in the fire ant antenna (González et al. 2009). We expressed four of these proteins using recombinant DNA methods and purified them by chromatography. We have begun binding studies, using an ultrafiltration method (Leal et al. 2005) modified with cyclodextrins to disperse and transfer the water-insoluble lipids (González et al. 2009). Preliminary results show several different polar lipid components bind to the two major antennal chemosensory proteins.

References Cited González, D., Q. Zhao, C. McMahan, D. Velasquez, W. E. Haskins, V. Sponsel, J. A. Cassill, and R. Renthal. 2009. The major antennal chemosensory protein of red imported fire ant workers. Insect Mol. Biol. 18: 395−4044. Leal, W. S., A. M. Chen, Y. Ishida, V. P. Chaing, M. L. Erickson, T. I. Morgan, and J. M. Tsuruda. 2005. Proc. Natl. Acad. Sci. USA 102: 5386−5391. Ozaki, M., A. Wada-Katsumata, K. Fujikawa, M. Iwasaki, F. Yokohari, Y. Satoji, T. Nisimura, and R. Yamaoka. 2005. Ant nestmate and non-nestmate discrimination by a chemosensory sensillum. Sci. 309: 311−314. Renthal, R., D. Velasquez, D. Olmos, J. Hampton, and W. P. Wergin. 2003. Sensilla structures and patterns on the antenna of the red imported fire ant. Micron 34: 405−413.

Figure: HPLC analysis of S. invicta cuticular lipids. Pentane wash of cuticle was fractionated on silica and polar lipids were applied to C-18 reversed phase column. Eluted with water-acetonitrile-isopropanol- chloroform gradient using evaporative light- scattering detection.

Sublethal Dose Insecticides Affect Fire-ant Foraging and Recruiting Efficiency

Xing Ping Hu and Zhonglin Yuan Department of Entomology and Plant Pathology Auburn University, Auburn AL 36849, email: [email protected]

Abstract Fire ants were exposed to Amdro bait (hydramethylnon, ai) for 12 h before replacing bait with a food source (crushed potato chips). The post-treatment foraging and recruiting behaviors of foraging workers were examined to determine the sublethal effects on foragers’ food foraging efficacy and locomotor. Exposed workers removed significantly smaller amount of food chips compared with unexposed workers. The time for the exposed workers to walk the distance from harborage to food source was twice longer than for the unexposed workers. At the food source, the exposed workers spent much longer time on examining the food before picking it up and carrying it to harborage. Additionally, it took a significantly longer time (3 times more) for the exposed workers to carry the food to harborage than the unexposed workers. These results indicate that at sublethal dose, Amdro bait significantly reduced fire ant food foraging efficacy by reducing their locomotor activity not by impairing their food perception or olfactory learning ability.

Influence of Genetic Variation on Colony-Level Foraging Behavior of the Red Imported Fire Ant

Alison A. Bockoven and Micky D. Eubanks Texas A&M University, Department of Entomology, College Station, TX 77843

Abstract: Among social insects, genetic variation in a behavior can allow a colony to exhibit more flexible, rapid responses to environmental changes and promote homeostasis. Such variation enables adaptive evolution and can increase colony fitness in multilineage colonies such as polygynous red imported fire ants (Solenopsis invicta). Our preliminary research demonstrated significant and persistent colony-level variation in fire ant foraging behavior. Variation in foraging behaviors and division of labor in some social insects has been associated with variation in expression of the foraging gene (for). We examined the effects of cGMP-dependent protein kinase G (PKG), the enzyme product of for, on foraging behavior by treating lab colonies with 8- Br-cGMP to artificially activate PKG. Behavioral assays revealed that this treatment had a significant effect on fire ant recruitment and predation compared to control colonies. Notably, changes in fire ant foraging behavior had significant indirect effects on plant damage due to herbivory. We founded single lineage colonies and assayed colony-level variation in foraging. Genetic lineage explained approximately 50% of variation observed in foraging behaviors. Currently, we are quantifying colony-level variation in expression of the fire ant foraging gene. This study suggests that significant variation in foraging behavior exists among fire ant colonies and may underlie variation in the ecological effects of fire ants and variation in methods necessary for their control.

“Fighting Texas’ Fire Ants: The Team Approach” 10 Years and Counting – Renewing Interest in the Lago Santa Fe Fire Ant Project

Paul R. Nester and Bastiaan “Bart” M. Drees Texas AgriLife Extension Service

Managing the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) through broadcast applications of fire ant bait products has been demonstrated to dramatically reduce the cost, insecticide use, maintain control of fire ants, and help eliminate problems caused by the fire ant (Riggs et.al, 2002). The Lago Santa Fe Fire Ant Project is a good example of a successful community-wide fire ant management program where the bi-annual broadcasting of a fire ant bait product (Extinguish® Plus) has resulted in continued suppression of a fire ant population. Lago Santa Fe is a Private Lake Community, situated 25 miles, South/Southwest of Houston, TX, in Galveston Co. The Community consists of 100 acres with four, ½ mile long X 200 ft wide lakes, with 48, one acre lots that border the lakes and 12 lots that do not border the lakes (Nester et.al, 2003). Historical Perspective, 2001 – 2002. In 2001, Lago Santa Fe (Figure 1) was chosen as the site to host the 2002 National Water Ski Championships and the 2002 U.S. Open Water Ski Championships. This meant that in August of 2002, 1000 participants from all over the U.S. and the World, with up to 4,000 spectators would invade this 100 acre area for 7 days. Initial fire ant mound activity evaluations showed over 160 large (> 12” diameter) active fire ant mounds per acre (Table 1). The Lago Santa Fe Community realized they needed a coordinated approach to manage these pests. The Texas AgriLife Extension Service decided to work with the residents of Lago Santa Fe and develop an annual repeatable process for the management of the fire ants in this community. The Lago Santa Fe Fire Ant Project gave The Texas AgriLife Extension Service a chance to “showcase” various ideas, practices, products, and product uses developed by the Texas Imported Fire Ant Research and Management Plan which included:

1) Evaluation of the effectiveness of the "hopper blend" treatment (50:50 hydramethylnon plus s-methoprene ant bait later released as Extinguish® Plus Fire Ant Bait) at 1.5 lb blended product/acre. 2) Demonstration of application methods such as the truck-mountable industrial "bait blower"; and the ATV mounted Herd Broadcast Spreader. 3) Demonstration of the practicality of scheduling fire ant bait treatments to reach a goal of maximum control for the athletic events being hosted by the Lago Santa Fe Community, i.e., the 2002 and 2003 National Water Ski Championships, and U.S. Open Water Ski Championships. 4) Demonstration of how coordinating the efforts of the entire community resulted in an efficient process for managing fire ants over the Lago Santa Fe property.

Fire Ant mound activity counts showed that after a single spring 2002 hopper blend treatment, fire ant activity was reduced 85% in the community of Lago Santa Fe before the scheduled 2002 water ski events (Table 2). Full report can be found at http://www.extension.org/sites/default/files/w/0/03/2003_IFA_Conference_Proceedings.pdf. 2002 – 2004 ongoing program treatments: This community was also chosen to host these same events in the summer of 2003. In the succeeding years after the successfully

implementation of the Lago Santa Fe fire ant project the community reduced the highs and lowered the lows of the yearly fluctuations in fire ant numbers (Table 2). After an additional fall 2002 and spring 2003 hopper blend treatment, fire ant mound activity was down 95%, before the scheduled 2003 water ski events. The community has continued to manage their fire ants. After 5 scheduled baiting events (April 2002 through May 2004) the fire ant mound activity reduction remained above 90% when compared to initial fire ant activity recorded in the spring of 2002 (Table 2). 2004 – 2008 ongoing program treatments: Lago Santa Fe residents continue their efforts to manage the fire ant with bi-annual applications of a fire ant bait product. Lago Santa Fe residents pay quarterly dues to the Property Owners Association (POA). Fire ant baiting is funded from the general dues just like mowing, lake dye, insurance, electricity etc. Fire ant bait is a line item in the budget that is approved annually by the Board of Directors (BOD). The most recent (2011) fire ant budget consists of seven 25 pound bags of fire ant bait, miscellaneous supplies and fuel for ATV on which a Herd G-77 Broadcast Seeder is mounted. It is estimated that each property owner will pay about $20 for fire ant management every 6 months for a total cost of $40/year/property (Table 3). In a 2008, the Lago Santa Fe Fire Ant Project was re-visited and the residents surveyed as to their satisfaction of this project over the past years (Nester et.al, 2008). The residents (11 responses from 40 property owners, 28%) all responded (100%) that they felt the dollars spent by the POA for the Lago Santa Fe Fire Ant Project was cost-effective and all responded (100%) that they wanted it to continue. All (100%) ranked the project from good to excellent and 10 of the residents (91%) felt very positive about this fire ant baiting program being supported by the Texas AgriLife Extension Service. One of the residents was somewhat positive. One comment from a resident was, “Prior to moving to TX / Lago Santa Fe a few years ago, we had never lived anywhere that had a fire ant problem. We’ve only been exposed to the problem and solutions since living here but have heard numerous stories from neighbors as to how bad it was and how it is today. We certainly appreciate all the efforts made by Texas AgriLife Extension Service and Jay and Lydia Gilbert” (Note: all comments and survey results can be viewed at: http://fireant.tamu.edu/research/projects/pdf/ipmmanual08a1.pdf ). 2010 to present program activities: Extenuating circumstances kept the original organizers from coordinating the baiting event in the fall of 2010. Since other community members did not pick up the effort, the fall 2010 community-wide baiting event did not happen. As the year ended and the new (2011) year began, there was chatter in the community indicating that fire ant populations were increasing on the various properties, in numbers not seen in previous years (personal communication with Jay Gilbert, Lago Santa Fe resident, fall 2010). When communicating with Jay Gilbert, one of the original organizers of the Lago Santa Fe Fire Ant Project, it was decided to try and renew interest in the community-wide project by making a concerted effort in the spring of 2011 (10 years after the first fire ant baiting event) to make all residents aware of the importance of the bi-annual baiting by organizing an event and taking before and after observations of fire ant activity.

Materials and Methods A date to spread the Extinguish® Plus Fire Ant Bait product (hydramethylnon + s- methoprene), donated by Central Life Sciences, Dallas, TX, over the Lago Santa Fe community was set for April 27, 2011. These are the only fire ant bait active ingredients that had been used on this property in previous years. Pre-treatment fire ant mound activity observations were taken using the minimal disturbance method April 25, 2011 (Table 4) from 0.25 acre circles from 6 properties within the Lago Santa Fe community (a reduction in plot numbers due to houses constructed on previously vacant lots). As in previous years, fire ant activity observations and ant mound counts were also taken from four 0.25 acre circles in an adjacent untreated pasture to serve as a control plot area. These same treated and untreated plot locations were checked 90 days later for fire ant mound activity. At the same time we conducted active ant mound counts in plots, we measured fire ant foraging activity by placing 20 hot dog food lures (0.25 inch thick slices of Bar-S Jumbo Franks) in the front yards of 20 of the interior properties that border the lakes. After 60 minutes, the total estimated number of fire ants per lure was recorded (Table 5). Food lures were also used 90 days later to check for fire ant foraging activity. This time the Lago Santa Fe Fire Ant Project gave The Texas AgriLife Extension Service a chance to again:

1) Showcase The Community –Wide fire ant baiting concept promoted by the Texas AgriLife Extension Service. 2) Demonstrate how coordinating the efforts of the entire community can result in an efficient process for managing fire ants over the Lago Santa Fe property. 3) Evaluate if tolerance or resistance (behavioral or physiological) of the fire ants in this community to the active ingredients hydramethylnon and s-methoprene was occurring.

Results and Discussion What started out to be a promising year for obtaining fire ant management data turned into a record setting year for Texas by having the hottest average temperature for June through August according to National Weather climatologists. The spring and summer of 2011 was extremely dry in south Texas. One would assume that the irrigation of lawns would attract fire ants. The lawns of Lago Santa Fe were no exception. Good fire ant activity was seen April 25, 2011 (Table 5). Of the 20 food lures, 19 (95%) of them had an average of 54 fire ants per lure. In April 2002 over 160 large (> 12” diameter) active fire ant mounds per acre were observed. The mounds (136/acre) observed in April 2011 were not overly large but they were present (Table 1). Fire ant mound activity counts taken in an adjacent pasture also showed good activity at 112 active fire ant mounds per acre. The dry conditions did have an effect on mound survivability as seen by the 36% reduction in activity with no bait application in the adjacent pasture. Greater than 90% reduction of the fire ant mound activity was observed in the treated properties of Lago Santa Fe when compared to pre-treatment counts (Table 4), and a 74% reduction in food lure hits was observed (Table 5). Were the residents of Lago Santa Fe satisfied with the 2011 effort? An email from Jay Gilbert, one of the originators of the Lago Santa Fe Fire Ant Project said, “Fall baiting was in October 2011 and was facilitated with 4 block captains. Utilizing block captains reduced the burden on the 2 of us and we are more willing to continue overseeing the semi-annual baiting.

The Fall baiting appeared successful - as there were no complaints compared to lots of negative chatter last year. You will recall we skipped the fall baiting that year (2010) and the ants really were a problem. I have only spotted a couple of nuisance mounds following a 6.5" rain event Jan 9, 2012. We will go after them again in the Spring.” Block captains were chosen to help facilitate future fire ant management events. (Appendix I). Jay summed it up when he said, “We will go after them in the spring!”

Conclusions: • The number of observation sites (sample size) was reduced for 2011 counts due to construction on previously empty lots but did not limit fire ant activity assessment. • Despite drought, populations in untreated area (pastureland) remained unchanged, statistically. • Percent reduction from April 25, 2011 to July 27, 2011 was 94.7% (versus 85.5% reduction after initial treatment, April 18, 2002 to July 7, 2002), indicating that after annual multiple application, Extinguish® Plus continues to perform over 10 years with no indication of resistance by imported fire ant populations. • The Community-Wide fire ant management concept as promoted by the Texas AgriLife Extension Service remains a viable solution to the management of the red imported fire ants in a community setting.

