IPM Award Nomination 1 James Cuda

Cover Page

2017

James P. Cuda, Ph.D.

Professor and Fulbright Scholar

Charles Steinmetz Hall

UF/IFAS Entomology & Nematology Dept.

Bldg. 970, Natural Area Drive

PO Box 110620

Gainesville, FL 32611-0620

(352) 273-3921

[email protected]

IPM Award Nomination 2 James Cuda

College of Agricultural and Life Sciences Steinmetz Hall, Bldg. 970 Entomology and Nematology Department 1881 Natural Area Drive P.O Box 110620 Gainesville, FL 32611-0620 352-273-3901 352-392-0190 Fax January 24, 2017

Southeastern Branch of the ESA Awards Committee Dear Committee:

Although I have only recently joined the Entomology and Nematology Department at the University of Florida, I have quickly come to learn of Dr. Jim Cuda’s accomplishments and passion for research and education in in biocontrol and integrated pest management. As a consequence, I have decided to nominate him for the ESA SEB Recognition Award in IPM and believe he is deserving of your strongest consideration. Jim has developed an internationally recognized program in biocontrol of invasive weeds and has become a globally recognized authority in identifying and evaluating potential biocontrol agents of invasive weeds. He has made significant contributions to the successful management of important invasive weed species in both aquatic and terrestrial environments. He also has made important discoveries in understanding the attributes of successful introduction of exotic biocontrol agents in a manner that successfully mitigates the invasion without disruption of native species. Information from this work has been critical to the management of important invasive plant species such as the tropical soda apple. He has recently identified an integrated approach to management of the important aquatic weed, Hydrilla verticllata that combines integrating different low risk control tactics including insect herbivory, a native pathogen and newly developed herbicide, effective control of hydrilla can be achieved. More importantly, combinations of a diversity of tactics in an IPM approach will lengthen the useful life of our valuable yet limited number of aquatic herbicides.

More recently, he has taken a leadership role in revision of the interagency management plan for Brazilian Peppertree in which an IPM section was included for the first time. In this plan, the interaction of natural ecological processes such as selective herbivory and plant parasitism as well as allelopathy and plant competition could be used in conjunction with conventional control practices to effectively restore sensitive natural areas. Importantly, he has recently been recognized as a Fulbright Scholar and has traveled multiple times to Brazil to continue his research on biocontrol of this important weed species. Dr. Cuda has published over 120 peer-reviewed journal articles and book chapters, and has received nearly $2 million in external research funding from state and federal funding agencies since 2004.

I have also become aware of Jim’s contributions to our teaching and extension missions. In the annual offering of his course, “Consequences of Biological Invasions,” Jim has developed novel teaching methods for distance delivery of course materials and in contributing to one of the first on- line majors developed at the University of Florida. He actively mentors graduate students and sets IPM Award Nomination 3 James Cuda

high standards for both his students and for himself, and I believe that the students respond positively to his mentoring style. His students and post-docs have gone on to important positions in our profession and it is clear that he is a talented and dedicated mentor. He has also shown an enduring commitment to undergraduate education and mentoring by employing students in his laboratory and instilling in them the excitement and joy of discovery that comes with learning to do research.

Additional details of his accomplishments can be found in the supporting materials and letters that accompany this nomination. I believe strongly that Dr. Cuda deserves your strongest consideration for this important and prestigious award.

Sincerely,

Blair D. Siegfried Professor and Chairman

The Foundation for The Gator Nation An Equal Opportunity Institution IPM Award Nomination 4 James Cuda

Academic Record and Professional IPM Experience

Dr. James P. Cuda is a Professor and Fulbright Scholar in the Department of Entomology & Nematology at the University of Florida. He grew up in Chicago Heights, IL and attended Southern Illinois University, Carbondale, where he obtained his BS (1973) and MS (1976) degrees in Zoology. He earned his Ph.D. in entomology at Texas A&M University in 1983, was a postdoc/research entomologist with the USDA in Texas and Montana from 1984-1993, and joined the University of Florida in 1994. He currently has a split appointment- 65 percent research, 25 percent extension, and 10 percent teaching. At UF, he is an affiliate faculty member for the School of Natural Resources and the Environment and Center for Aquatic and Invasive Plants. His research involves the sustainable management of invasive weeds with a focus on biological control. Dr. Cuda has published over 150 peer-reviewed journal articles and book chapters, and has received nearly $2.5 million in external research funding from state and federal funding agencies over past 10 years. He has chaired 12 graduate student committees, served as a member or co-chair on an additional 7 committees, and has mentored 14 undergraduate honors students. He also developed/teaches an online course on biological invasions.

RESEARCH

• Classical biological control of invasive aquatic and terrestrial weeds.

• Development of bio-based IPM practices for the aquatic weed hydrilla.

EXTENSION

• Demonstration and implementation of bio-based weed management strategies.

• Develop IPM training materials for county faculty, Master Naturalists, general public.

TEACHING

• ENY 4162/6935, Consequences of Biological Invasions

• ENY 6934, Biological Weed Control

• Guest lecturer, IFAS graduate/undergraduate courses

• Advising and mentoring graduate students IPM Award Nomination 5 James Cuda

Statement of IPM Accomplishments

Since its inception, numerous definitions have been proposed for the concept of Integrated Pest Management, or simply IPM. However, they all have a common theme: IPM is “. . . an ecologically based, environmentally conscious method that combines, or integrates, biological and nonbiological control techniques to suppress weeds, insects and diseases.” (Frisbee and Luna 1989)*. Biological control by natural enemies (predators, herbivores, parasitoids, and pathogens) should be the foundation of any IPM program because of its broad applicability to virtually all groups of pest organisms (Rosen et al. 1996)*. Throughout my professional career, I have worked tirelessly to promote biological control as the basis for IPM in my extension, teaching and research programs. In 2001, I was awarded a $93,949 grant from the Southern Region Sustainable Agriculture Research and Education Program titled, “Delivery of Biological Control Information and Technology in Florida”. This grant facilitated practical training in biological control and IPM as the preferred pest management strategy for conventional and organic growers, Master Gardeners and Master Naturalists, and other pest consultants. It also provided critical resources to improve the knowledge base of Florida’s extension professionals through the development and implementation of in-service training programs/educational tools in biological control techniques and IPM protocols. For example, we developed two image galleries of Beneficial Arthropods (vol. 1-Predators, vol. 2-Parasitoids) to assist UF/IFAS county and state faculty in identifying natural enemies of Florida’s key pests. The grant also provided first year funding for IPM Florida (http://ipm.ifas.ufl.edu/index.shtml), which today serves as clearinghouse for a wealth of up-to-date information on biological control and IPM technologies. These technologies are needed to reduce the risk of environmental contamination, food poisoning, and human and animal illness resulting from the misuse of pesticides in Florida. ______*Frisbee, R.E. and J.M. Luna. 1989. Integrated pest management systems: protecting profits and the environment. Farm Management: the 1989 Yearbook of Agriculture, p. 226. NAL Call No. 1Ag84y. Rosen, D., F.D. Bennett, and J.L. Capinera (eds.). 1996. Pest management in the subtropics integrated pest management- a Florida perspective. Andover, UK.