Acknowledgements The authors want to thank the residents of Lago Santa Fe for allowing them access to their community over the past years to document events and follow the effects of their fire ant management attempts. Special thanks go to Jay and Lydia Gilbert for their willingness to meet with the authors when necessary and help with fire ant activity evaluations. Central Life Sciences (Doug Van Gundy) is thanked for the generous donation of fire ant bait product when it was needed.

References Cited Nester, P. R., C. P. Bowen, B. M. Drees. 2003. The Lago Santa Fe Fire Ant Project: An example of Community-wide imported fire ant management in Texas. 2003 Proc. Red Imported Fire Ant Conf. Nester, P. R., C. P. Bowen, B. M. Drees. 2008. “Fighting Texas’ Fire Ants: the Team Approach” A six year review of the Lago Santa Fe Fire Ant Project. Pg 36-42. Urban IPM Summary report. Texas AgriLife Extension Service posted at: http://fireant.tamu.edu/research/projects/pdf/ipmmanual08a1.pdf Riggs, N. L., L. Lennon, C. L. Barr, B. M. Drees, S. Cummings, and C. Lard. 2002. Community-wide red imported fire ant programs in Texas (B. M. Drees, ed.). Southwestern Entomol. Supplement No. 25:31-42 posted at: http://www.extension.org/sites/default/files/w/0/03/2003_IFA_Conference_Proceedings.pdf.

Table1: Average active mounds per acre in 2011 before baiting (pre) and after baiting (post) based on number of active mounds in 0.25 acre circles, as compared to counts taken in 2002.

Average Active Mounds per Acre (Percent Reduction) Locations 4/18/2002 7/17/2002 4/25/2011 7/27/2011 (pre) (post) Lago properties 169 25 136 8 (94%) Untreated adjacent pasture 87 75 112 72 (36%)

Table 2. Red imported fire ant mounds per 0.25 acre circle plot, Lago Santa Fe, Galveston Co., Texas, treated with a hopper blend of hydramethylnon fire ant bait (Amdro®Pro or Probait™) plus s-methoprene fire ant bait (Extinguish™) at 0.75 lbs of each product per acre through fall of 2003 or Extinguish® Plus Fire Ant Bait (s-methoprene + hydramethylnon) at 1.5 lb product/acre in 2004*. Observations on active fire ant mounds were taken at indicated intervals (weeks after treatment = WAT). Number of Red Imported Fire Ant Mounds/0.25 Acre Lot Number I* II III IV V Treated area: 32 38 8 1 2 13 25 48 15 1 5 14 24 32 11 3 8 20 23 29 11 5 8 17 20 41 16 8 14 7 2 55 23 9 3 23 1 48 18 4 4 26 46 47 8 6 5 11 Mean + Stand. Dev. 42.25 + 8.88 13.75** + 5.23 4.62** + 2.97 6.13** + 3.83 16.38** + 6.37 T = 7.8243 11.3686 10.5679 6.6994 n = 8; d. f. = 14; P = 0.0000 0.0000 0.0000 0.0000 Percent reduction: -67.46% -89.07% -85.50% -61.23%

Untreated area (plot): 1 34 27 28 - 24 2 27 28 17 - 39 3 12 10 13 - 15 4 14 15 17 - 15 Mean + Stand. Dev. 21.75 + 10.53 20.00***+ 8.91 18.75*** + 6.44 23.25*** + 11.32 T = 0.2537 0.4859 -0.1940 n = 4; d. f. = 6; P = 0.4041 0.3221 0.4263 Percent reduction: -8.10% -13.8% -6.8%

*I = 4/18/02 (pre-treatment), II = 5/28/02 (6 WAT), III = 6/12/02 (8 WAT), IV = 7/17/02 (12 WAT), V = 9/26/02 (22 WAT, pre- fall treatment) ** Mean significantly different (P < 0.05) from pre-treatment count (4/18/02) mean using the Student T test (Microstat). *** No significant reduction in mean number of fire ant mounds per plot

Number of Red Imported Fire Ant Mounds/0.25 Acre Lot Number VI* VII VIII IX X Treated area: 32 0 0 6 3 0 25 4 0 2 0 1 24 13 0 4 0 0 23 13 0 3 7 2 20 5 1 20 3 2 2 8 1 20 46 10 1 2 0 8 9 5 46 5 0 11 4 2 Mean + Stand. Dev. 6.25 + 4.77 0.25 + 0.46 9.25 + 7.23 9.00 + 15.27 2.75 + 3.33 T = -10.1033 -13.3654 -8.155 5.325 11.786 n = 8; d. f. = 14; P = 0.00** 0.00** 0.00** 0.00** 0.00** Percent reduction: -85.2% -99.4% -78.1% -78.7% -93.5%

Untreated area (plot): 1 43 11 15 41 23 2 38 12 12 26 15 3 16 10 14 17 6 4 14 16 19 24 15 Mean + Stand. Dev. 27.75*** + 14.89 12.25*** + 2.63 15.00 ***+ 2.94 27.00***+10.10 14.75***+6.95 T = 0.6581 -1.7503 -1.2345 -0.720 1.110 n = 4; d. f. = 6; P = 0.2674 0.0653 0.1316 0.499 0.310 Percent reduction: 0.8% -43.7% -31.0% +19.4% -32.2%

* VI = 5/2/03 (pre spring treatment, 31 WAFT), VII = 7/1/03 (60 days after spring treatment, 5/3/03), VIII = 9/19/03 (pre fall application, 9/11/03), IX = 4/23/04 (pre spring application), X=9/17/04 (pre fall application) ** Mean significantly different (P < 0.05) from pre-treatment count (4/18/02) mean using the Student T test (Microstat). *** No significant reduction in mean number of fire ant mounds per plot from pre-treatment counts (4/18/02)

Table 3: Example of total actual costs paid by Lago Santa Fe POA for fire ant management: (Includes 25# bags of Extinguish® Plus ant bait, misc. supplies, fuel for ATV). Year Actual Amount of product Comments Cost 2007 $2,015 2 X 6 bags 2008 $2,042 2 X 6 bags 2009 $2,043 2 X 6 bags 2010 $1,217 1 X 7 bags Spring treatment only, fall treatment skipped 2011 $2,528 2 X 7 bags Estimated cost, increased rate from 1.5# to 2#/acre

Table 4. Red imported fire ant mounds per 0.25 acre circle plot, Lago Santa Fe, Galveston Co., Texas, treated with Extinguish® Plus Fire Ant Bait (s-methoprene + hydramethylnon, 1.5 lb product/acre) on 4/27/11*. Observations on active fire ant mounds were taken 91 days after application. Number of Red Imported Fire Ant Mounds/0.25 Acre Lot Number XI* XII Treated area: 32 4 0 28 11 0 22 39 3 11 63 3 2 31 5 46 58 0 Mean + Stand. Dev. 34.33 + 24.00 1.83 + 2.14 T = 0.0867 10.817 n = 6; d. f. = ; P = 0.403 0.000** Percent reduction: 18.74 95.67

Untreated area (plot): 1 37 28 2 27 16 3 21 12 4 27 17 Mean + Stand. Dev. 28.00 + 6.63 18.25 +6.85 T = -1.004 0.557 n = 4; d. f. = 6; P = 0.354*** 0.598*** Percent reduction: 0.00 16.09

* XI = 4/25/11 (pre spring 2011 application), XII = 7/27/11 (91 days after spring 2011 application) ** Mean significantly different (P < 0.05) from pre-treatment count (4/18/02) mean using the Student T test (SPSS 0.19). *** No significant reduction in mean number of fire ant mounds per plot from pre-treatment counts (4/18/02)

Table 5: Observations from 20 food lure stations (0.25 inch hot dog slices) placed in front yards of 20 properties lining the interior lakes of Lago Santa Fe Private Lake Community, Santa Fe, TX, Galveston County.

Evaluation Total Avg. RIFA % RIFA Total Avg # % dates RIFA on per Lure reduction RIFA hits RIFA on reduction lures on lures on lures hit lures lure hits 4/25/2011 1085 54 -- 19 57 -- 7/27/2011 300 15 72 5 60 74

Figure 1. Aerial view of Lago Santa Fe subdivision, Galveston Co., TX, site of a community wide fire ant management program, 2002-2012. Locations of 6 evaluation sites and 4 check sites for 2011 activity assessments are identified in image.

Appendix I: Example of advisory email sent to Lago Santa Fe Residents to alert them of the community fire ant baiting event in fall of 2011.

Email text:

Next weekend (Oct 14-16, 2011) is the community wide fall fire ant bait spreading time. Thanks again to our Fire Ant Captains: Jimmy Baker, Cathie Johnson, Danny LeBourgeois and David Pratt. Please contact them directly if you can help with some of the common areas or absentee neighbor lots. Keep in mind you may volunteer for an area with a different captain than the one from whom you receive your bait.

The captains are responsible for distributing the bait and lining up volunteers; they are not personally treating all the common areas and empty lots. We have had good support from the community volunteers in the past and the more volunteers we have, the more likely it is that all areas are treated and the baiting will be successful. If you have not volunteered previously, please consider signing up, more helpers makes the job easier for all.

Thanks,

Lydia and Jay Gilbert

Please look at the list and baiting tips below. If you are a lot owner or will be absent, please email your Captain to advise if you will arrange baiting on your own or if a volunteer needs to be arranged. Everyone else, please see if there is a specific empty lot or common area that you could treat, and email Jimmy, Cathie, Danny or David.

Jimmy Baker - Fire Ant Bait Captain-

Empty Lots 223 2 Lago Cove 9 Lago Cove 226

Common Areas Peninsula between Lake 2 & 3 Lake 4 Dock Bridge

Homeowners 1 6 10 13 218 222

303 306 307 314 318 403 ======Cathie Johnson – Fire Ant Bait Captain -

Empty Lots 135 – 142 - 214 –

Common Areas Lake 2 Islands Lake 2 Dock Strip between 147 & 203

Homeowners 127 130 131 134 138 139 143 Crook 146 147 202 203 206 210 211 426 ======Danny LeBourgeois – Fire Ant Bait Captain -

Empty Lots 311 315

Common Areas Lake 3 & 4 Islands Triangle Lake 3 Dock

Homeowners 319 322 323 326 Wehe 327 330 331 334 335 402 406 407 410 411 ======

David Pratt – Fire Ant Bait Captain -

Empty Lots 102 – 122

Common Areas Next to Cemetery Rd Front Gate Flower Beds Strip between 114 Gass & 423 Gonzalez Lake 1 Islands

Homeowners 106 107 110 114 115 119 123 415 418 419 422 423

Remember the following tips: * It is better not to mow or otherwise disturb mounds 48 hours before baiting as ants will rebuild their mound rather than take the bait. You can mow the following day.

* Bait should go on and stay dry - turn off sprinklers (lawn and septic) or place buckets over the heads. Please keep dry for 24 hrs if possible but 12 hours is OK. Wait for morning dew to clear before application. * Ants generally eat in the evenings so it's optimal to put out bait later in the day so it does not spoil in the sun/heat before they take it that night. *The opening of your hand held spreader (push spreader will not work) should be set at about 1/4 to 1/3 of an inch which is #1 or #2 generally.

Dr Nester confirmed that it is proper to bait near your vegetable gardens but not directly into the garden itself. He also advises starting by baiting the flower beds around the perimeter of your house. Next bait your property line and work in a spiral towards the center of your lot. This will make sure that if you run out of bait before you finish that you have all areas covered. We are providing 2# bags of Extinguish Plus ant bait, the recommended application is 1.5 to 2 lbs per acre.

Death by 1000 cuts: Continuing to Assemble Communities of Phorid Fly Parasitoids for the Biological Control of Imported Fire Ants

Edward G. LeBrun,1 Robert M. Plowes,1 and Lawrence E. Gilbert2 1 Brackenridge Field Laboratory, Section of Integrative Biology, University of Texas at Austin, 2907 Lake Austin Blvd, Austin, TX 78703. 2 Organizational Director, Section of Integrative Biology, University of Texas at Austin, 1 University Station #C0930, Austin, TX 78712

In its native range in Northern Argentina and southern Brazil, imported fire ants host a wide variety of natural enemies. In total there are over 41 species of organisms known to exploit S. invicta and its close allies. 23 of these natural enemy species, 53%, are phorid flies in the genus Pseudacteon (summarized from Tschinkel 2006, Briano et al. 2012). To date, 6 Pseudacteon species have been successfully imported, released, and established in North America from South America source populations (Porter et al. 2004, Gilbert et al. 2008, LeBrun et al. 2008, Porter 2010, Plowes et al. 2011, Porter et al. 2011). In the over 90 years that South American Saevissima complex fire ants have been present in the United States, these flies represent the only natural enemies to have been successfully tested for specificity, reared, released and established in North America. Thus the importation, culture, and release operations operating out of the Brackenridge Field Laboratory in Austin Texas and out of the USDA ARS laboratory in Gainesville Florida constitute the only mature form of biological control technology available globally for imported fire ants. In this summary we will briefly describe the progress to date in establishing Pseudacteon fly species in Texas and outline the direction our organization is taking with respect to further introductions of these parasitoids. Phorid flies are parasitoids of adult fire ant workers. Over long distance, they locate their hosts using olfaction exploiting the semio-chemicals that fire ants use to organize their labor. They primarily exploit the pheromones associated with foraging and nest defense. Flies oviposit on the wing by injecting an egg into the thorax of fire ant workers. The larval developmental window takes about 2 weeks. Larvae migrate to the head capsule and the first couple of instars feed primarily on hemolymph. Shortly before pupariation the larvae induce the ant to leave the colony and seek out an appropriate place for pupariation (Henne and Johnson 2007). They then release an enzyme which decapitates the ant and use the head capsule of their host as a puparium. The pupal stage lasts another 2 weeks for a total immature life cycle of about a month. Adults live for only a few days (reviewed in Porter 1998). Twenty-three described species of Pseudacteon attack imported fire ants in their native range. Up to 15 species are documented to co-occur in long-term samples of a single site (Folgarait et al. 2007) and up to 9 species in short term samples (Calcaterra et al. 2005). These high levels of alpha diversity are maintained, at least in part, through a wide variety of niche partitioning mechanisms. Species differ in the size of fire ant workers they parasitize, time of day and season of the year in which they are active, their mating behaviors, their ability to tolerate abiotically stressful conditions, and, mediated by the types of pheromones they exploit for host location, and the context in which they locate their hosts (Porter 1998). Depending on the semio-chemicals they rely upon phorids locate and attack their hosts either at foraging trails or disturbed mounds. All species will sometimes locate their hosts in either of these contexts, however the relative preference and thus prevalence of species at disturbed mounds versus foraging trials varies widely (Orr et al. 1997). In their entirety, they create an assemblage of