. IPM Award Nomination 6 James Cuda

Earlier in my career at the University of Florida, I facilitated the screening of the leaf beetle Gratiana boliviana that eventually led to its release for classical biological control of tropical soda apple, Solanum viarum in Polk County, Florida, in August 2003. To date, more than 220,000 beetles have been released in 39 counties in Florida, three counties in Georgia, two counties in Alabama, and one county in Texas. The beetles, which established at all sites in central and south Florida, cause extensive defoliation (20-100%), and have spread from ~2 to 16 km/year from the initial release sites. High populations of G. boliviana provided effective control of tropical soda apple in 1-2 years post-release. Furthermore, non-target plants growing in close proximity to tropical soda apple have not been attacked by the beetle post-release. The leaf beetle was incorporated into a successful IPM program for tropical soda apple. Although the economic impact of tropical soda apple on Florida grazing land ranged from $6.5 to $16 million annually, this impact has been reduced substantially by the introduction of the biological control agent. For example, cattle ranchers in Polk and Okeechobee counties have reduced or completely eliminated the herbicide applications on their properties where the beetles have been released. This program also provided Florida’s $430 million dairy cattle industry with a new biological method for controlling TSA in pastures where conventional herbicide use was prohibited.

IPM Award Nomination 7 James Cuda

Assessment of Nominee’s Accomplishments During Past 10 Years

My research program, which is in the area of environmental IPM, emphasizes the importance of biological control in invasive plant management. In 2006, I revised the Interagency Brazilian Peppertree (Schinus terebinthifolia) Management Plan for Florida (http://www.fleppc.org/Manage_Plans/2006BPmanagePlan5.pdf ), which received the University of Florida/IFAS Extension Gold Image Award in 2007. This revision included for the first time a section on IPM. In this management plan, we illustrated how natural ecological processes such as selective herbivory by biological control agents and plant parasitism (top-down regulation) as well as allelopathy and plant competition (bottom-up regulation) can be used in conjunction with conventional control practices to effectively restore sensitive natural areas in Florida invaded by Brazilian peppertree. This approach will enable land managers to use chemical and mechanical controls in a more sustainable manner for removing standing biomass while minimizing environmental impacts. In 2010, we discovered the leaflet galling psyllid Calophya latiforceps attacking Brazilian peppertree in northeastern Brazil. Because of the host specificity and impact on Brazilian peppertree exhibited by C. latiforceps in laboratory tests, a petition (TAG No. 15-02) was prepared requesting approval for its release in Florida. The USDA APHIS federal interagency Technical Advisory Group for Biological Control Agents of Weeds recommended field release on 8 April 2016. Pending review of biological and environmental assessments by APHIS PPQ, C. latiforceps will be the first biological control agent released against Brazilian peppertree in the continental United States. In 2010, I also was the recipient of a USDA NIFA IPM RAMP grant ($311,684) to develop and demonstrate a reduced risk solution for control of the invasive aquatic weed hydrilla, Hydrilla verticllata, which had developed resistance to the herbicides fluridone and more recently endothall. We showed that selective insect herbivory, a native disease organism, and low concentrations of a new herbicide recently registered for aquatic use are compatible with each other. By integrating these different low risk control tactics, effective control of hydrilla can be achieved. More importantly, combinations of a diversity of tactics in an IPM approach will lengthen the useful life of our valuable yet limited number of aquatic herbicides.

IPM Award Nomination 8 James Cuda

Professional Activities During Past 10 Years

Entomological Society of America, SEB Everglades Cooperative Invasive Species Management Area (ECISMA) Member, Research Subcommittee Florida Aquatic Plant Management Society Florida Association of Natural Resource Extension Professionals Florida Entomological Society Florida Exotic Pest Plant Council- Chair, Brazilian peppertree Task Force. Chair, Research Grant Committee Florida Lake Management Society International Organization for Biological Control, Nearctic Regional Section International Weed Science Society Weed Science Society of America Biological Control Committee Member

In the profession, Dr. Cuda has been an associate editor of the Journal of Aquatic Plant Management since 2013. He currently represents the Florida Agricultural Experiment Station on the S1058 regional project on biological control of arthropod pests and weeds. In the policy arena, Dr. Cuda is actively involved in Florida’s invasive plant management issues by serving on the UF/IFAS Invasive Plant Working Group and Invasive Species Leadership Team.

IPM Award Nomination 9 James Cuda

List of Patents/Publications During Past 10 Years

Overholt,W.A., P. Hidayat, B. LeRu, K. Takasu, J.A. Goolsby, A. Racelis, A. M. Burrell, D. Amalin, W. Agum, M. Njaku, B. Pallangyo, P.E. Klein, and J.P. Cuda 2016. Potential biological control agents for management of cogongrass [Imperata cylindrica (L.) P. Beauv. (Poaceae)] in the southeastern USA. Florida Entomologist 99: 734-739.

Cuda, J.P. 2016. The Brazilian peppertree thrips Pseudophilothrips ichini (Thysanoptera: Phlaeothripidae) as a biological control agent: a reappraisal of the timeline of events and attribution of credit. Florida Entomologist 99: 799-800.

Baniszewski, J., J.P. Cuda, S.A. Gezan, S. Sharma, and E.N.I. Weeks. 2016. Stem fragment regrowth of Hydrilla verticillata following desiccation. Journal of Aquatic Plant Management 54:53-60.

Mukherjee, A., D. Williams, M.A. Gitzendanner, W. A. Overholt and J. P. Cuda. 2016. Microsatellite and chloroplast DNA diversity of the invasive aquatic weed Hygrophila polysperma in native and invasive ranges. Aquatic Botany 129: 55-61. http://dx.doi.org/10.1016/j.aquabot.2015.12.004

Cuda, J.P., J. F. Shearer, E.N.I. Weeks, E. Kariuki, J. Baniszewski and M. Giurcanu. 2016. Compatibility of an insect, a fungus and a herbicide for IPM of dioecious hydrilla. Journal of Aquatic Plant Management 54: 20-25. http://apms.org/wp/wp- content/uploads/2015/02/japm-54-01-020.pdf

Cuda, J.P., J.L. Gillmore, A.O. Mitchell, J. Bricker, R.A. Watson, B.R. Garcete-Barrett, and A. Mukherjee. 2016. Laboratory biology and impact of a stem boring weevil Apocnemidophorus pipitzi (Faust) (Coleoptera: Curculionidae) on Schinus terebinthifolia. . Biocontrol Science and Technology 26: 1249-1266. http://dx.doi.org/10.1080/09583157.2016.1193844

Cuda, J.P. 2016. Novel approaches for reversible field releases of candidate weed biological control agents: Putting the genie back into the bottle, Chapter 7, pp. 137-152. In: J.F. Shroder and R. Sivanpillai (eds.), Biological and Environmental Hazards, Risks and Disasters. Elsevier, Inc., Amsterdam. http://dx.doi.org/10.1016/B978-0-12-394847- 2.00010-3

Prade, P, R. Diaz, M. D. Vitorino, J. P. Cuda, P. Kumar, B. Gruber, W.A. Overholt. 2015. Galls induced by Calophya latiforceps Burckhardt (Hemiptera: Calophyidae) reduce leaf performance and growth of Brazilian peppertree. Biocontrol Science and Technology 26: 23-34. DOI: 10.1080/09583157.2015.1072131

Baniszewski, J., E.N.I. Weeks, and J.P. Cuda. 2015. Impact of refrigeration on eggs of the hydrilla tip miner Cricotopus lebetis: larval hatch rate and subsequent development. J. Aquatic Plant Management 53: 209-215.

IPM Award Nomination 10 James Cuda

Coon, B.R., N.E. Harms, J.P. Cuda, and M.J. Grodowitz. 2014. Laboratory biology and field population dynamics of Trichopria columbiana (Hymenoptera: Diapriidae), an acquired parasitoid of two hydrilla biological control agents. Biocontrol Science and Technology 24: 1243-1264.

Le Ru, B. P., C. Capdevielle-Dulac, E. F. A. Toussaint, D. E. Conlong, J. Van den Berg, B. Pallangyo, G. Ong’amo, R. Molo, W. Overholt, J. Cuda and G. J. Kergoat. 2014. Integrative taxonomy of Acrapex stem borers (Lepidoptera: Noctuidae: Apameini): combining morphology and Poisson Tree Process analyses. Invertebrate Systematics 28: 451-475.

Stratman, K.N., W.A. Overholt, J.P. Cuda, A. Mukherjee, R. Diaz, M.D. Netherland, and P.C. Wilson. 2014. Temperature-dependent development, cold tolerance, and potential distribution of Cricotopus lebetis, a tip miner of Hydrilla verticillata. Journal of Insect Science 14(153): DOI: 10.1093/jisesa/ieu015.

Baniszewski, J., B.R. Coon, J.P. Cuda, N.E. Harms, M.J. Grodowitz, D.H. Habeck, J.E. Hill, J. Russell, E.N.I. Weeks, 2014. Insects and fish associated with hydrilla, pp. 77-124. In J.L. Gillett-Kaufman, V. Ulrike- Lietze, E.N.I Weeks, Hydrilla Integrated Management. UF|FAS. Gainesville, FL.

Cuda, JP. 2014. Chapter 5: Aquatic plants, mosquitoes and public health, pp. 31-36. In, Gettys LA, Haller WT Petty DG (eds.), Biology and Control of Aquatic Plants: A Best Management Practices Handbook, 3rd edition. Aquatic Ecosystem Restoration Foundation, Marietta, GA.

Cuda, JP. 2014. Chapter 8: Introduction to biological control of aquatic weeds, pp. 51-58. In Gettys LA, Haller WT, Petty DG (eds.), Biology and Control of Aquatic Plants: A Best Management Practices Handbook, 3rd edition. Aquatic Ecosystem Restoration Foundation, Marietta, GA.

Cuda, JP. 2014. Chapter 9: Insects for biocontrol of aquatic weeds, pp. 59-66. In Gettys LA, Haller WT, Petty DG. (eds.), Biology and Control of Aquatic Plants: A Best Management Practices Handbook, 3rd edition. Aquatic Ecosystem Restoration Foundation, Marietta, GA.

Medal, J., Gandolfo, D., Gaskalla, R., Overholt, W., Diaz, R., Charudattan, R., Bustamante, N., 77 Davis, B.J., Hibbard, K., Fox, A., Díaz, J., Roda, A., Amalin, D., Hight, S., Stansly, P., Gioeli, K., Osborne, L., Seller, B., McKay, F., Usnick, S., Sudbrink, D., Cuda, J., Pitelli, R., Santana, A., Vitorino, M., Beal, L., Buss, A., Pedrosa, J.H., Bredow, E., Ohashi, D., Wikler, C. and Gravena, R. 2014. Biological control of Solanum viarum in Florida, USA: a successful project, p. 77-81. Proceedings of the XIV International Symposium on Biological Control of Weeds, F.A.C. Impson, C.A. Kleinjan and J.H. Hoffmann (eds), 2-7 March 2014, Kruger National Park, South Africa.

IPM Award Nomination 11 James Cuda

Manrique, V., J.P. Cuda and W.A. Overholt. 2013. Brazilian peppertree: a poster child for invasive plants in Florida landscapes. Journal of Florida Studies http://www.journaloffloridastudies.org/0102peppertree.html

Mukherjee, A. M.R. Khan, W.T. Crow, and J P.Cuda. 2013. Phytoparasitic nematodes associated with the rhizosphere of the aquatic weed Hygrophila polysperma. J. Aquatic Plant Manage. 50:84-91.

Mukherjee, A., C.A. Ellison, J. P. Cuda, and W. A. Overholt. 2013. Biological control of hygrophila: Foreign exploration for candidate natural enemies, pp. 142-152. In: Y. Wu, T. Johnson, S. Sing, S. Raghu, G. Wheeler, P. Pratt, K. Warner, T. Center, J. Goolsby, and R. Reardon (eds.). Proc. XIII Int’l Symp Biol Control of Weeds, Waikiloa, HI, USA, September 11-16, 2011, FHTET- 2012-07, Jan 2013. USDA Forest Service, Forest Health Technology Enterprise Team, Morgantown, WV, USA.

Christ, L.R., J.P. Cuda, W.A. Overholt, M.D. Vitorino, and A. Mukherjee. 2013. Biology, host preferences, and potential distribution of Calophya terebinthifolii (Hemiptera: Calophyidae), a candidate for biological control of Brazilian peppertree, Schinus terebinthifolius, in Florida. Florida Entomol. 96: 137-147.

Stratman, K.N., W.A. Overholt, J.P. Cuda, M.D. Netherland, and P.C. Wilson. 2013. Host range and searching behaviour of Cricotopus lebetis (Diptera: Chironomidae), a tip miner of Hydrilla verticillata (Hydrocharitaceae). Biocontrol Sci. & Tech. 23: 317-334.

Stratman, K.N., W.A. Overholt, J.P. Cuda, M.D. Netherland, and P.C. Wilson. 2013. Toxicity of fipronil to the midge, Cricotopus lebetis Sublette. Journal of Toxicology and Environmental Health, Part A, 76: 716-722.

Stratman, K.N., W.A. Overholt, J.P. Cuda, M.D. Netherland, and P.C. Wilson. 2013. Diversity of Chironomidae (Diptera) associated with hydrilla in Florida. Florida Entomol 96: 654- 657.

Cuda, J.P., L.R. Christ, V. Manrique, W.A. Overholt, G.S. Wheeler, and D. A. Williams. 2012. Role of molecular genetics in identifying ‘fine tuned’ natural enemies of the invasive Brazilian peppertree, Schinus terebinthifolius: a review. BioControl 57: 227-233.

Overholt, W.A., J.P. Cuda and L. Markle. 2012. Can novel weapons favor native plants? Allelopathic interactions between Morella cerifera (L.) and Schinus terebinthifolius Raddi. J. Torrey Botanical Society 139: 356-366.