complementary parts that impacts fire ant populations in an additive manner although with some functional overlap between species. A putative additional role played by flies may be vectoring of certain pathogens between colonies (Valles and Porter 2007). The long-term goal of our laboratory is to recreate the most functionally critical parts of this complex system in North America. Pseudacteon parasitoids exert two types of costs on fire ant colonies. They kill workers directly reducing colony growth rates and they alter the behavior of fire ants in ways that compromise colony function thereby indirectly reducing colony growth rates. Measures of direct loss of workers to parasitism range from 0 – 3% of workers within a colony are infected at any given time (Morrison et al. 1997, Calcaterra et al. 2008). This is equivalent to a cropping of 0- 3% of the worker population every 2 weeks. The variability in this estimate is likely primarily driven by the very high level of variability in Pseudacteon population abundance. The primary way that Pseudacteon indirectly impact colony growth rates is by disrupting foraging (Orr et al. 1995). Over short periods, they can reduce resource harvesting rate by up to 84% in a field setting (Feener and Brown 1992). This short-term reduction in harvesting is accompanied by an increase in resource harvesting by competitor ant species (Orr et al. 1995). In the laboratory, long-term exposure to flies can reduce colony protein intake by up to 50% (Mehdiabadi and Gilbert 2002). These effects of phorid attack likely exert a larger cost on fire ant colony growth rates than do the loss of worker tissue to parasitism. These costs are also likely to be proportionately greater on small and founding colonies than on larger colonies, and on colonies undergoing other biotic and abiotic stresses. To maximize the impacts of introduced Pseudacteon on imported fire ants, we must create a Pseudacteon assemblage containing species that exploit a wide variety of worker size classes, that locate and attack them both at mound disturbances and foraging trails, are active during most of the year, and that can tolerate a wide variety of climatic conditions. As the result of ongoing efforts since 1995, we currently have 4 species established in Texas: P. tricuspis, P. curvatus, P. obtusus, and the recently established P. nocens. P. tricuspis, and P. curvatus are widespread, occupying most of the area of Texas that is invaded by imported fire ants. We have established P. obtusus at 11 sites around Texas, it is spreading locally and will soon be regionally widespread {Plowes, 2011 #557}. P. nocens is currently established at a single site. Table 1 summarizes the biology of these 4 established species and the niche axes that they currently fill in Texas. Two critical niche axes remain to be filled. First, currently there is no species established in North America that locates fire ants at foraging trails and attacks small fire ant workers. From the perspective of the functional impacts of Pseudacteon on fire ant populations, this is a huge gap in our North American assemblage. The vast majority of fire ants in the colony are small, and these ants are the primary foragers within the colony and thus largely responsible for colony energy intake (Tschinkel 2006). While these small foraging workers may be close to the end of their lifespan (polyethism), any parasitoid induced limitation of their abilities to gather resources may have important colony level impacts. Thus the agents thought responsible for propagating the most significant indirect effects on colony growth rate are currently missing in North America. Second, among the currently established species, in typical years in Central Texas, none reach high abundance before the late spring and none maintain a presence through the winter. To fill these functionally significant and currently empty North American niche axes, we are targeting two species of small bodied, trail orienting phorid flies for importation and release

in Texas: P. nudicornis and P. small obtusus (Table 1). P. small obtusus is a discrete but much smaller species than P. obtusus (Kronforst et al. 2007) that possesses similar but not identical morphological traits. Successfully establishing either of these species in North America would represent an important step forward in limiting the foraging efficiency of S. invicta. Other species are being evaluated for their potential to buffer the gaps in the currently highly variable population abundances of the established Pseudacteon assemblage. In particular, species like P. borgmeieri that are abundant during the cool season would allow for more continuous pressure annually and attack imported fire ants during the critical early spring period when their colonies are gathering resources to be allocated towards reproduction. Of the species introduced to date, an important dynamic has been observed where in central Texas P. tricuspis has been displaced by P. curvatus due to indirect competition (LeBrun et al. 2009). In contrast, preliminary data indicate that P. obtusus is fully complementary with the currently established, synthetic assemblage. We also observe regional variation in the relative abundances of species in the assemblages. Large scale community dynamics are also observed in the home range, with swings in composition and relative abundances across time. The temporally and environmentally dynamic nature of these species assemblages means that adding species is not typically a zero-sum game with respect to impacts on fire ant colonies even if the species interferes under some conditions with established taxa.

Conclusions S. invicta is host to a wide suite of natural enemies in its native range. None of them alone appear to be responsible for regulating fire ant populations and differences in densities between native and introduced ranges may be exacerbated by differences in the local assemblages of competing ant species. Overall S. invicta abundances are likely governed by the interactions of habitat, disturbance and natural enemies. The premise for our research program is that sustainable control will arise from the concerted impacts of antagonistic organisms on colony growth rates, mortality rates, and reproductive output. As would be expected in a system where abiotic conditions, disturbance and succession play a large role in governing population densities, fire ant populations exhibit remarkable fluctuations in local abundance through time. In this sort of non-equilibrial, dynamic system, dampening growth rates is equivalent, over the long-term, to reducing average abundance. Pseudacteon species assemblages act on fire ant populations through the accumulation of impacts on colony growth rate. Maximizing impact requires assembling communities of flies comprised of species that exhibit differential responses to abiotic variation and impact fire ants through a variety of pathways. We are not there yet. And some of the most promising candidate species remain to be introduced.

Acknowledgements For their long-term and continued financial support we thank the Lee & Ramona Bass Foundation and the Helen C. Kleberg and Robert J. Kleberg Foundation. For its past financial support ,we thank the State of Texas Fire Ant Initiative. The efforts of the staff of the Brackenridge Field Laboratory and Integrative Biology and many technicians have made this work possible.

Table 1: The niche axes occupied by currently established Pseudacteon and species being investigated for introduction (Folgarait et al. 2003, Folgarait et al. 2005, Folgarait et al. 2007). Species in bold are currently established in North America. Underlined species are current targets for importation and establishment. ??? indicates an absence of Pseudacteon species known to fill this niche. Early Hot and Fly size Late spring Dusk, Foraging spring - Mid-day dry / host – fall dawn niche winter active conditions size abundant active abundant active P. Mound P. tricuspis, P. Large 1 2 tricuspis, P. nocens ??? Disturbances P. litoralis borgmeieri and P. litoralis1 Very Large Foraging P. Flies P. obtusus 2 P. obtusus ??? P. obtusus Trails borgmeieri

P. P. curvatus, Mound curvatus, P. ??? ??? Disturbances P. ??? cultellatus Small cultellatus Flies P. P. Foraging nudicornis, nudicornis, P. small ??? ??? Trails P. small P. small obtusus obtusus obtusus 1 Currently established in Alabama, this is a low priority fly for south & central Texas, as it utilizes only the absolute largest workers as hosts and would therefore exclusively impact monogyne S. invicta colonies (Porter et al. 2011). 2 A common parasite of S. richteri, field collections suggest it may parasitize S. invicta in Santiago del Estero province (Folgarait et al. 2007). Further study in Argentina is required to identify a suitable source population.

References Cited Briano, J., L. A. Calcaterra, and L. Varone. 2012. Fire ants (Solenopsis spp.) and their natural enemies in southern South America. Psyche 2012: doi:10.1155/2012/198084. Calcaterra, L. A., S. D. Porter, and J. A. Briano. 2005. Distribution and abundance of fire ant decapitating flies (Diptera : Phoridae : Pseudacteon) in three regions of southern South America. Ann. Entomol. Soc. Am. 98: 85-95. Calcaterra, L. A., A. Delgado, and N. D. Tsutsui. 2008. Activity patterns and parasitism rates of fire ant-decapitating flies (Diptera: Phoridae: Pseudacteon spp.) in their native Argentina Ann. Entomol. Soc. Am. 101: 539-550. Feener, D. H., Jr., and B. V. Brown. 1992. Reduced foraging of Solenopsis geminata (Hymenoptera: Formicidae) in the presence of parasitic Pseudacteon spp. (Diptera: Phoridae). Ann. Entomol. Soc. Am. 85: 80 - 84. Folgarait, P. J., O. A. Bruzzone, and L. E. Gilbert. 2003. Seasonal patterns of activity among species of black fire ant parasitoid flies (Pseudacteon : Phoridae) in Argentina explained by analysis of climatic variables. Biol. Cont. 28: 368-378.

Folgarait, P. J., R. J. W. Patrock, and L. E. Gilbert. 2007. The influence of ambient conditions and space on the phenological patterns of a Solenopsis phorid guild in an arid environment. Biol. Cont. 42: 262-273. Folgarait, P. J., O. Bruzzone, S. D. Porter, M. A. Pesquero, and L. E. Gilbert. 2005. Biogeography and macroecology of phorid flies that attack fire ants in south-eastern Brazil and Argentina. J. Biogeography 32: 353-367. Gilbert, L. E., C. L. Barr, A. A. Calixto, J. L. Cook, B. M. Drees, E. G. Lebrun, R. J. W. Patrock, R. M. Plowes, S. D. Porter, and R. T. Puckett. 2008. Introducing phorid fly parasitoids of red imported fire ant workers from South America to Texas: Outcomes vary by region and by pseudacteon species released. Southwestern Entomol. 33: 15-29. Henne, D. C., and S. J. Johnson. 2007. Zombie fire ant workers: behavior controlled by decapitating fly parasitoids. Insectes Sociaux 54: 150-153. Kronforst, M., P. J. Folgarait, R. J. W. Patrock, and L. E. Gilbert. 2007. Genetic differentiation between body size biotypes of the parasitoid fly Pseudacteon obtusus (Diptera: Phoridae). Mol. Phylogen. Evol. 43: 1178-1184. LeBrun, E. G., R. M. Plowes, and L. E. Gilbert. 2008. Dynamic expansion in recently introduced populations of fire ant parasitoids (Diptera: Phoridae). Biol. Invasions 10: 989-999. LeBrun, E. G., R. M. Plowes, and L. E. Gilbert. 2009. Indirect competition facilitates widespread displacement of one naturalized parasitoid of imported fire ants by another. Ecol. 90: 1184-1194. Mehdiabadi, N. J., and L. E. Gilbert. 2002. Colony-level impacts of parasitoid flies on fire ants. Proceedings of the Royal Society of London Series B-Biological Sci. 269: 1695- 1699. Morrison, L. W., C. G. Dall'aglio-Holvorcem, and L. E. Gilbert. 1997. Oviposition behavior and development of Pseudacteon flies (Diptera: Phoridae), parasitoids of Solenopsis fire ants (Hymenoptera: Formicidae). Environ. Entomol. 26: 716-724. Orr, M. R., S. H. Seike, and L. E. Gilbert. 1997. Foraging ecology and patterns of diversification in dipteran parasitoids of fire ants in south Brazil. Ecol. Entomol. 22: 305- 314. Orr, M. R., S. H. Seike, W. W. Benson, and L. E. Gilbert. 1995. Flies suppress fire ants. Nature 373: 292. Plowes, R. M., E. G. LeBrun, and L. E. Gilbert. 2011. Introduction of the fire ant decapitating fly Pseudacteon obtusus in the United States: factors influencing establishment in Texas. Biocontrol 56: 295-304. Porter, S. D. 1998. Biology and behavior of Pseudacteon decapitating flies (Diptera: Phoridae) that parasitize Solenopsis fire ants (Hymenoptera: Formicidae). Florida Entomol. 81: 292- 309. Porter, S. D. 2010. Distribution of the Formosa Strain of the Fire Ant Decapitating Fly Pseudacteon Curvatus (Diptera: Phoridae) Three and a Half Years after Releases in North Florida. Florida Entomol. 93: 107-112. Porter, S. D., L. A. Nogueira de Sá, and L. W. Morrison. 2004. Establishment and dispersal of the fire ant decapitating fly Pseudacteon tricuspis in North Florida. Biol. Cont. 29: 179-188.

Porter, S. D., L. C. Graham, S. J. Johnson, L. G. Thead, and J. A. Briano. 2011. The Large Decapitating Fly Pseudacteon Litoralis (Diptera: Phoridae): Successfully Established on Fire Ant Populations in Alabama. Florida Entomol. 94: 208-213. Tschinkel, W. R. 2006. The Fire Ants. The Belknap Press of Harvard University Press, Cambridge. Valles, S. M., and S. D. Porter. 2007. Pseudacteon decapitating flies: potential vectors of a fire ant virus? . Florida Entomol. 90: 282-283.

Impact of the Invasion of the Imported Fire Ant, Does Identification Matter?

S. Bradleigh Vinson Department of Entomology, Texas A&M University, Collage Station TX 77843.

The IFA appears to have evolved in South America and over time these ants have evolved into a number of species. It appears that several species over the years have migrated (or in a few cases “Imported”, presumably accidently) to North America and some of these appear to have further evolved into distinct species or forms. All of these species and forms have an impact on the ecosystem, particularly animals and plants, as well as, people and the infrastructure of modern society. However the severity of their impact is influenced by the species and form (one example is the polygyne form of fire ant). But the impact that these fire ants have in different situations appears to be influenced by the species and form. A problem is that over the years the publications regarding fire ants has either assumed they were “imported “ or in some more recent years they used some morphological characteristics to identify the ant involved as imported. However recent evidence suggests that molecular methods may be more accurate than morphology which suggests that the impact may in some cases be attributed to the wrong species or form. Further, the impact if the different fire ant species also depends on the system under study and the approach that the investigator takes in regards to the study As a result the ants impact can be very negative or in other cases very positive and this designation often depends on the species and the aspect of the impact that was studied or emphasized. Here I briefly review the impact that the “Imported Fire Ant” has on various biological systems, as well as, their impact on humans and their infrastructure.