Benda, N, J., Possley, D. Powell, C. Buchanan-McGrath, and J. Cuda. 2012. New host plant record for the poison ivy sawfly, Arge humeralis (Beauvois) (Hymenoptera: Argidae), and its performance on two host plant species. Florida Entomol. 95: 529-531.

Copeland, R. S., B. Gidudu, F. Wanda, J. H. Epler, J. P. Cuda and W. A. Overholt. 2012. Chironomidae (Insecta: Diptera) associated with Hydrilla verticillata (Alismatales: IPM Award Nomination 12 James Cuda

Hydrocharitaceae) and other submersed aquatic macrophytes in Lake Bisina and other Ugandan lakes, with a new country list. Journal of East African Natural History 101: 29- 66.

Copeland, R. S., E. Nkubaye, B. Nzigidahera, J. H. Epler, J. P. Cuda and W. A. Overholt. 2012. The diversity of Chironomidae (Diptera) associated with Hydrilla verticillata (Alismatales: Hydrocharitaceae) and other aquatic macrophytes in Lake Tanganyika, Burundi. Annals of the Entomological Society of America 105: 206-224.

Paraiso, O., M.T.K. Kairo, S.D. Hight, N.C. Leppla, J.P. Cuda, M. Owens, M.T. Olexa. 2012. Opportunities for improving risk communication during the permitting and importation process of entomophagous biological control agents: a review of current systems. BioControl. DOI 10.1007/s10526-012-9464-0.

Mukherjee, A., and J. P. Cuda. 2012. Biological control prospects for hygrophila. Aquatics 34: 13-14.

Cuda, J.P. 2012. Can nematodes control tropical soda apple (Solanum viarum)?, pp. 77-79. In: Medal, J., W. Overholt, R. Charudattan, J. Mullahey, R. Diaz and J. Cuda, Tropical Soda Apple Management Plan: Recommendations from the Florida Tropical Soda Apple Implemenation Management Team. University of Florida, Gainesville.

Cuda, J.P. 2012. Can native Leptinotarsa beetles control tropical soda apple (Solanum viarum)?, pp. 80- 85. In: Medal, J., W. Overholt, R. Charudattan, J. Mullahey, R. Diaz and J.Cuda, Tropical Soda Apple Management Plan: Recommendations from the Florida Tropical Soda Apple Implemenation Management Team. University of Florida, Gainesville.

Cuda, J. P., L. R. Christ, V. Manrique, W. A. Overholt, G. S. Wheeler, and D. A. Williams. 2012. Role of Molecular Genetics in Identifying ‘Fine Tuned’ Natural Enemies of the Invasive Brazilian Peppertree, Schinus terebinthifolius: A Review. BioControl 57: 227- 232.

Mukherjee, A., D. A. Williams, G. S. Wheeler, J. P. Cuda, S. Pal and W. A. Overholt. 2012. Brazilian peppertree (Schinus terebinthifolius) in Florida and South America: Evidence of a niche shift driven by hybridization. Biol Invasions 14: 1415-1430 http://www.springerlink.com/content/w416h04tk82778q3/fulltext.pdf

Mukherjee, A., J.W. Jones, J. P. Cuda, G. Kiker and W. A. Overholt. 2012. Effects of simulated herbivory on growth and biomass accumulation of the invasive weed hygrophila and its mathematical modelling Biological Control 60: 271-279.

Mukherjee, A. R. Diaz, M. Thom, W. A. Overholt and J. P. Cuda. 2012. Niche based prediction of establishment of biocontrol agents: an example with Gratiana boliviana and tropical soda apple. Biocontrol Science and Technology 22: 447-461.

IPM Award Nomination 13 James Cuda

Lewis, D. S., J.P Cuda, and B. R. Stevens. 2011. A novel biorational pesticide: Efficacy of methionine against Heraclides (Papilio) cresphontes, a surrogate of the invasive Princeps (Papilio) demoleus (Lepidoptera: Papilionidae). J. Econ. Entomol. 104: 1986-1990.

Cuda, J.P., B. R. Coon, Y. M. Dao and T. D. Center. 2011. Effect of an herbivorous stem mining midge on the growth of hydrilla. J. Aquatic Plant Manage. 49: 83-89.

Wanda, F., B. Gidudu, S. Wandera, R. S. Copeland, J. P. Cuda and W. A. Overholt. 2011. Herbivory of Hydrilla verticillata by cichlid fish in Lake Bisina, Uganda. Journal of East African Natural History 100: 113-121.

Copeland, R. S., E. Nkubaye, B. Nzigidahera, J. P. Cuda and W. A. Overholt. 2011. The African burrowing mayfly, Povilla adusta (Ephemeroptera: Polymitarcyidae), damages Hydrilla verticillata (Alismatales: Hydrocharitaceae) in Lake Tanganyika. Florida Entomol. 94: 669-676.

Burckhardt, D., J. P. Cuda, V. Manrique, R. Diaz, W. A. Overholt, D. A. Williams, L. R. Christ, and M. D. Vitorino. 2011. Calophya latiforceps, a new species of jumping plant lice (Hemiptera: Calophyidae) associated with Schinus terebinthifolius (Anacardiaceae) in Brazil. Florida Entomol. 94(3): 489-499.

Medal, J., N. Bustamante, E. Bredow, H. Pedrosa, W. Overholt, R. Diaz, and J. Cuda. 2011. Host specificity of Anthonomus tenebrosus (Coleoptera: Curculionidae), a potential biological control agent of tropical soda apple (Solanaceae) in Florida. Florida Entomol 94: 214- 225.

Manrique,V. R. Diaz, J.P. Cuda, W.A. Overholt. 2011. Suitability of a new invader as a target for biological control in Florida. Invasive Plant Science and Management 4: 1-10.

Mukherjee, A., M. C. Christman, W. A. Overholt, J. P. Cuda. 2011. Prioritizing areas in the native range of hygrophila for surveys to collect biological control agents. Biological Control 56: 254-262.

Vitorino, M.D., L.R. Christ, G. Barbieri, J.P. Cuda and J.C. Medal. 2011. Calophya terebinthifolii (Hemiptera: Psyllidae), a candidate for biological control of Schinus terebinthifolius (Anacardiaceae): Preliminary host range, dispersal, and impact studies. FL Entomol. 94(3): 694-695.

Macedo, Davi M. de, Danilo. B Pinho, Robert W. Barreto, Olinto L. Pereira & James P. Cuda. 2010. Black mildew fungi (Meliolaceae) associated with Schinus terebinthifolius (Brazilian pepper tree) in Brazil. Mycotaxon 114: 429–437.

Christ, L., J. Cuda, W. Overholt, and M. Vitorino. 2010. New candidate for biological control of Brazilian peppertree? Wildland Weeds 13: 12-13.

IPM Award Nomination 14 James Cuda

Medal, J., N. Bustamante, W. Overholt, R. Diaz, P. Stansly, A. Roda, D. Amalin, K. Hibbard, R. Gaskalla, B. Sellers, S. Hight and J. Cuda. 2010. Biological control of tropical soda apple (Solanaceae) in Florida: Post-release evaluation. Florida Entomologist 93: 130- 132.

Medal, J., N. Bustamante, M. Vitorino, L. Beal, W. Overholt, R. Diaz and J. Cuda. 2010. Host specificity tests of Gratiana graminea (Coleoptera: Chrysomelidae), a potential biological control agent of tropical soda apple, Solanum viarum (Solanaceae). Florida Entomol. 93: 231-242.

Medal, J.C. and J.P. Cuda. 2010. Establishment and initial impact of Gratiana boliviana (Chrysomelidae), first biocontrol agent released against tropical soda apple in Florida. Florida Entomol. 93: 493-500.

Schmid, T.A., J.P. Cuda, G.E. MacDonald, and J.L. Gillmore. 2010. Performance of two established biological controls agents on susceptible and fluridone resistant genotypes of the aquatic weed hydrilla. J. Aquat. Plant Manage 49: 102-105.

Crow, W.T., J.P. Cuda, and B.R. Stevens. 2009. Efficacy of methionine against ectoparasitic nematodes on golf course turf. J. Nematol. 41: 217-220.

Van Driesche, R.G., Carruthers, R.I., Center, T., Hoddle, M.S., Hough-Goldstein, J., Morin, L., Smith, L., Wagner, D.L., Blossey, B., Brancatini, V., Casagrande, R., Causton, C.E., Coetzee, J. A., Cuda, J., Ding, J., Fowler, S.V., Frank, J.H., Fuester, R., Goolsby, J., Grodowitz, M., Heard, T.A., Hill, M.P., Hoffmann, J.H., Huber, J., Julien, M., Kairo, M.T.K., Kenis, M., Mason, P., Medal, J., Messing, R., Miller, R., Moore, A., Neuenschwander, P., Newman, R., Norambuena, H., Palmer, W.A., Pemberton, R., Perez Panduro, A., Pratt, P.D., Rayamajhi, M., Salom, S., Sands, D., Schooler, S., Sheppard, A., Shaw, R., Schwarzländer, M., Tipping, P.W., van Klinken, R.D., 2010. Classical biological control for the protection of natural ecosystems: past achievements and current efforts. Biological Control 54, Supplement 1.

Cuda, J.P. 2009. Book review: Muniappan, Reddy, G.V.P., and Raman, A. (eds.). Biological control of tropical weeds using arthropods. Cambridge University Press, Cambridge, UK, ISBN 978-0-521-87791-6. FL Entomol. 92: 675-676.

Cuda, J.P., Medal, J.C., Gillmore, J.L., Habeck, D.H., Pedrosa-Macedo, J.H. 2009. Fundamental host range of Pseudophilothrips ichini sensu lato (Thysanoptera: Phlaeothripidae), a candidate biological control agent of Schinus terebinthifolius (Sapindales: Anacardiaceae) in the USA. Environ. Entomol. 38: 1642-1652.

Cuda JP, Gordon DR, DiTomaso, JM. 2009. Cultivating non-native plants in Florida for biomass production: Hope or harm? Wildland Weeds 12: 21.

Cuda, JP. 2009. Chapter 5: Aquatic plants, mosquitoes and public health, pp. 31-34. In Haller WT, Gettys LA, Bellaud M (eds.), Best Management Practices Manual for Aquatic IPM Award Nomination 15 James Cuda

Plants. Aquatic Ecosystem Restoration Foundation, Marietta, GA. http://plants.ifas.ufl.edu/misc/pdfs/AERF_handbook.pdf.

Cuda, JP. 2009. Chapter 8: Introduction to biological control of aquatic weeds, pp. 47-54. In Haller WT, Gettys LA, Bellaud M (eds.), Best Management Practices Manual for Aquatic Plants. Aquatic Ecosystem Restoration Foundation, Marietta, GA. http://plants.ifas.ufl.edu/misc/pdfs/AERF_handbook.pdf.

Cuda, JP. 2009. Chapter 9: Insects for biocontrol of aquatic weeds, pp. 55-60. In Haller WT, Gettys LA, Bellaud M (eds.), Best Management Practices Manual for Aquatic Plants. Aquatic Ecosystem Restoration Foundation, Marietta, GA. http://plants.ifas.ufl.edu/misc/pdfs/AERF_handbook.pdf.

Diaz, R., W.A. Overholt, J.P. Cuda. P.A. Pratt, and A. Fox. 2009. Host specificity of Ischnodemus variegatus, an herbivore of West Indian marsh grass (Hymenachne amplexicaulis). Biocontrol 54: 307-321.

Grajczyk, A., D. Williams, W. Overholt, J. Cuda, and S. Brown. 2009. Characterization of microsatellite loci in Hydrilla verticillata. Molecular Ecology Resources 9: 1460-1559.

Manrique, V., J. P. Cuda, W. A. Overholt, and S.M.L. Ewe. 2009. Influence of host plant quality on the performance of Episimus unguiculus, a candidate biological control agent of Brazilian peppertree in Florida. BioControl 54: 475-484.

Manrique, V. J. P. Cuda, and W. A. Overholt. 2009. Effect of herbivory on growth and biomass allocation of Brazilian peppertree (Sapindales: Anacardiaceae) seedlings in the laboratory. BioControl Sci. & Tech. 19: 657-667.

Manrique, V., J. P. Cuda, W. A Overholt and S.M.L. Ewe. 2009. Synergistic effect of insect herbivory and plant parasitism on the performance of the invasive tree Schinus terebinthifolius (Anacardiaceae) Entomologia Experimentalis et Aplicata. 132: 118-125.

McKay, F., Oleiro, M., Walsh, G. C., Gandolfo, D., Cuda, J. P., and Wheeler, G. S. 2009. Natural enemies of Brazilian peppertree (Schinus terebinthifolius: Anacardiaceae) from Argentina: their possible use for biological control in the USA." Florida Entomol. 92: 292-303.

Medal, J., N. Bustmante, J. Barrera, O. Avila, J. Monzon, and J. Cuda. 2009. Host specificity of Anthonomus elutus (Coleoptera: Curculionidae), a potential biological control agent of wetland nightshade (Solanaceae) in Florida. Florida Entomol. 92: 458-469.

Moeri, O.E. J. P. Cuda, W.A. Overholt, S. Bloem, and J. E. Carpenter. 2009. F1 Sterile Insect Technique: a Novel Approach for Risk Assessment of Episimus unguiculus (Lepidoptera: Tortricidae), a Candidate Biological Control Agent of Schinus terebinthifolius in the Continental USA. BioControl Sci. & Tech. 19, Supplement 1: 303-315.

Diaz, R., W.A. Overholt, J.P. Cuda. P.A. Pratt, and A. Fox. 2008. Temperature-dependent IPM Award Nomination 16 James Cuda

development, survival and potential distribution of Ischnodemus variegatus (Hemiptera: Blissidae), an herbivore of West Indian marsh grass (Hymenachne amplexicaulis). Ann. Entomol. Soc. Am. 101: 604-612.