Reinventing Fire Ant eXtension

Kathy Flanders1 and Bart Drees2 1Department of Entomology and Plant Pathology, Auburn University, AL 36849 2Department of Entomology, Texas A&M University, College Station, TX 77843-2475

The Imported Fire Ant eXtension Community of Practice was formed in 2005 with the goal of providing unbiased, science-based information on fire ants. Fire Ant eXtension is now one of over 60 eXtension Communities of Practice (see about.extension.org). Fire Ant eXtension has evolved over the years in response to changes in the way clients seek information. Information delivery platforms now include our flagship web site, www.extension.org/fire+ants; Fire Ant Info on Facebook; FireAntInfo on Twitter; eXfireants on YouTube; and the Ant Detective Game on Second Life. The Entomological Society of America also hosts an eXtension Fire Ant network (see: http://www.entsoc.org/esa-networks). The Community of Practice currently has 222 members. Land Grant, State, and Federal employees are encouraged to join the Fire Ant eXtension Community of Practice at www.extension.org/people, and to register to answer questions on fire ants at http://www.extension.org/aae/prefs. As a member of the Community of Practice you can join various work teams dedicated to developing fire ant content for the web site and social media sites. Everyone who works on fire ants is encouraged to link to fire ant eXtension web pages, and to follow us at the various social media sites. All are encouraged to promote these sites whenever possible. Researchers are encouraged to work with the Fire Ant eXtension CoP to highlight key research findings in eXtension content. Anyone with new images or videos of fire ants are encouraged to submit them to Ms. Neal Lee, for inclusion in the fire ant section of bugmugs.org. Changes in the past year include a new content development system. Content for the fire ant eXtension page is now developed at create.extension.org. All of fire ant eXtension content is being apprised to enhance discoverability by internet search engines. The community just completed a self-assessment on how it functions according to various criteria for CoP optimization (create.extension.org/node/89255). The Community conducts annual webinars on fire ants designed for the general public. It recognizes the Scholarship of eXtension. Recent improvements in the eXtension system allow participants access to information on their eXtension activities. In 2011 the Community began to use Mini-Surveys to assess the impact of the most popular fire ant content located on www.extension.org. It encourages researchers to partner with eXtension as they develop competitive grant proposals.

Toxicity of Alkaloids of Red Imported Fire Ant, Solenopsis invicta Buren Against the Green Peach Aphid, Myzus persicae (Sulzer)

Tahir Rashid,1 Paul J. McLeod,2 and Jian Chen3 1Department of Agriculture, 1000 ASU Drive, #330 Alcorn State University, Alcorn State, MS 39096 2Department of Entomology, University of Arkansas, Fayetteville, AR 72701 3 USDA-ARS, Biological Control of Pests Research Unit, Stoneville, MS 38776

Introduction The polyphagous green peach aphid, Myzus persicae (Sulzer) can directly damage the host plants by feeding on the leaves and extracting sap or indirectly by transmitting viruses (Flanders et al. 1991, Tagu et al. 2008). It also secretes honeydew which attracts fungus causing smutting of leaves and fruit (Gray and Gildow 2003). M. persicae has very high reproductive potential and can cause substantial damage to young plants thus causing eventual death (Petitt and Smilowitz 1982). Systemic insecticides are frequently used to suppress M. persicae populations but are less effective in cooler temperatures (McLeod 1991). Over time, M. persicae populations have developed resistance against synthetic insecticides (Blackman and Devonshire 1978, Bauernfeind and Chapman 1985). Anstead et al. (2005) have reported development of resistance in M. persicae to more insecticides than any other insect. Plant alkaloids, such as neem oil and nicotine are increasingly used as insecticides (Sharp et al. 2009). Certain mineral and essential oils were comparable to chemical insecticides in toxicity against M. persicae (Yankova et al. 2009). Alkaloid-based venom of the red imported fire ants, Solenopsis invicta (Buren) has been reported to possess antibacterial, antifungal and insecticidal activities (Jones et al. 1982). The venom alkaloids extracted from S. invicta have two groups of alkaloids, piperidines and piperideines. Chen et al. (2009) found six new piperideine alkaloids in S. invicta. This study investigates the biological activity of newly reported piperideine alkaloid component of S. invicta venom against M. persicae.

Material and Methods Pepper cv ‘Jalapeno’ plants were produced in the greenhouse. About six weeks old plants that had eight to ten true leaves were moved to the laboratory and infested with M. persicae from a laboratory culture maintained on pepper. Infested leaves were cut from the culture plants and placed on each test plant. Aphids were allowed to move to the test leaves. After a one-week establishment period plants were searched for aphids. Those containing leaves with at least 20 adult apterous aphids were selected for testing. Immediately prior to testing, individual leaves were removed from the plant with a razorblade. Immature aphids were removed with a camel hair brush. Extract source: Workers (2.5 g) of S. invicta were extracted in 15 mL hexane in a 50 mL beaker for three times. The pooled extract was then concentrated to about 0.5 mL under air flow. A flash chromatography (Isolera Four, Biotage , LLC, Charlotte, North Carolina, USA) with a SNAP silica gel cartridge (10 g) was used to isolate and purify the piperideines and piperidines. Bioassay: Each of the two extracts, i.e., piperideines (18.2 mg) and piperidines (20.6 mg), was dissolved in 0.5 ml acetone to produce the initial concentrations of 46008 and 52014 ppm extract:acetone, respectively. Subsequent serial dilutions were made with acetone and

ranged from 4600 to 23 ppm for the piperideines and 2600 to 5 ppm for the piperidines. Each concentration was applied with an ISCO Model M microapplicator, ISCO Company, Lincoln, NE 68507, fitted with a glass B-D syringe and 27 gauge needle (Becton, Dickinson and Company, Rutherford, NJ 07070). The volume of the application was 0.337µl. Applications were made with the aid of a desk magnifying light. Although the application was applied directly to the dorsal surface of each aphid, some of the mixture spread from the aphid to the leaf surface due to the small aphid size. After treatment, leaves were placed in 28-ml plastic cups, Comet Products, Inc., Chelmsford, MA 01824, with a 2.5-cm diameter disk of moist blotter paper. Cups were capped and held in an environmental chamber set at 26o C and 12:12 h photo phase. Mortality was assessed at 24 and 48 h by prodding each aphid with a camel hair brush. Lack of movement was used as the criterion for judging death. Additional observations were made at hourly intervals to establish the time of death. Data were analyzed with Proc Probit (SAS 9.1, SAS Institute, Cary, NC).

Results & Discussion Both piperideines and piperidines were toxic to M. persicae (Table 1). Chi Square values were significant for both extracts at one and two days after treatment (DAT) indicating that increasing dosage caused an increase in mortality. Aphid death occurred rapidly. In as few as four hours, approximately 50% mortality was observed with aphids treated with the high dosages of piperideines (4600 ppm) and piperidines (2600 ppm). At six hours post treatment 100% of the aphids receiving the high dosages were dead. Counts taken 1 DAT produced LC50 values of 116.6 and 91.5 ppm for the piperideine and piperidine extracts, respectively. These values did not statistically differ based on overlap of the 95% fiducial limits. The 1 DAT LC95 values were 2480 and 824.8 ppm for the piperideine and piperidine extracts, respectively. Although the LC95 value for the piperideine extract was approximately three times higher than that for the piperidine extract, the fiducial limits overlapped indicating no significant difference. Toxicity was not greatly increased in the 2 DAT observation. The LC50 values were 89.3 and 48.5 ppm for the piperideine and piperidine extracts, respectively (Table 1). Again, based on fiducial limit overlap, the two LC50 values did not statistically differ. The LC95 value for the piperidine extract was 396.8 ppm and this was statistically lower indicating more toxicity to the green peach aphid than the piperideine extract. Further research will investigate the field persistence and any phytotoxicity of piperideine and piperidine alkaloids extracted from S. invicta. The high and rapid mortality of M. persicae exposed to elevated dosages of both piperideines and piperidines indicates that these alkaloids may provide an environmentally safe method of aphid control.

Table 1. Dosage response of green peach aphid to piperideine and piperidine extracts 1 DAT LC LC Extract n slope (±SE) 50 95 χ2 (95%FL) (95%FL)

116.3 2480 piperideines 877 1.24 (0.08) 233.08 (92.3-143.1) (1792-3713)

91.5 824.8 piperidines 933 1.72 (0.22) 63.28 (54.5-146.3) (425.9-2803)

2 DAT LC LC Extract n slope (±SE) 50 95 χ2 (95%FL) (95%FL)

89.3 1404 piperideines 877 1.37 (0.16) 78.23 (49.0-139.5) (749.9-4118)

48.5 396.8 piperidines 933 1.80 (0.12) 229.48 (41.1-56.8) (308.4-542.8)

References Cited Anstead, J. A., M. S. Williamson, and I. Denholm. 2005. Evidence for multiple origins of identical insecticide resistance mutations in the aphid Myzus persicae. Insect Biochem. Mol. Biol. 3:249-256. Bauernfeind, R., and R. Chapman. 1985. Nonstable parathion and endosulfan resistance in green peach aphids (Homoptera: Aphididae) J. Econ Entomol. 78(3): 516-522. Chen, J., C. Cantrell, Han-Wu Shang, and M. Rojas. 2009. Piperideine alkaloids from the poison gland of the red imported fire ant (Hymenoptera: Formicidae). J. Agric. Food Chem. 2009, 57, 3128–3133. Flanders, K., E. Radcliffe, and D. Ragsdale. 1991. Potato leafroll virus spread in relation to densities of green peach aphid (Homoptera: Aphididae): implications for management thresholds for Minnesota seed potatoes. J. Econ. Entomol. 84: 1028-1036. Gray, S. M., and F. Gildow. 2003. Luteovirus-aphid interactions. Annu. Rev. Phytopathol. 41:539-566. Jones, T. H., M. S. Blum, and H. M. Fales. 1982. Ant venom alkaloids from Solenopsis and Monomorium species. Recent developments. Tetrahedron. 38: 1949-1958. McLeod, P. 1991. Influence of temperature on translaminar and systemic toxicities of aphicides for green peach aphid (Homoptera: Aphididae) suppression on spinach. J. Econ. Entomol. 84: 1558-1561. Petitt, F., and Z. Smilowitz. 1982. Green peach aphid feeding damage to potato in various plant growth stages. J. Econ. Entomol. 75: 431-435.

SAS Institute. 2003. SAS version 9.1. SAS Institute, Cary, NC. Sharp, D. N., A. J. Lentz-Ronning, J. Barron, and L. S. Adler. 2009. The effect of larval diet and sex on nectar nicotine feeding preferences in Manduca sexta (Lepidoptera: Sphingidae). Fl. Entomol. 92(2):374-376. Yankova, V., D. Markova, and V. Todorova. 2009. Biological activity of certain oils in control of green peach aphid (Myzus persicae Sulz.) on pepper. Acta Hort. 830:619-625.

Managing the Red Imported Fire Ant on a Green Roof in Friendswood, TX Paul R. Nester,1 Anthony W. Camerino,1 and Alejandro A. Calixto2 1Texas AgriLife Extension Service, Houston, TX 2Texas AgriLife Research, College Station, TX

The use of green roof technology is gaining popularity among many real estate groups because of the marketable benefits which include energy conservation, storm water management, air pollution mitigation, scenic landscapes, wildlife habitat, and added recreational areas. In 2003 the United States Environmental Protection Agency cited reduced urban heat-island effects and lowered cooling costs as benefits for buildings utilizing this technology (USEPC 2003). Jacob White Construction Company, a Friendswood, TX based company (Figure 1), is a leader in the design and construction of green roofs atop of new “green” building projects in and around the Houston area. During the early spring of 2011 while checking plant trials on a green roof at the Jacob White Construction Company Headquarters (2000 West Parkwood, Friendswood, TX) Dr. Camerino noticed active red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae). In response, Dr. Nester and Dr. Camerino planned a fire ant management demonstration at the site. This report outlines some of the strategies used for the management of this pest.

Materials and Methods The green roof located atop the Jacob White Construction Headquarters was assessed May 3, 2011 and nine subsequent dates (Figure 2) for the presence of the red imported fire ant. Foraging ant activity was checked using individual hot dog slice food lures (0.25 inch thick hot dog slices, Bar-S Jumbo Franks) that were placed in a grid across the green roof (Figure 3). Twenty three lures were used on 5/3/2011, while 34 lures were used on subsequent assessment dates. Food lures were checked after 60 minutes and total ants present on the lures were recorded (Figure 4). DuPont™ Advion® ant bait arenas (30 arenas, 0.1% indoxacarb) were positioned in a grid pattern within the confines of the green roof (Figure 5). Bait stations were used so as not to directly apply a pesticide to the green roof growing media (Figure 6). Through a rainwater catchment system, all irrigation water applied to the roof is recycled and reapplied on site. The selection of bait arenas was to reduce the chance of pesticide movement from the target site. Additionally, irrigation water is applied several times per day as an energy saving passive cooling method. The frequent irrigation may have disrupted the integrity of a “unprotected” bait product. Since the roof was 11,000 sq. ft., the total active ingredient (0.059 g) contained within the 30 bait arena’s was approximately equal to the active ingredient (0.052 g) in a 1.0 pound product per acre broadcast application of the DuPont™ Advion® fire ant bait (0.045% indoxacarb). Since assessments of fire ant activity on the green roof indicated the continued presence of a population of fire ants (Figure 2), a fall broadcast application of the DuPont™ Advion® fire ant bait (2 pounds product/acre) was planned for the grounds around the Jacob White Headquarters (Figure 7). Total mound counts were taken on September 22, 2011 (Figure 8) before fire ant bait applications and on 4 subsequent dates (Figure 8). To determine if a mound was active, visible fire ant mounds were checked using the minimal disturbance method, i.e., mounds were probed with a shovel and if no fire ants appeared after 15 seconds, the mound was

considered inactive. The fire ant bait product was evenly spread with Scotts® HandyGreen® II Hand-Held Spreader set on smallest opening (Figure 9). In addition to the broadcast application on October 21, 2011, DuPont™ Advion® fire ant bait (0.5 oz/mound) was uniformly distributing around the active mounds with active brood. We used a T-test statistical analysis to compare the mean numbers of worker ants observed at lures before and after the arena bait station treatment. We also estimated the mean and 95% Confidence Intervals (CI) for each sampling and display on a time series graph (Figure 2). No overlap among 95% CI indicates significant differences, and overlap indicates no significant differences. This approach allow us to compare post-treatment dates to pre-treatment numbers which in this case are consider a Control.