Faria, A.B.V., R.W. Barreto and J.P. Cuda. 2008. Fungal pathogens of Schinus terebinthifolius from Brazil as potential classical biological control agents, pp. 270-277. In: M.H. Julien, R. Sforza, M.C. Bon, H.C. Evans, P.E. Hatcher, H.L. Hinz and B.G. Rector (eds.), Proc. XII Intern. Symp. Biol. Contr. Weeds. CAB International, Wallingford, UK.

Gandolfo, D., J.C. Medal, and J.P. Cuda. 2008. Effects of temperature on the development and survival of Metriona elatior (Coleoptera: Chrysomelidae) immature. Florida Entomol. 91: 491-493.

Cuda, J.P. 2008. Biological control of weeds, pp. 501-506. In Capinera, J.L. (ed.), Encyclopedia of Entomology, Second Edition, Kluwer Academic Publishers. Dordrecht, Netherlands.

Cuda, J.P., J.L. Gillmore, J.C. Medal and J.H. Pedrosa-Macedo. 2008. Mass rearing of Pseudophilothrips ichini (Thysanoptera: Phlaeothripidae), an approved biological control agent for Brazilian peppertree, Schinus terebinthifolius (Sapindales: Anacardiaceae). Florida Entomol. 91: 338-340.

Cuda, J. P., R. Charudattan, M. J. Grodowitz, R. M. Newman, J. F. Shearer, M. L. Tamayo, and B. Villegas. 2008. Recent advances in biological control of submersed aquatic weeds. J. Aquat. Plant Manage. 46: 15-32.

Manrique, V., J. P. Cuda, W. A Overholt, and D. A. Williams. 2008. Effect of host-plant genotypes on the performance of three candidate biological control agents of Brazilian peppertree in Florida. Biological Control: Theory and Applications in Pest Management 47: 167-171.

Manrique, V., J. P. Cuda, and W. A. Overholt. 2008. Temperature-dependent development and potential distribution of Episimus utilis (Lepidoptera: Tortricidae), a candidate biological control agent of Brazilian peppertree (Sapindales: Anacardiaceae) in Florida. Environ. Entomol. 37: 862-870.

Iponga, D. M., Cuda, J. P., Milton, S. J.and Richardson, D. M. 2008 Megastigmus wasp damage to Schinus molle (Peruvian pepper tree) seeds across a rainfall gradient in South Africa: Implications for invasiveness. African Entomol. 16: 127-131.

Mukherjee, A., J. P. Cuda, W.A.Overholt, and C. Ellison. 2008. Biological control of Hygrophila polysperma: Searching for natural enemies in India- first trip report. Aquatics: 30: 20-22.

Legaspi, J.C., C. Gardner, G. Queeley, N. Leppla, J. Cuda, and B.C. Legaspi, Jr. 2007. Effect of organic and chemical fertilizers on growth and yield of hot pepper, and insect pests and their natural enemies. Subtropical Plant Science 59: 74-84.

IPM Award Nomination 17 James Cuda

Cuda, J.P., Dunford, J.C., and Leavengood Jr., J.M. 2007. Invertebrate fauna associated with torpedograss, Panicum repens (Cyperales: Poaceae), in Lake Okeechobee, Florida and prospects for biological control. Florida Entomol. 90: 238-248.

Gandolfo, D., F. McKay, J. C. Medal, and J. P.Cuda. 2007. Open-field host specificity test of Gratiana boliviana (Chrysomelidae), a biocontrol agent of tropical soda apple in the USA. Florida Entomol. 90: 223-228.

Treadwell, L.W. and Cuda, J.P. 2007. Effects of defoliation on growth and reproduction of Brazilian peppertree (Schinus terebinthifolius). Weed Science 55: 137-142.

Wessels, F.J, Cuda, J.P., Johnson, M.T. and Pedrosa-Macedo, J.H. 2007. Host specificity of Tectococcus ovatus (Hemiptera: Eriococcidae), a potential biological control agent of the invasive strawberry guava, Psidium cattleianum (Myrtales: Myrtaceae), in Florida. BioControl 52: 439-449.

Williams, D.A., Muchugu, E., Overholt, W.A. and Cuda, J.P. 2007. Colonization patterns of the invasive Brazilian peppertree, Schinus terebinthifolius, in Florida. Heredity 98: 284-293.

Pedrosa-Macedo, J.H., W. Poulmann, L. Stolle, D. Ukan, J.P. Cuda, and J.C. Medal. 2006. Greenhouse mass rearing of a defoliating sawfly for biological control of Brazilian peppertree. Floresta 36: 371-378.

Tifft, K.H., Leppla, N.C., Osborne, L.S., and Cuda, J.P. 2006. Rearing Diomus terminatus (Coleoptera: Coccinellidae) on the corn leaf aphid, Rhopalosiphum maidis (Homoptera: Aphididae). Florida Entomol. 89: 263-265.

IPM Award Nomination 18 James Cuda

List of Scholarly Presentations During Past 10 Years

Bini, L.A., J.P. Cuda, and M.D. Vitorino. 2016. Controle Biologico da Aroeira (Schinus terebinthifolia Raddi)", at the FURB Interacao held in Blurmenau, 21 September.

Boeno, M.M., L.A. Bini, J.P. Cuda, and M.D. Vitorino. 2016. "Availacao dos danos causados por Calophya terebinthifolii (Hemiptera: Psyllidae): Potencial agente de controle biological de Schinus terebinthifolia (Anacardiaceae) (Aroeira-vermelha). 10th Mostra Teaching Integrated Research, Extension and Culture Conference (MIPE) held on the FURB campus, 28 -30 September 2016.

Prade, P., J.P. Cuda, and W.A. Overholt. 2016. Host specificity of the psyllid Calophya terebinthifolii (Hemiptera: Calophyidae), a potential biological control agent against Brazilian peppertree in Florida, USA. 2016 XXV International Congress of Entomology, Orlando, FL, USA, 25-30 September.

Kariuki, E.M., J.P. Cuda, R.L. Hix, J. L. Gillett-Kaufman, and S. Hight. 2016. Field host specificity of a potential hydrilla biological control agent, Cricotopus lebetis Sublette (Diptera: Chironomidae). 2016 XXV International Congress of Entomology, Orlando, FL, USA, 25-30 September.

Cuda, J.P. 2016. Recent advances in biological weed control: a Florida perspective. Invited Seminar, Department of Entomology, Purdue University, 27 October 2016.

Prade, P., R. Diaz, M.D. Vitorino, W.A. Overholt, and J.P. Cuda. 2016. Calophya latiforceps: A Potential Biological Control Agent of Brazilian Peppertree in Florida. 38th Annual Meeting of the Florida Weed Science Society, Haines City, FL, 29 February- 1 March.

Cuda, J.P., E.N.I. Weeks, J. F. Shearer, M.A. Jackson and M.V. Hoyer. 2016. New IPM approach for hydrilla management: Research update. 40th Annual Meeting of the Florida Aquatic Plant Management Society, Daytona Beach, FL, 17-20 October.

Cuda, J.P., W.A. Overholt, R. Diaz. V. Manrique, and P. Prade. 2016. Brazilian peppertree biological control: Update. 2016 Everglades Invasive Species Summit, Davie, FL, 12-13 July.