Results and Discussion Results of the T-test (P <0.000, df: 327, F= 26.270) indicated that the mean number of worker ants recorded on food lures over the assessment period were significantly reduced compared to the initial pre-treatment values (Figure 10). The DuPont™ Advion® ant bait arenas did successfully reduce the ant population at all assessment dates (Figure 2), based on food lures, on the green roof atop the Jacob White Construction Company Headquarters. Since some fire ant foraging activity was observed during the assessment period, and active fire ant mounds were found on the grounds surrounding the Jacob White Headquarters (Figure 7), DuPont™ Advion® fire ant bait was broadcast to the grounds and a 76% reduction in active fire ant mounds was observed 14 days after the treatment. Subsequent assessments of active fire ant mounds showed a continued increase in mound activity with no discernible reduction in activity after the additional single mound treatments with DuPont™ Advion® fire ant bait. A spring 2012 application of fire ant bait will be planned along with continued monitoring for fire ants on the green roof with placement of DuPont™ Advion® ant bait arenas as needed for continued management of the red imported fire ant on the green roof.

References Cited United States Environmental Protection Agency. 2003. Cooling summertime temperatures: strategies to reduce urban heat islands. Publication No. 430-F-03-014.

Acknowledgements The authors wish to thank Jeff Mickler, President /CEO, Jacob White Construction Company for allowing us total access to his facility to conduct this study and for DuPont for their gracious support of the project.

Figure 1. Headquarters of Jacob White Construction Company, Friendswood, TX, Galveston Co. 2011.

Figure 2. Mean number of red imported fire ants (RIFA) per food lure at various assessment dates atop green roof in Galveston County, 2011. No overlap among 95% CI indicates significant differences, and overlap indicates no significant differences.

Figure 3. Approximate locations of 34 food lures for the assessment of red imported fire ant foraging on green roof, Galveston, Co. 2011.

Figure 4. Representative food lure with foraging fire ants as found on green roof during fire ant activity assessments. Galveston County, 2011.

Figure 5. Approximate locations of 30 DuPont™ Advion® ant bait arenas on green roof, Galveston County, 2011.

Figure 6. Example of DuPont™ Advion® ant bait arenas placement atop green roof, Galveston Co., 2011.

Figure 7. Grounds around Jacob White Construction Headquarters where the broadcast application of the DuPont™ Advion® fire ant bait was applied, Galveston, Co. 2011.

Figure 8. Total number of fire ant mounds found on grounds around Jacob White Construction Company Headquarters, before and after broadcast application of DuPont™ Advion® fire ant bait at 2.0 pounds product/acre, Galveston Co., 2011.

Figure 9. Scotts® HandyGreen® II Hand-Held Spreader used for broadcasting DuPont™ Advion® fire ant bait on grounds surrounding Jacob White Construction Headquarters, Friendswood, TX, Galveston County, 2011.

Figure 10. Analysis of mean number of red imported fire ants (RIFA) on food lures over nine assessment periods (May 10, 2011 – January 27, 2012), Galveston County, 2011.

T-test: P <0.000, df: 327, F= 26.270

Trolling for Red Imported Fire Ant (Solenopsis invicta) Control Efficacy Data in the Lone Star State's Record-Setting Hottest, Driest Year in U. S. History!

Paul R. Nester,1 Bastiaan “Bart” M. Drees,2 and Alejandro A. Calixto3 1Texas AgriLife Extension Service, Houston, TX 2Extension Entomologist, Texas AgriLife Extension Service, College Station, TX 3Department of Entomology, Texas A&M University, College Station, TX

Introduction Each year applied field research is conducted to develop performance data as support documentation for the development of red imported fire ant insecticides as required by the Environmental Protection Agency. This effort is, in many ways, similar to a fishing expedition. The year, 2011, proved to be one year where these efforts were curtailed in Texas by having the hottest average temperature for June through August according to National Weather climatologists. Texas experienced the hottest, driest year for any state in U. S. history! Data from a large replicated trial in Beaumont, TX and a smaller replicated trial in College Station, TX document the demise in fire ant activity and mound numbers in untreated control plots through the summer months. A lot of effort was involved to set up these trials to assure plots with above minimum (10 mounds/plot) in the spring.

Materials and Methods Beaumont, TX This trial evaluated “hopper blend” treatments combining faster-acting (hydramethylnon, indoxacarb or fipronil) and a slower-acting insect growth regulator (IGR) active ingredient (s-methoprene or pyriproxyfen) into single treatments (Table 1). It was established on managed Bermuda grass turf in the soccer fields of the Cris Quinn Memorial Soccer Complex, Beaumont, TX, (Figure 1). The soccer complex consists of fifteen acre, comprised of 30 large and small soccer fields, concession stands and restrooms. Fifteen soccer fields were used, allowing 30 plot areas. Active ant mound counts were taken from 0.25 acre circles from middle of plots. Thirty 200 ft × 250 ft (1.15.acre) plots were established, across this sports field complex on April 26, 2011. The area was mowed and trimmed once per week, weather permitting. Low rainfall occurred during the testing period. No supplemental irrigation was provided. All treatments (Table 1) were broadcast applied April 26, 2011, in the late afternoon, with an ATV (Kawasaki Prairie 700) mounted Herd GT-77 ATV Broadcast Seeder (Kasco Manufacturing Company, Shelbyville, IN. The Herd GT-77 was calibrated to deliver 1.5 lb fire ant bait with a 20 ft swath while the ATV traveled at 10 mph. The Herd GT-77 was fitted with a Herd Seeder Co. #1 plate covering the agitator. After all the replications of each treatment were applied, the broadcaster hopper was vacuumed clean with a small industrial vacuum before the next treatment. A pre-treatment assessment of the number of active red imported fire ant mounds within a 0.25 acre circle was conducted (Table 2). Plots were then arrayed in order from the plot containing the highest to the lowest number of ant mounds per plot. Replications were established by dividing the array into four blocks and randomly assigning eight treatments to each block and then adjusting to assure that pre-treatment mean differences between treatments in all replications or blocks were minimal. Untreated controls had to be located outside of the soccer field area. We adhered to an agreement with the grounds maintenance crew that all

playing services would be treated. Extinguish® Plus was used on all fields and border areas not involved in the actual study. Fifteen soccer fields were subdivided for this study. Each soccer field could be divided into two plots. Based on the array results, the treatments were replicated within 28 of the subdivisions (Figure 1). At 0, 2, 4, 8, 12 and 16 weeks after treatment (WAT) fire ant mound activity was assessed. To determine if a mound was active, visible fire ant mounds were checked using the minimal disturbance method, i.e., mounds were probed with a shovel and if no fire ants appeared after 15 seconds, the mound was considered inactive. Total active fire ant mounds in each plot were counted, and the data was recorded as the number of active fire ant mounds per acre (Table 2). Data were analyzed using Analysis of Variance (ANOVA) with means separated using Duncan’s Multiple Range Test (P≤0.05) (SPSS 18.0). College Station, TX A field trial was conducted on a field site at Texas A&M University, initiated May 24, 2011. Treatments were applied to 18 0.25 acre field plots with a 42 ft radius circular, 0.125 acre, sub-plot monitored within each plot. These were arrayed by highest to lowest pre-treatment active mound count numbers taken from sub-plots and blocked into six replicates or blocks to which treatments were randomly assigned. Treatments were a broadcast application of Esteem® Ant Bait (0.5% pyriproxyfen) at 2 lbs/acre using a hand held seeder or “belly bumper” and an untreated control. Plots were monitored for active ant mounds prior to and periodically following (June 29, July 20, and Sept. 20 or 4, 8, 16 WAT) May 24, 2011 application.

Results and Discussion Beaumont, TX What started out to be a promising year for obtaining fire ant management data turned into a record setting year for Texas by having the hottest average temperature for June through August according to National Weather climatologists. Even though reductions in fire ant mound activity looked good (Figure 2), the untreated areas also showed reduction in fire ant mound activity. Not until the September observation date did the untreated control plots show a significant higher number of active fire ant mounds than the treated plots (Table 2). Mean daily temperatures remained high during the assessment period (Figure 2). In summary, there are a few pertinent points that can be made about the data from this study: 1) There were no pre-count differences in means (analysis included check plots) between treatments. 2) Advion® + Distance® ant bait products did not provide significantly faster control (mound reduction) than Extinguish® Plus at the early evaluation dates. 3) There were no treatment differences for the first 4 weeks after treatment, including no difference compared to untreated plots. 4) At 8 WAT Extinguish® Plus, Distance® and the Distance® hopper blends performed significantly better than MaxForce® FC but otherwise there few only numerical treatment differences, even from the untreated control. 5) Only at the September assessment did bait treatments result in mound numbers significantly lower than untreated plot numbers and the hopper blends with Distance® performing significantly better than MaxForce® FC, but with no other statistical differences between treatments (Figure 2)

College Station, TX Due to extremely hot dry weather during the summer months, active ant mound numbers declined in all field plots (Figure 3), separation of treatment means was obscured.

Conclusion By late summer, no differences occurred between treated and untreated control plot means, rendering researchers unable to effectively document long-term product performance normally provided by slow-acting, long-lasting treatments such as IGR granular ant bait formulations. Thus, we have documented that bad data happens! Hopefully our efforts to “troll” for efficacy data will improve in the year(s) ahead after more “normal” environmental conditions return to the state.

Table 2. Mean number of red imported fire ant mounds per 0.25 acre circle subplot before and following broadcast-applied fire ant bait product treatments, Cris Quinn Memorial Soccer Complex, Beaumont, TX initiated April 26, 2011, Jefferson Co., TX.

* Means followed by the same letter are not significantly different using Analysis of Variance (ANOVA) with means separated using Duncan’s Multiple Range Test (P≤0.05) (SPSS 18.0).

Figure 2. Average number of imported fire ant mounds per 0.25 acre circle at 0, 2, 4, 8, 12 and 16 weeks before and following broadcast-applied fire ant bait product treatments applied April 26, 2011, Cris Quinn Memorial Soccer Complex, Beaumont, TX.

Figure 3. Number of active red imported fire ant mounds per plot following May 24, 2011 applications of Esteem® Ant Bait applied as a broadcast application (2 lbs/acre).

Acknowledgments The authors want to thank the managers and grounds maintenance crew of the Cris Quinn Memorial Soccer Field for allowing us to use the facility for this study and the timely mowing of the soccer field area. We would also like to recognize the representatives of the following companies, BASF, Bayer, DuPont, Central Life Sciences, and Valent U. S. A. for the donation of all bait products. Special thanks goes to the Jefferson County Master Gardener volunteers who helped with the assessment of fire ant mound activity.

A Novel Use of Microsatellite Markers for Distinguishing Two Imported Fire Ant Species and Their Hybrid

David C. Cross,1 Rajesh B. Garlapati,2 Juliet D. Tang,3 Pummi Singh,1 and Michael A. Caprio1 1Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University 2Genesis Laboratories, Inc., Patna, India 3Forest Products, Mississippi State University

Introduction The hybrid imported fire ant, HIFA, is a reproductively viable hybrid of Solenopsis invicta, the red imported fire ant, RIFA, and S. richteri, the black imported fire ant, BIFA. In the U.S., HIFA occurs from mid Tennessee, south through the northern half of Mississippi and to the east through northern Alabama and northwestern Georgia. The RIFA effectively limits the HIFA range from the latter’s southwestern to southeastern extent. The BIFA is limited to a shrinking area in northeastern MS and western TN. Because of a reported gradient for the degree of hybridization in HIFA’s range, there is no single genetic assay for positive identification for pure BIFA or RIFA adjacent to or intermixed in this region. As a consequence, GC/MS analysis has been a tool of choice for a definitive identification of species/hybrid status. We compared the allele frequency of polymorphic microsatellite repeat regions of the two pure species and HIFA, particularly where the three types have the greatest overlap in the southeastern U.S. Marked differences in the relative frequencies and most notably, complete absences of some of the alleles in one versus the other were indicated. This variability can be used as a powerful genetic assay aiding species identification.

Materials and Methods Capillary Electrophoresis: Ants were collected from 286 HIFA colonies (or isolated queen ants) mostly in Oktibbeha, Lauderdale, and Noxubee Co.’s in MS, and 141 RIFA colonies in Lauderdale, Yazoo and Copiah Co.’s, MS and Mobile Co. in AL. Only 4 mounds of BIFA were sampled in Chester and Madison Co.’s, TN. Ants collected for genotyping were kept in chilled 100% ethanol and transferred to -80 °C. Queens were dissected and examined for sperm presence. After removing the sperm, male and female derived tissues were extracted for DNA. DNA samples were genotyped for five loci of microsatellite repeat regions: Sol11, Sol20, Sol42, Sol49 and Sol55, using modified PCR primers from Krieger and Keller, (1997). Alleles were identified through capillary electrophoresis (Beckman Coulter CEQ 8000).

Agarose Gel Electrophoresis of Sol49: An additional 117 samples of RIFA , 9 of HIFA and 16 of BIFA were made from 36 counties or parishes in MS (17 Co.’s), AL (3), TN (6), LA(1), GA(5), SC(1), FL(2) and CA(1). PCR products of the Sol49 locus were electrophoresed on a 3% agarose gel (see Fig. 5).

Molecular diagnostic tests for species/hybrid status: For newly mated queens, we used one or a combination of RAPD assays, (random amplified polymorphic DNA), and for some samples, a mitochondrial DNA marker, digested with TaqαI (Goodisman et al., 1998). For most mounds, though, we determined species/hybrid status by collecting >100 workers in 7 ml of hexane for identification using cuticular hydrocarbons and venom alkaloids. The hydrocarbon/alkaloid samples were analyzed by GC/MS at the National Biological Control Lab., USDA-ARS, Stoneville, MS.

Results and Discussion Of the five loci, Sol11, Sol42, Sol49 and Sol55 all showed an absence/presence difference between HIFA and RIFA for at least one allele that was present a minimum of 30 percent of the time in this hybrid or pure species form (Fig.'s 1-4). Like the HIFA genotypes, those of the 4 BIFA colonies showed the Sol49 126 allele, the Sol55 232 allele and the Sol42 208 allele (data not shown), all which were absent from the RIFA DNA. We conclude this component of HIFA genotype is derived from BIFA.