Cuda, J.P., E.N.I. Weeks, J. Shearer, M.A. Jackson and M.V. Hoyer. 2016. Field testing a new IPM approach for hydrilla management: Preliminary results. 27th Annual Technical Symposium, Florida Lake Management Society, Daytona Beach Shores, FL, 7-10 June (Invited paper). IPM Award Nomination 19 James Cuda

Cuda, J.P., W.A. Overholt, R. Diaz. V. Manrique, P. Prade, and J.C. Medal. 2016. BioControl of Brazilian peppertree- thrips and psyllids on the horizon. 2016 Central Florida Invasive Species Workshop, Lakeland, FL, 24 May.

Prade, P., W.A. Overholt, and J.P. Cuda. 2016. Host specificity of Calophya terebinthifolii, a biological control candidate for Brazilian peppertree in Florida. 2016 Annual Conference, Florida Exotic Pest Plant Council, Melbourne, FL, 9-11 March.

Cuda, J.P., M.D. Vitorino, M. Boeno, F. M. dos Santos, P. Prade, and W.A. Overholt. 2016. Native range assessment of the impact on Brazilian peppertree by a potential biological control agent, Calophya terebinthifolii (Hemiptera: Calophyidae): Preliminary results. 2016 Annual Conference, Florida Exotic Pest Plant Council, Melbourne, FL, 9-11 March.

Cuda, J.P., M.D. Vitorino, M. Boeno, F.M. dos Santos, and P. Prade. 2016. Calophya terebinthifolii (Hemiptera: Calophyidae), a Potential Biological Control Agent of Brazilian Peppertree: Preliminary results of a field impact study. 90th Annual Meeting of the ESA-SEB, Raleigh, NC, 13-16 March.

Cuda, J.P., B.R. Stevens, N. Denslow, K. Kroll, J. Baniszewski, and E.N.I. Weeks. 2016. Non- target aquatic organism toxicity testing of a biopesticide for mosquito management. 90th Annual Meeting of the ESA-SEB, Raleigh, NC, 13-16 March.

Stachowiak, J. Baniszewski, J. P. Cuda and E. N. I. Weeks. 2016. Influence of Competition and Predation on Success of Cricotopus lebetis as a Biological Control Agent. 90th Annual Meeting of the ESA-SEB, Raleigh, NC, 13-16 March.

Cuda, J.P., E.N.I. Weeks, J. L. Gillett-Kaufman, M.A. M.V. Hoyer and Jackson. 2016. Testing a new IPM approach for hydrilla management: an udpate. 20th Annual Southwest Florida Invasive Species Workshop, Ft. Myers, FL, 24 February (Invited paper).

Cuda, J.P. and W.A. Overholt. 2015. Is poisonwood vulnerable to attack by biological control agents of Brazilian peppertree? 19th Annual Southwest Florida Invasive Species Workshop, Ft. Myers, FL, 22 January (Invited paper).

Cuda, J.P. and W.A. Overholt. 2015. Screening of a new candidate biological control agent of Brazilian peppertree. 14th Activities Board of Directors Meeting, Florida Industrial and Phosphate Research Institute, Bartow, FL, 23 January (Invited paper).

IPM Award Nomination 20 James Cuda

Cuda, J.P., W.A. Overholt, R. Diaz, and V. Manrique. 2015. Recent advances in biological control of Brazilian peppertree, Schinus terebinthifolia. S1058 Regional Project Symposium, ESA-SEB Annual Meeting, Biloxi, MS, 15-18 March (Invited paper).

Cuda, J.P. and W.A. Overholt. 2015. Will Biological Control Agents of Brazilian Peppertree Impact Poisonwood? 2015 Annual Conference of the Florida Exotic Pest Plant Council: Knocking ‘Em Out of the Park, Melbourne, FL, 8-10 April (Invited paper).

Prade, P., R. Diaz, M.D. Vitorino, J.P. Cuda and W.A. Overholt. 2015. Damage by Calophya latiforceps (Hemiptera: Calophyidae) results in reduction of photosynthesis, chlorophyll and growth of Brazilian peppertree. 2015 Annual Conference of the Florida Exotic Pest Plant Council: Knocking ‘Em Out of the Park, Melbourne, FL, 8-10 April (Contributed paper).

Cuda, J.P., W.A. Overholt, R. Diaz, V. Manrique, A.M. Berro, P. Prade, and J. Medal. 2015. Recent advances in biological control of Brazilian peppertree, Schinus terebinthifolia, Greater Everglades Ecosystem Restoration: Science in Support of Everglades Restoration, Coral Springs, FL, 21-23 April (Invited paper).

Medal, J., W. Overholt, R. Diaz, A. Roda, K. Hibbard, R. Charudattan, N. Bustamante, S. Hight, and J. Cuda. 2015. Biological control of tropical soda apple, Solanum viarum (Solanaceae) in Florida: A successful project, Greater Everglades Ecosystem Restoration: Science in Support of Everglades Restoration, Coral Springs, FL, 21-23 April (Invited paper).

Overholt, W.A., J.P. Cuda, J.A. Goolsby, A.M. Burrell, B. Le Ru, K. Takasu, P.E. Klein, and A. Recelis. 2015. Prospects for classical biological control of cogongrass, Greater Everglades Ecosystem Restoration: Science in Support of Everglades Restoration, Coral Springs, FL, 21-23 April (Invited paper).

Cuda, J.P., J. Shearer, J.L. Gillett-Kaufman, V. Ulrike- Lietze, and E.N.I. Weeks. 2015. Testing a new IPM approach for hydrilla management: an update. 26th Annual Technical Symposium, Florida Lake Management Society, Naples, FL, 8-11 June (Invited paper).

Bennett, B.O. and J.P. Cuda. 2015. Biological control of the Brazilian peppertree. 57th Student Science Training Program, University of Florida, 23 July.

Kariuki, Eutychus, James Cuda, Jennifer Gillett-Kaufman, Stephen Hight, and Raymond Hix. 2015. Field Host Specificity of a Potential Hydrilla Biological Control Agent, Cricotopus lebetis Sublette (Diptera: Chironomidae), 98th Annual Meeting Florida Entomological Society, Ft. Myers, FL, 2-5 August.

IPM Award Nomination 21 James Cuda

Cuda, J.P. J.L. Gillmore, J.C. Medal, J. Bricker, and B.R. Garcete-Barrett. 2015. Biology of a stem boring weevil Apocnemidophorus pipitzi (Faust) (Coleoptera: Curculionidae) And its impact on Brazilian peppertree, Schinus terebinthifolia. 98th Annual Meeting Florida Entomological Society, Ft. Myers, FL, 2-5 August.

Cuda, J.P., A.M. Berro, and W.A. Overholt. 2014. Update on the leaflet galling psyllid Calophya terebinthifolii, a candidate biocontrol agent of Brazilian peppertree. FWC-IFAS Research Review for Invasive Plants in Florida held at the Plant Science Research and Education Unit, Citra, FL, 12-13 March 2014.