Fig.'s 1-4. Shown above are the most frequently occurring microsatellite alleles within the four most polymorphic loci. The alleles with the most divergent frequency between HIFA and RIFA are also represented. Fig. 2 of Sol49 gene locus also represents BIFA allele frequency.

• Agarose gel data also indicates the absence of the Sol49 126 allele in U.S. RIFA. The closest RIFA allele in size found is 116 and is infrequent.

• The combined Sol49 data for 20 BIFA colonies (in 8 counties) showed them all to possess the 126 allele. It should be noted that the few HIFA collected at greatest distances from BIFA range lacked the 126 allele.

Application of this information: For imported fire ant populations in the U.S., genetic assays using simple PCR may delineate the pure species types. One assay in particular, for the Sol49 126 allele, is strongly indicative (89%) of HIFA colonies, yet excludes all pure S. invicta - RIFA. It differs in size from the next smallest RIFA allele by 10 base pairs, a difference easily detectable on an agarose gel (Fig. 5).

Fig. 5. Three percent agarose gel showing different sized microsatellite alleles amplified from locus Sol49. Eighth and last lanes are molecular weight markers.

• Tests using allelic differences add inexpensive tools for even a simply equipped genetics lab. Data reported in Caldera et al., 2008, for MS and LA RIFA populations support our findings, with the exception of locus Sol11. They report a RIFA 169 (equivalent) allele which we did not observe except in HIFA.

• Additional testing of ants, from other locations within the introduced range likely to have species/hybrid overlap will be done to check the effectiveness of these genetic markers for determining accurate identification.

References Cited Caldera, E. J., K. G. Ross, C. J. DeHeer, and D. D. Shoemaker. 2008. Putative native source of the invasive fire ant Solenopsis invicta in the USA. Biol. Invasions 10: 1457-1479. Fritz, G. N., R. K. Vander Meer, and C. A. Preston. 2006. Selective male mortality in the red imported fire ant, Solenopsis invicta. Genetics 173: 207-213.

Goodisman, M. A. D., D. D. Shoemaker, and M. A. Asmussen. 1998. Cytonuclear theory for haplodiploid species and X-linked genes and application to a fire ant hybrid zone. Evolution 52: 1423-1440. Krieger, M. J. B., and L. Keller. 1997. Polymorphism at dinucleotide microsatellite loci in fire ant Solenopsis invicta populations. Molecular Ecol. 6: 997-999. Menzel, T. O., D. C. Cross, J. Chen, M. A. Caprio, and T. E. Nebeker. 2008. A survey of imported fire ant (Hymenoptera: Formicidae), species and social forms across four counties in east-central Mississippi. MidSouth Entomologist online journal 1: 3-10.

Acknowledgements Special thanks to Andie Brace, Anny Guthrie and Natalie Barker for collection, DNA isolation and molecular testing. Thanks to John Munger for data compilation. Jian Chen, USDA- ARS-BCPRU, Stoneville, MS, provided pivotal data by performing the GC/MS analyses. Joe MacGown, JoVonn Hill and Tim Menzel supplied additional ant collections. Fragment analysis samples were processed at LSBI, Mississippi State University. This work was made possible by funding through USDA-ARS, Management of Imported Fire Ants Project.

Fig. 5

Cytochrome P450 Gene Expression Profile in the Red Imported Fire Ant, Solenopsis invicta

Nannan Liu,* Feng Liu, and Ting Li Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849 *Corresponding author

Abstract The expression levels of 82 P450 genes were characterized using quantitative real-time PCR in the red imported fire ant, Solenopsis invicta. Most of these P450 genes belong to clans 3 and 4. Highest expression levels of these genes were determined in the small workers and queens. We identified that several P450 genes were differentially up-regulated in different life stages and castes, including 23% of the P450 genes up-regulated in 3rd-4th instar larvae in comparison with 1st-2nd instar larvae, 22% and 33% of the P450 genes over-expressed in alate pupae and worker pupae, respectively, in comparison with 3rd-4th instar larvae, as well as 39% and 23% of the P450 genes up-regulated in female alates and female pupae, respectively compared with male pupae and male alates. In the comparison with female alates, 75% of the P450 genes were over-expressed in the queens.

Introduction The red imported fire ant, Solenopsis invicta, is a widespread exotic species that results in a very high pressure for the local ecosystem in America. Moreover, this species has complex social system and displays insect ployphenism. Gene expression difference related to phenotypic variation has been studied in fire ants (Robinson 2008). Cytochrome P450 genes have been identified to be involved in the processes of insect growth, development and reproduction (Feyereisen 1999). To determine the potential biological and physiological roles of P450s associated with development, caste differentiation and reproduction in the fire ant, we characterized the expression profiles of P450s in different life stages and castes of fire ants. The results revealed that P450 genes may play an important role in development and reproduction of the fire ant.

Materials and Methods Solenopsis invicta. The red imported fire ant samples with different life stages and castes were collected from the field of Alabama. RNA Extraction and cDNA Preparation. Total RNAs were extracted from different life stages and castes, including 1st-2nd instar larvae, 3rd-4th instar larvae, small workers, big workers, male alates, female alates, queens, worker pupae, alate pupae, male pupae and female pupae of the fire ant. DNA was relieved from total RNAs. The first-stranded cDNA was synthesized. RNA extraction was repeated for 3 times from different colonies. Quantitative Real-time PCR (qRT-PCR) and P450 Gene Expression Analysis, qRT- PCR was performed by ABI 7300 Real-time PCR system (ABI Biosystems). Relative expression levels of P450 genes were calculated by the 2-∆∆Ct method using SDS RQ software. The 18S ribosome RNA gene was used as endogenous control. Each experiment was repeated 3 times with different preparations of the RNA samples.

Results Cytochrome P450 genes in S. invicta. The S. invicta genome sequence data base revealed 82 putative P450 genes. Sixty five of them were annotated while the other 17 P450 genes were unnamed. The S. invicta P450 genes fell into four major clans, CYP2, CYP3, CYP4, and mitochondrial clan. Of the 65 S. invicta P450 genes, the majority of them are assembled in clans 3 and 4. Twenty five P450s were found in the clan CYP3 with 6 in the CYP9 family, 16 in the CYP6 family, and 3 in the CYP336 family. Twenty-six P450 genes were in the clan 4 with all of them distributed in the CYP4 family. Ten P450 genes belonged to clan 2 and the remaining 7 P450 genes were in mitochondrial clan within 5 P450 families of CYP12, CYP301, CYP302, CYP314 and CYP315. In comparison with other insect species, S. invicta showed a clear expansion of P450 genes in clans 3 and 4, especially in the CYP4 and CYP6 families. P450 gene expression in S. invicta. The P450 genes with highest levels of expression were compared among the life stages and castes. The small workers and queens had highest number of P450 genes, with 23 and 20 P450 genes, respectively, that showed highest expression levels among all castes and developmental stages tested. About half of these highly expressed P450 genes belonged to CYP6 in small workers and CYP4 in queens, suggesting that P450s in these families may have functional importance associated with development and reproduction. Different P450 gene expression in the larvae and pupae of S. invicta. Our results (Fig. 1) showed that 23% of the P450 genes were up-regulated in the 3rd-4th larvae compared to the 1st-

8.5% 1.2% 3.7% A. 7.3% 7.3% B. 17.1% 34.1% 32.9% 20.7% 3.7% 8.5% 18.3% 30.5% 6.1% up-regulated >10-fold up-regulated 10-5-fold up-regulated 5-2-fold down-regulated 5-2-fold down-regulated 10-5-fold down-regulated >10-fold Similar D. 2.4% C. 15.9% 4.9% 14.6% 48.8% 17.1% 56.1% 12.2% 11.0% 4.9% 7.3% 4.9%

Fig. 1 Programmatic representation of the analysis of the P450 gene expression profiles in: A. 3rd-4th larvae st nd rd th compared with 1 -2 larvae; B. alate pupa compared with 3 -4 larvae; C. worker pupae compared with 3rd-4th larvae; D. alate pupae compared with worker pupae.

2nd larvae. 22% of the P450 genes were up-regulated in the alate pupae compared to the 3rd-4th larvae (Fig. 1) 1.However, nearly 33% of the P450 genes were up-regulated in worker pupae compared to the 3rd-4th larvae. Moreover, 22% of the P450 genes were up-regulated in the alate pupae compared to the worker pupae. Taken together, these results indicated that the up- regulated P450 genes in 3rd-4th larvae, alate and worker pupae may be involved in the process of development. The comparison between alate pupae and worker pupae indicated that the up- regulated P450 genes may play an important role in the development of different castes of fire ants.

P450 genes expression in pupae and alates in the S. invicta. To determine the P450 genes involving in the sex determination, we tested the expression levels of all P450 genes in male and female pupae, and male and female alates. Overall, 23% of the P450 genes were up- regulated in the female pupae compared to the male pupae. Furthermore, 39% of the P450 genes were up-regulated in the female alates compared to the male alates (Fig. 2), among which 6% of these genes were over-expressed for >10-fold higher in the female alates than in the male alates. In addition, 10% of the P450 genes were down-regulated in the female alates compared to the male alates. These results indicated that some of P450 genes may involve in sex determination in the fire ant.

Female pupa 2% 5% 16%

54% 17%

5% 1% up-regulated >10-fold up-regulated 10-5-fold up-regulated 5-2-fold down-regulated 5-2-fold down-regulated 10-5-fold down-regulated >10-fold 6% Female alate 12% Similar

51% 21%

3% 6%

Fig. 2 Programmatic representation of the analysis of the P450 gene expression profiles in female pupa and female alate compared with male pupa and male alate, respectively.

P450 genes expression between the queen and female alates of S. invicta. To determine whether P450 genes have the functions on reproduction system in fire ants, gene expression levels were characterized between queen and female alates. Results showed that 75% of P450 genes were up-regulated in the queen compared to the female alates (Fig. 3). A total of 10% of these genes were over-expression >10-fold higher than female alates. Meanwhile, 20% and 45% of P450 gene expression was 5-10 and 2-5 fold higher in queen than female alates, respectively. In addition, 3.6% of all genes tested were down-regulated in the queen compared to the female alates (Fig. 3). The results suggested that some of P450 genes may play crucial roles on development of reproduction in the fire ant.

9.8% 22.0% 1.2% 19.5% up-regulated >10-fold 1.2% up-regulated 10-5-fold 1.2% up-regulated 5-2-fold 45.1% down-regulated 5-2-fold down-regulated 10-5-fold down-regulated >10-fold Similar

Queen

Fig. 3 Programmatic representation of the analysis of the P450 gene expression profiles in the queen compared with female alate.

Discussion The P450 genes with highest levels of expression were compared among the life stages and castes. It was showed that the small workers and queens had the highest number of P450 genes that were highly expressed, half of which belonged to CYP6 in small workers and to CYP4 in queens. One of major roles of P450 genes in CYP6 family is involved the metabolism of exogenous compounds (Scott 1999), suggesting a possible function of small workers in response to the exogenous chemicals. The P450 genes in CYP4 family have been suggested to be involved in the metabolism of odorant and endogenous compounds, such as the juvenile hormones or pheromones (Sutherland and Feyereisen 1998), revealing the importance of queens in regulation of the development and reproduction. Overall, differential expression levels of P450 genes in life stages and morphological forms through the larva, worker, alate, pupa, and queens indicated the crucial role of P450 genes in fire ant development, caste variance, and reproduction.

Acknowledgement This study was supported by the Fire Ant Program, Alabama Department of Agriculture and Industries, and Hatch Project ALA015-1-10026.

References Cited Feyereisen, R. 1999. Annu. Rev. Entomol. 44: 507–33. Robinson, E. G. 2008. Sci. 322(5903): 896–900. Scott, J. G. 1999. Insect Biochem. Mol. Biol. 29: 757–777. Sutherland, T. D., and R. Feyereisen. 1998. PNAS. 95: 12884–12889.

Specialized Setae and Spongiform Lobes in Strumigenys and Pyramica Ants (Hymenoptera: Formicidae)

Joe A. MacGown and Richard L. Brown Mississippi Entomological Museum, Mississippi State University Box 9775, Mississippi State, MS 39762

Introduction Scales and squamiform setae are descriptive terms for non-homologous modifications of unicellular setae. Although "scales" are best known in Lepidoptera, flattened setae have evolved independently throughout other Hexapoda, including the Collembola, Apterygota, Homoptera, Psocoptera, Phthiraptera, Coleoptera, Diptera, and Hymenoptera (Ferris, 1934). Scales in Lepidoptera are involved in a variety of functions (Kristensen & Simonsen 2003), but functions of scales within other insect orders are largely unknown. In Hymenoptera scales are present in Pyramica Roger and Strumigenys Smith (Formicidae: Dacetini), but the functions of these scales in these ants are unknown. Strumigenys and Pyramica are a monophyletic clade of dacetine ants, but Pyramica has alternated between being synonymized with Strumigenys, treated as a subgenus, or as a valid genus (Bolton 1999, 2000, Baroni Urbani and De Andrade 2007, Deyrup and Cover 2007). Strumigenys includes 466 species worldwide, of which five occur in the Nearctic Region north of Mexico. Pyramica includes 327 species worldwide, of which 42 species in 11 species groups occur in the Nearctic Region north of Mexico (MacGown and Hill 2010, MacGown, unpublished data). Pyramica is most speciose in southeastern U.S., and in Mississippi, the genus includes 23 named species (MacGown and Hill 2010). The Strumigenys – Pyramica clade shares seven autapomorphies and nine apomorphies, but the two most distinctive characters of this clade, as described by Bolton (1999), are the "bizarre pilosity" on the head and other areas of the body and the presence of "lobes of spongiform tissue" on the petiole and postpetiole, the latter being lost in some species. The two genera have been differentiated by seven characters of the mouthparts (Bolton, 1999), and five of these involve the mandibles. The objectives of this research included the following questions. 1) What is the fine structure of the scales and "bizarre pilosity" and how does it vary between species groups of Pyramica? 2) Is the distinctness of Strumigenys and Pyramica supported by characters of the body pilosity? 3) What is the fine structure of "spongiform tissue?" Answers to these questions also can contribute to answering the general question of why flattened setae have evolved so many times among insects.

Materials and Methods A JEOL-JSM65600F SEM was used to examine P. angulata (Smith), P. clypeata (Roger), P. hexamera (Brown), P. ohioensis (Kennedy & Schramm), P. ornata (Mayr), P. reflexa (Wesson & Wesson), and P. rostrata (Emery), which represent seven species groups of Pyramica, and S. louisianae Roger. Although scales are present on the thorax and petiole of some species, SEM examinations concentrated on the heads for comparative purposes. Except for a pinned specimen of P. angulata, all other specimens were preserved in 90% ETOH, and the

latter were sonicated 10-15 seconds and air-dried. All specimens were mounted on aluminum stubs with carbon tape and coated with gold-palladium.

Results The forms of scales in the dacetine ants used in this study (Figs. 1-8) represent a transformation series from setiform with widened and dentate apices (Figs. 1b,c) to elongate and widened with dentate apices (Figs. 2c, 3c), to widened and rounded (Figs. 4c, 5c), to laterally rolled and basally fused (Figs. 6d-f, 8c-e), and to laterally rolled and fused for most of length to form a secondarily hollow scale (Figs. 7c,f). Longitudinal ridges are present on all scales, although those of some scales of P. reflexa are modified. Scales were present on head capsules and antennae of all species and on mandibles of all species, except P. ohioensis and P. rostrata, both of which have simplified and narrow scales on their head capsules (Figs. 1b, 2b). Mandibles of P. ornata have setiform scales with rolled lateral edges, but with the longitudinal support ridges exposed medially (Fig. 7b). Mandibles of S. louisianae have rolled setiform scales with the lateral edges nearly fused (Fig. 8c). The hollowed, basally fused scales of P. ornata are apically filled with an extruded substance (Figs. 7c, 7d). This indicates that these scales are secretory for some chemical substance that may have a defensive, attractive, or other function, a novelty not previously documented for these ants. Spongiform "tissue" may be a misnomer, because these structures appear to be sclerotized outgrowths of the integument and retain their integrity after being air dried from alcohol preserved specimens (Figs. 9, 10, 11b). Slight variations in the forms of these structures were found between the species of Pyramica, but the ventral lobes of P. hexamera were unique in being more rounded and with the cavities more closed (Fig. 10). No evidence of extruded materials associated with the lobes was found in these species. Spongiform lobes were mostly lacking in Strumigenys, with only a reduced amount present beneath the postpetiole.

Discussion The evolution of flattened setae may be related to a specific selection gradient, but once evolved, the flattened setae, or scales, can be further selected for a variety of functions. This may be one explanation for the "bizarre pilosity" present in Pyramica. No explanation can be given for the transformation of setae into flattened scales in Pyramica. However, once these flattened setae have evolved within a taxon, then they can be modified by other selection factors to assume other functions, as is evident in P. ornata that shows evidence of a substance being excreted by the scales. Scales with laterally rolled margins are hypothesized to be the derived condition from the flattened form, with those in P. ornata representing the most derived condition. Scales of S. louisianae are similarly rolled, indicating a derived state among dacetine ants with scales. It is unknown if the rolled scales in S. louisianae may have evolved independently from flat scales in that genus or if Strumigenys is be a highly derived taxon from within Pyramica.

References Cited Baroni Urbani, C., and M. L. De Andrade. 2007. The ant tribe Dacetini: Limits and constituent genera, with descriptions of a new species. Annali de Museo Civico di Storia Naturale Giacoma Doria (Genova). 99: 1-191. Bolton, B. 1999. Ant genera of the tribe Dacetonini (Hymenoptera: Formicidae). J. Nat. Hist.

33: 1639-1689. Bolton, B. 2000. The ant tribe Dacetini. Mem. Am. Entomol. Inst. 65:1-1028. Deyrup, M. 2006. Pyramica boltoni, a new species of leaf-litter inhabiting ant from Florida (Hymenoptera: Formicidae: Dacetini). Flor. Entomol. 89: 1-5. Ferris, G.F. 1934. Setae. Canadian Entomol. 66: 145-150. Kristensen, N.P., and T.J. Simonsen. 2003. ‘Hairs’ and scales. Pp. 9-22. In Kristensen, N.P. Lepidoptera, Moths and Butterflies. Vol. 2: Morphology, Physiology and Development. Walter de Gruyter, New York. MacGown, J.A., R.L. Brown, and J.G. Hill. 2005. An annotated list of the Pyramica (Hymenoptera: Formicidae: Dacetini) of Mississippi. J. Kansas Entomol. Soc. 78: 285-289. MacGown, J.A., and J.G. Hill. 2010. A new species of Pyramica Roger (Hymenoptera: Formicidae) from Mississippi, U.S.A. Florida Entomol. 93: 571-576.

Acknowledgments This research was supported by the USDA-ARS Areawide Management of Imported Fire Ant Project (R. L. Brown, Principal Investigator). Bill Monroe and Richard Kuklinski (Institute for Imaging and Analytical Technologies) facilitated coating of preparation and examination of specimens for SEM. Approved for publication by the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.

Figures 1-5. Heads, mandibles, and scales of Pyramica species. 1, P. ohioensis, a, head, b, mandibles, c, apex of clypeal scale; 2, P. rostrata, a, head, b, mandibles, c, frontal scales; 3, P. clypeata, a, head, b, mandibles, c, frontal scales; 4, P. angulata, a, head, b, mandibles, c, frontal scales; 5, P. hexamera, a, head, b, mandibles, c, frontal scales.

Figures 6-8. Heads, mandibles, and scales of Pyramica and Strumigenys species. 6, P. reflexa, a, head, b, mandibles, c, clypeal scale, d, antennal scale, e-f, frontolateral scales; 7, P. ornata, a, head, b, mandibles, c-d, clypeal scales with extruded material, e, clypeal scales, f, frontolateral scale; 8, S. louisianae, a, head, b, mandibles, c, mandibular scales, d, antennal scales, e, clypeal scales, f, postpetiolar scale.

Figures 9-10. Spongiform lobes of Pyramica. 9, P. ornata, a, petiole and postpetiole, b, portion of petiole; 10, P. hexamera, postpetiole venter.

Figure 11. Pyramica ornata. a, head, b, lateral view of body showing spongiform lobes (arrow) on petiole and postpetiole.

Efficacy of Hydramethylnon, Indoxacarb, and Methoprene Baits Against the Little Fire Ant Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae)

Susan K. Cabral, Arnold H. Hara, and Kris L. Aoki University of Hawaii at Manoa Komohana Research and Extension Center 875 Komohana Street Hilo, Hawaii 96720 email: [email protected]

Abstract Bioassays of several ant baits using lab-reared little fire ant (LFA) colonies in a no-choice test were conducted. Four of the baits were presented in a soybean oil-impregnated corn grit matrix: Probait (0.73% hydramethylnon), Advion (0.22% indoxacarb), Extinguish Professional (0.5% S-methoprene), and Extinguish Plus (0.365% hydramethylnon and 0.25% S-methoprene). A fifth bait, 0.18% indoxacarb was presented in a peanut butter matrix (Indoxacarb PB); all were compared to peanut butter (control). To ensure uptake and transmission of the toxicants, colonies were exposed to three days of fasting and two days of bait only. During this observation period, 95-100% worker and queen ant mortality were attained in the Probait and Extinguish Plus colonies at 7 weeks after treatment (WAT). This trial demonstrated that hydramethylnon and a mixture of hydramethylnon and methoprene formulated with soybean oil on corn grit were more effective than indoxacarb or methoprene on corn grit, or indoxacarb in peanut butter.

Introduction The little fire ant (LFA), Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae) has become one of the most damaging invasive pests to agriculture, landscapes, and residences, negatively impacting human and animal health to such an extent that workers have refused to harvest or tend fields, landscapes or orchards (Conant and Hirayama 2000, Conant et al. pers. comm.). A native to South America, LFA has had an outdoor latitudinal range from 32º40'S to 32º 20'N. More recently, this pest has become established on several Pacific and Atlantic islands, as well as Africa, the mainland US (Florida), and temperate regions under greenhouse conditions (Krushelnycky et al. 2005, Lubin 1984, Mikheyev et al. 2009, Vanderwoude 2007, Wetterer et al. 1999, Wetterer and Porter 2003). LFA is extremely difficult to control once established as it shares many traits of highly successful and destructive invasive ant species: 1) generalist feeding and nesting habits, 2) superficial nests in manmade and natural cavities, not limited to underground, 3) high colony mobility, 4) polygyny, 5) colony budding, 6) low intraspecific aggression, 7) high interspecific aggression, and 8) small physical size. Moreover, LFA is a quarantine pest, which hinders exportation of agricultural and floricultural products from Hawaii and other infested Pacific islands to other states and foreign countries. The most economically feasible and environmentally least damaging treatment for LFA over large areas is the use of the bait toxicant system, similar to those currently used to control the red imported fire ant (RIFA), Solenopsis invicta Buren. This minimizes the use of pesticides and provides effective colony-level control (Williams 1992). Hydramethylnon is a toxicant that has proved successful in controlling RIFA (Williams and Whelan 1992). However, this toxicant loses efficacy after exposure to light and moisture (Vander Meer et al 1982, Mallipudi et al 1986,

Taniguchi et al 2003). Effective bait formulations for LFA under tropical conditions would include the ability of the bait to remain attractive to ants under wet humid conditions, as well as exhibit delayed toxicity, good efficacy after trophylaxis, non-repellency, and ease of formulation with carriers. Because LFA are able to construct supercolonies and arboreal nests, an additional important characteristic would be the ability to apply the bait toxicant to arboreal nests. This study evaluated the efficacy of hydramethylnon, methoprene, a hydramethylnon/methoprene mixture, and indoxacarb as granular and paste baits.

Materials and Methods The trial was conducted at the University of Hawaii at Hilo, College of Agriculture, Forestry and Natural Resource Management (CAFNRM) instructional farm near Hilo, Hawaii. Baits tested were: Probait (0.73% hydramethylnon); Advion (0.22% indoxacarb); 0.18% indoxacarb in a peanut butter matrix (Indoxacarb PB, Vanderwoude et al 2010); Extinguish Professional (0.5% S-methoprene); Extinguish Plus (0.365% hydramethylnon and 0.250% S- methoprene); and an untreated peanut butter check. All treatments were replicated four times in a randomized design. Two weeks before exposure to baits, LFA workers were transferred to 5.7 L plastic boxes with fluon coated internal walls; an insect trapping adhesive was applied just inside the container rim to form a 0.5 cm wide barrier. Queens were transferred one week later. Colonies consisted of approximately 350 workers and one queen. A polystyrene petri dish (60 ID mm x 15 H mm) containing a water moistened cotton ball, used to maintain humidity, was placed at one end of each colony’s box to serve as a nest; the lid was darkened with black spray paint. Ants in all treatments were fed a diet of peanut butter, soybean oil, and sugar water (10% w/v); the feeding station was situated on the opposite end of the box from the nest. The colonies were maintained in a roofed, screen-house with natural sunlight. During the trial, the average temperature was 72.1°F (22.3°C) (range 65.6°F (18.7°C) to 82.2°F (27.9°C)); average relative humidity was 75% (range 40.8 to 100%). All food was removed three days prior to bait exposure; water was provided throughout this fasting period. Each bait treatment (1.2 ml) was measured onto a plastic vial lid (30 mm ID x 5 mm H) and placed at the feeding station end of each colony’s box. Food was returned to the feeding station 48 hours after bait introduction and changed weekly; original baits remained unchanged in the feeding station throughout the observation period. Observations of ant mortality were recorded 2 and 3 h after treatment (HAT), at 1, 2, 3, 4, 7, 9, and 11 d after treatment (DAT), then at weekly intervals from 2 to 7 weeks after treatment (WAT). Digital photographs of each colony documented live and dead ant counts which were completed in the lab. Ant mortality for each bait treatment was corrected using Abbot’s formula, then arcsine transformed prior to statistical analysis (ANOVA and Tukey’s Test).

Results and Discussion Probait (hydramethylnon) maintained a higher (P<0.05) level of LFA worker and queen mortality as compared to the other baits throughout the trial period (Fig. 1). Worker ant mortality of >50% was achieved at 2 weeks after treatment (WAT) for Probait, at 3 WAT for Extinguish Plus (hydramethylnon and methoprene), at 4 WAT for Extinguish Professional (methoprene), and at 6 WAT for Advion (indoxacarb). By 7 WAT, only Probait and Extinguish Plus achieved or exceeded 95% worker and queen ant mortality. Early mortality within colonies exposed to Extinguish Plus can be attributed to the efficacy of hydramethylnon within the bait rather than

the methoprene which would affect queen reproduction. The highest worker ant mortality achieved with Extinguish Professional was only 65.4% during the 7 wk trial period. This may be due in part to the repellency of the higher level of methoprene (0.5%) in Extinguish Professional to LFA workers as compared with Extinguish Plus (0.25% methoprene, 0.365% hydramyethylnon), which was demonstrated in field attractancy LFA trials (Cabral et al., unpublished data). Reproductive pupae, as opposed to worker pupae, were observed within 7 wk, which may be indicative of colony stresses on the queens due to methoprene’s growth regulator effect on reducing worker replacements in this no-choice bait trial. Extending trial observations to 8 to 12 WAT may have provided more insight into the effects of Extinguish Professional on LFA colonies. Worker ant mortality rates among Advion (indoxacarb in corn grit) and Indoxacarb PB-treated LFA colonies were 61.4% and 40.1% at 7 WAT, respectively. Advion did not provide faster control of LFA as compared to hydramethylnon, contrary to results observed with imported fire ants (IFA) (Solenopsis invicta Buren) (Hu and Song 2007). Moreover, colonies exposed to Indoxacarb PB continued to produce eggs and pupae during the trial (at 6 WAT), indicating the colonies were reduced but not eliminated with a single treatment. In addition, LFA possibly preferred the baits with hydramethylnon (Probait, Extinguish Plus) over either indoxacarb formulations; this preference was observed in field attractancy LFA trials (Cabral et al., unpublished data). Most fire ant baits are formulated as a granular form on corn grit, suitable for ground nesting ants, but cannot be applied during or soon after rainfall (Vander Meer et al. 1982). Previous efforts to control LFA in Hawaii under various conditions clearly demonstrate that this tramp ant species is difficult to control with commercially available ant bait products. Three factors contribute to this. First, the major agricultural area in the state receives an average annual rainfall of 300 cm (East Hawaii Island), making treatment with dry granules problematic (Souza et al. 2008). Second, LFA form nests in both the arboreal and ground strata, and ground-applied baits do not appear to be effective toward the arboreal component (Souza et al. 2008). Finally, even after repeated weekly or bi-weekly treatments with granular baits, ant activity quickly returns to pre-treatment levels after treatments cease (Souza et al. 2008, Taniguchi 2003). A paste or gel formulation may prove more effective for arboreal ants like LFA. Additional trials are needed to evaluate alternate methods of arboreal applications and to determine if a repeated bait application can achieve 100% mortality of LFA colonies with single and multiple queens.

100 Indoxacarb 90 PB (0.18% 80 indoxacarb)

60 Advion (0.22% 50 indoxacarb)

Corrected Mortality Corrected Mortality (%) 30 Extinguish 20 Professional (0.5% S- 10 methoprene) 0 1 2 3 4 5 6 7 Interval (weeks after treatment)

Figure 1: Efficacy of Ant Baits on LFA Colonies

*Treatment means with different letters were different (p<0.05) within week after treatment. Control mortality (%) was less than 10% for the duration of the trial.

References Cited Causton, C. E., C. R. Sevilla, and S. D. Porter. 2005. Eradication of the little fire ant Wasmannia auropunctata, (Hymenoptera: Formicidae) from Marchena Island, Galapagos: on the edge of success? Florida Entomol. 88: 159-168. Conant, P., and C. Hirayama. 2000. Wasmannia auropunctata (Hymenoptera: Formicidae): established on the Island of Hawaii. Bishop Museum Occasional Papers 64: 21-22. Hu, X. P., and D. Song. 2007. Field trial ant bait products for controlling fire ants, pp. 13-16. In D. H. Oi (ed). Proc. 2007 Annual Imported Fire Ant Conf., Gainesville, FL. Krushelnycky, P. D., L. L. Loope, and N. J. Reimer. 2005. The ecology, policy and management of ants in Hawai’i. Proc. Hawaiian Entomol. Soc. 37:11-25. Lowe, S., M. Browne, S. Boudjelas, and M. De Poorter. 2000. 100 of the world’s worst invasive alien species: A selection from the Global Invasive Species Database. The Invasive Species Specialist Group (ISSG) a specialist group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN), World Conservation Union (IUCN), Auckland, New Zealand, 12pp. Lubin, Y. D. 1984. Changes in native fauna of the Galapagos Islands following invasion by the little red fire ant, Wasmannia auropunctata. Biol. J. Linnaean Soc. 21:229-242. Mikheyev, A. S., S. Bresson, and P. Conant. 2009. Single-queen introductions characterize regional and local invasions by the facultatively clonal little fire ant Wasmannia auropunctata. Molecular Ecol. 18: 2937-2944.

Souza, E., P. A. Follett, D. K. Price, and E. A. Stacey. 2008. Field suppression of the invasive ant Wasmannia auropunctata (Hymenoptera: Formicidae) in a tropical fruit orchard in Hawaii. J. Econ. Entomol.101:1068-1074. Stringer, C. E., Jr, C. S. Lofgren, and F. J. Bartlett. 1964. Imported fire ant toxic bait studies: evaluation of toxicants. J. Econ. Entomol. 57: 941-945. Taniguchi, G., A. K. Asher, M. K. Kawate, K. G. Rohrbach, and G. K. Uchida. 2003. Preliminary field tests on the suitability of Amdro and Distance in ant bait container for control of the big-headed ant, Pheidole megacephala (Hymenoptera: Formicidae). Proc. Hawaiian Entomol. Soc. 36: 129-133. University of Hawai‘i at Mānoa, College of Tropical Agriculture and Human Resources. 2008. Stop the little fire ant. IP-24. UH CTAHR Office of Communication Services, Honolulu, HI. Vander Meer, R. K., D. F. Williams, and C. S. Lofgren. 1982. Degradation of the toxicant AC 217,300 in Amdro imported fire ant bait under field conditions. J. Agric. Food Chem. 30: 1045Ð1048. Vanderwoude, C. 2007. Little fire ant (Wasmannia auropunctata) in Port Vila: Report to Secretariat of the Pacific Community on activities 5-14th October 2007, and recommendations for future management. VCL New Zealand. Vanderwoude, C. 2008. Operational plan for management of Wasmannia auropunctata (little fire ant) in East Sepik Province, Papua New Guinea. VCL New Zealand. Vanderwoude, C., K. Onuma, and N. Reimer. 2010. Eradicating Wasmannia auropunctata (Hymenoptera: Formicidae) from Maui, Hawaii: the use of combination treatments to control an arboreal invasion. Proc of the Hawaii Entomol. Soc. 42: 23-31. Wetterer, J. K., P. D. Walsh, and L. J. T. White. 1999. Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae), a destructive tramp ant, in wildlife refuges of Gabon, West Africa. African Entomol. 7: 292-294. Wetterer, J. K., and S. D. Porter. 2003. The little fire ant, Wasmannia auropunctata: distribution, impact and control. Sociobiology 41: 1-41. Williams, D. F., and P. M. Whelan. 1992. Bait attraction of the introduced pest ant, Wasmannia auropunctata (Hymenopter: Formicidae) in the Galapagos Islands. J. Entomol. Sci. 1:29-34.

2012 List of Registrants

Vicky Bertagnolli Alison Bockoven Clemson Extension Texas A&M University 1555 Richland Avenue East TAMU MS 2475 Aiken, SC 29801 College Station, TX 77843 [email protected] [email protected]

Alicia Bray Anne-Marie Callcott Tennessee State University USDA-APHIS-PPQ-CPHST College of Agric., Human, & Natural Sciences 3505 25th Ave. Otis L. Floyd Nursery Research Center Gulfport, MS 39501 472 Cadillac Lane [email protected] McMinnville, TN 37110 [email protected]

Man-Yeon Choi David Cross USDA-ARS CMAVE Mississippi State University 1600 SW 23rd Drive Department of Entomology & Plant Pathology Gainesville, FL 32608 100 Old Hwy 12 [email protected] Mississippi State, MS 39762 [email protected]

Ron Davis Kimberly Dickinson The Scotts-Miracle-Gro Company Central Garden & Pet P O box 2177 7625 Woodhaven Drive Apopka, FL 32704 North Richland Hills, TX 76182 [email protected] [email protected]

Bobbye Dieckmann John Drake Coachella Valley MVCD O. C. Vector Control District 43420 Trader Place 13001 Garden Grove Blvd. Indio, CA 92201 Garden Grove, CA 92843 [email protected] [email protected]

Bart M. Drees Ed FitzHarris Texas AgriLife Extension Service Central Life Sciences Mail Stop 2475, TAMU 12111 Ford Rd. College Station, TX 77843-2475 Dallas, TX 75234 [email protected]

Kathy Flanders Geneva Ginn Auburn University Coachella Valley MVCD 201 Extension Hall 43420 Trader Place Auburn University, AL 36849-5624 Indio, CA 92201 [email protected] [email protected]

Gina Goodwyn Fudd Graham Virginia Dept. of Agriculture Auburn University 1206 Gillette Ct. 301 Funchess Hall Chesapeake, VA 23323 Auburn, AL 36849 [email protected] [email protected]

Rachel Greenman Ellie Groden Central Garden & Pet University of Maine 2030 Powers Ferry Road, Suite 370 5751 Murray Hall Atlanta, GA 30339 Orono, ME 04469-5751 [email protected] [email protected]

Frank Hale Arnold Hara The University of Tennessee University of Hawaii 5201 Marchant Drive 875 Komohana St. Nashville, TN 37211-5122 Hilo, HI 96720 [email protected] [email protected]

Tracy Harris III JoVonn Hill Central Life Sciences Mississippi Entomological Museum P.O. Box 70 P.O. Box 9775 Red Banks, MS 38661-0070 MSU, MS 39768 [email protected] [email protected]

John D. Hopkins Xing Ping Hu University of Arkansas Auburn University 2301 S. University Ave. 203 Extension Hall Little Rock, AR 72204 Auburn University, Auburn AL 36849 [email protected] [email protected]

Rong-Nan Huang Brenda Johnson-Asnicar National Taiwan University Virginia Department of Agriculture & No. 27, Lane 113, Sec. 4, Roosevelt Rd., Consumer Services Taipei, Taiwan 1100 Armory Drive [email protected] Franklin, VA 23851 [email protected]

Phil Kaster Steve Kelly Kasco Mfg./Herd Seeder The Scotts Company 170 W. 600N P.O. Box 2187 Shelbyville, IN 46176 Apopkas, FL 32704 [email protected] [email protected]

Maggie Lawrence Edward LeBrun Alabama Cooperative Extension System University of Texas at Austin 232 Duncan Hall Annex 2907 Lake Austin Blvd. Auburn University AL 36849 Austin, TX 78703 [email protected] [email protected]

Kelly Loftin Joe MacGown University of Arkansas Mississippi State University 2601 N. Young Ave. Box 9775 Fayetteville, AR 72704 MSU, MS 39768 [email protected] [email protected]

Mike Martinez Collin Cutrone McMichael Coachella Valley MVCD Texas A&M University 43420 Trader Place 3200 Woodcrest Dr. Indio, CA 92201 Bryan, TX 77802 [email protected] [email protected]

Karl Morris Paul R. Nester Bayer Environmental Science Texas AgriLife Extension Service 981 NC Highway 42 East 3033 Bear Creek Drive Clayton NC 27527 Houston, TX 77084 [email protected] [email protected]

Ryan Newcomer David Oi The Scotts Company USDA-ARS CMAVE 14111 Scottslawn Rd. 1600 SW 23rd Drive Marysville, OH 43041 Gainesville, FL 32608 [email protected] [email protected]

Jason Oliver Michael Owen Tennessee State University DuPont Professional Products College of Agric., Human, & Natural Sciences 7490 Summerhill Drive Otis L. Floyd Nursery Research Center Summerfield, NC 27358 472 Cadillac Lane [email protected] McMinnville, TN 37110 [email protected]

Kelly Palmer Doug Petty Auburn University University of Arkansas 301 Funchess Hall 400 Laurel, Suite 215 Auburn, AL 36849 Texarkana, AR 71854 [email protected] [email protected]

Sanford Porter Steve Powell USDA-ARS, CMAVE Tennessee Department of Agriculture 1600 SW 23rd Av. P. O. Box 40627 Gainesville, FL 32608 Nashville, TN 37204 [email protected] [email protected]

Tahir Rashid Mike Reeves Alcorn State University Alabama Cooperative Extension System 294 Grainger Dorsey Road 3120 Hwy 36W Mound Bayou, MS 38762 Hartselle, AL 35640 [email protected] [email protected]

Neil Reimer Robert Renthal Hawaii Department of Agriculture University of Texas at San Antonio 1428 S. King St. 1 UTSA Circle Honolulu, HI 96814 San Antonio, TX 78249 [email protected] [email protected]

Cynthia Ross Pete Schultz O. C. Vector Control District Virginia Tech 13001 Garden Grove Blvd. Hampton Roads Agric. Res. & Extension Ctr. Garden Grove, CA 92843 1444 Diamond Springs Rd. [email protected] Virginia Beach, VA 23455 [email protected]

Lawrence Shaw Jimmy Smitherman O. C. Vector Control District Alabama Cooperative Extension 13001 Garden Grove Blvd. 400 Eastern Blvd. Suite 107 Garden Grove, CA 92843 Montgomery, AL 36117 [email protected] [email protected]

Victor Teran Karen M Vail Coachella Valley MVCD University of Tennessee 43420 Trader Place, Indio, CA 92201 Entomology and Plant Pathology [email protected] 2431 Joe Johnson Dr., 205 Ellington Plant Sci. Knoxville, TN 37996 [email protected]

Robert K. Vander Meer Doug VanGundy USDA-ARS CMAVE Central Life Sciences 1600 SW 23rd Drive 12111 Ford Rd. Gainesville, FL 32608 Dallas, TX 75234 [email protected] [email protected]

Matthew VanWeelden Roger Vines Louisiana State University Alabama Cooperative Extension System 404 Life Sciences Bldg. Coosa County Extension, PO Box 247 Department of Entomology Rockford, AL 35136 Baton Rouge, LA 70803 [email protected] [email protected]

S. Bradleigh Vinson Ron Weeks Texas A&M University USDA-APHIS, PPQ Department of Entomology 920 Main Campus Drive College Station, TX 77843-2475 Raleigh, NC 27606-5210 [email protected] [email protected]

Greg White Jennifer Williams Coachella Valley MVCD 8810 Tenth Ave. N 43420 Trader Place Minneapolis, MN 55427 Indio, CA 92201 [email protected] [email protected]

Chin-Cheng (Scotty) Yang Julian Yates Department of Entomology University of Hawaii National Taiwan University 3050 Maile Way No. 1, Sec. 4, Roosevelt Road Honolulu, HI 96822 Taipei, 10617 Taiwan [email protected] [email protected]

Nadeer Youssef Pat Zungoli Tennessee State University Clemson University College of Agric., Human, & Natural Sciences 118 Long Hall, Entomology Otis L. Floyd Nursery Research Center Clemson University 472 Cadillac Lane Clemson, SC 29634-0315 McMinnville, TN 37110 [email protected] [email protected]

Conference Sponsors

APPENDIX 1 – 2012 Program Agenda

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APPENDIX 2 – Conference Photos

Welcome Session speakers, including Tennessee Commissioner of Agriculture, Julius Johnson (top photo), Tennessee State University College of Agriculture, Human & Natural Sciences Dean and Director of Research / Administrator of Extension, Dr. Chandra Reddy (middle photo), and Commissioner Johnson receiving a gift from Dr. Reddy (bottom photo).

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Speakers for the Special Session on Invasive Ants receiving a speaker gift. From left to right: Dr. Patricia Zungoli (Clemson University), Dr. Karen Vail (The University of Tennessee), and Dr. David Oi (USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology). Pictured at the far right is Dr. Jason Oliver (Conference Chairman, Tennessee State University)

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Conference reception and poster viewing room.

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Conference room and meeting participants interacting during a break.

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