Cuda, J.P. and W.A. Overhololt. 2014. Update: screening of a new candidate biocontrol agent of Brazilian peppertree, Schinus terebinthifolia. Board of Directors, Florida Industrial and Phosphate Research Institute, Bartow, Florida, 31 January 2014.

Cuda, J.P., R. Diaz, V.Manrique, M. Vitorino, W. Overholt, A. Berro and P. Prade. 2014. Gall- forming psyllids (Hemiptera: Calophyidae): new biological control agents for Brazilian peppertree? 29th Annual Everglades Coalition Conference held in Naples, FL, 9-11 January 2014. (Poster)

Cuda, J.P., J.L. Gillmore, J.C. Medal, B.R. Garcete-Barrett, and W.A. Overholt. 2014. Effect of plant sex (dioecism) on the performance of Apocnemidophorus pipitzi (Coleoptera: Curculionidae), a stem boring weevil of Brazilian peppertree, Schinus terebinthifolia. 97th Annual Meeting of the Florida Entomological Society held in Jupiter, FL, 3-6 August 2014. (Poster)

Cuda. J.P. 2014. Does plant sex affect the performance of the stem boring weevil Apocnemidophorus pipitzi (Coleoptera: Curculionidae) on its host plant Brazilian peppertree? .Florida Chapter of the Wildlife Society and the Florida Exotic Pest Plant Council, Safety Harbor, 28 April-1 May 2014;

Diaz, R., V. Manrique, W.A. Overholt, Patricia Prade, and J.P. Cuda. 2014. Host specificity reveals that the gall-forming psyllid, Calophya latiforceps (Hemiptera: Calophyidae), is safe to release for biological control of Brazilian peppertree (Anacardiaceae). Florida Chapter of the Wildlife Society and the Florida Exotic Pest Plant Council, Safety Harbor, 28 April-1 May 2014.

Gioeli, K. J.L. Gillett-Kaufman, and J.P. Cuda. 2014. Hydrilla IPM: Starting a statewide extension project. Florida Chapter of the Wildlife Society and the Florida Exotic Pest Plant Council, Safety Harbor, 28 April-1 May 2014.

IPM Award Nomination 22 James Cuda

Cuda, J.P. 2014. Invasive Plant Management, the other IPM. Florida Pest and Lawn Care Expo, Kissimmee, FL., 23 January 2014.

Overholt, W.A., J.P. Cuda, J.A. Goolsby, A.M. Burrell, B. Le Ru, K. Takasu, P.E. Klein, A. Racelis and P. Hidayat. 2013. Partnerships in weed biological control: the quest for Old World natural enemies of cogongrass. Symposium presentation at the ESA annual meeting held in Austin, Texas, November 10th to 13th, 2013. Member Symposium: Making Connections Across Disciplines to Combat Alien Invaders.

Bradshaw, J.P., J. P. Cuda, J. L. Gillett-Kaufman, K. Gioeli, R. L. Hix, V. Ulrike Lietze, W. A. Overholt, and J. F. Shearer. 2013. Spreading the word: New strategies for hydrilla IPM are investigated. EPAF (Extension Professionals Association of Florida) Annual Conference, Ponte Vedra Beach, FL, August 2003. (Poster)

Overholt, W.A., B. Le Ru, J.P. Cuda, 2013. Biological control of cogongrass (Imperata cylindrica): preliminary results of natural enemy surveys in East Africa. 40th Annual Natural Areas Conference , Chicago, IL, October 1st to the 4th 2013. (Poster)

Cuda, J.P., J.L. Gillmore, J.C. Medal, B.R. Garcete-Barrett, and W.A. Overholt. 2013. Proposed Release of the Stem Boring Weevil Apocnemidophorus pipitzi for Biological Control of Brazilian Peppertree, Schinus terebinthifolia, in Florida. Annual meeting of the USDA Technical Advisory Group for the Biological Control Agents of Weeds, Washington DC, June 18th to the 19th, 2013.

Cuda, J.P., J.L. Kaufman, V. Lietze and W.A. Overholt. 2013. Combining control tactics to reduce hydrilla biomass. 37th Annual Training Conference of the Florida Aquatic Plant Management Society, St. Augustine, FL, October 14th to the 17th, 2013.

Cuda, J.P. 2013. Is it possible to find a biological control agent that will slow or impair the growth rate of BP? Brazilian pepper biological control workshop- Co-sponsored by SFWMD and FWC, Ft. Lauderdale REC, 23 January 2013.

Cuda, J.P., L.W. Treadwell, and W.A. Overholt. 2013. Effects of defoliation on growth and reproduction of Brazilian peppertree (Schinus terebinthifolius). Joint Annual Symposium of the Florida Exotic Pest Plant Council and Southeast Exotic Pest Plant Council, Panama City Beach, FL, 20-23 May 2013.

Cuda, J.P., W.A. Overholt, and B.P. Le Ru. 2013. Exploration in East Africa for Potential BioControl Agents for the Invasive Cogongrass, Imperata cylindrica (Poaceae).96th Annual Meeting of the Florida Entomological Society Meeting held in Naples, FL, 14-17 July 2013. (Poster)

IPM Award Nomination 23 James Cuda

Cuda, J., J. Gillett-Kaufman, W. Overholt, K. Stratman, R. Hix, E. Kariuki, J. Shearer, E. Weeks, J. Bradshaw, K. Gioeli, V. Ulrike- Lietze. 2013. Progress on developing a novel IPM research and demonstration project for the aquatic weed hydrilla in Florida. Student Symposium on Invasive Species, SEB-ESA Meeting. Baton Rouge, LA, 3-6 March 2013. Also participated in the new SDC351 (now S1058) regional project meeting on Biological Control of Arthropod Pests and Weeds.

Cuda, J.P. 2012. Reversible field testing of host plant specificity in the U.S.A. 60th Annual Meeting of the Entomological Society of America, Knoxville, TN, 11-14 November.

Cuda, J.P., W. A. Overholt, and L. Markle. 2012. Reciprocal allelopathy: the wax myrtle and Brazilian peppertree story. 2012 Annual Meeting of the Florida Weed Science Society, Haines City, FL, 27-28 February.

Cuda, J.P. 2012. Biological control of Florida’s invasive weeds: Progress and Perspectives. Annual Meeting of the Magnolia Chapter, Florida Native Plant Society, Tallahassee, FL, 3 May.

Cuda, J.P., W.A. Overholt, J. Gillett-Kaufman, O.U. Onokpise, D.A. Williams, and B. Le Ru. 2012. Integrated management of cogongrass, Imperata cylindrica (Poaceae): Prospects for biological control. 38th Annual Natural Areas Conference: Adaptations and Protection of Biodiversity in a Changing World, Tallahassee, FL, 1-4 November.

Cuda, J.P., Christ, L.R., Overholt, W.A., Vitorino, M.D. 2012. Biology, impact and field host specificity of Calophya terebinthifolii (Hemiptera: Calophyidae), a candidate for biological control of Brazilian peppertree, Schinus terebinthifolius (Sapindales: Anacardiaceae). Florida Chapter of the Wildlife Society | Florida Exotic Pest Plant Council Joint 2012 Spring Conference, Ocala, Florida, 16-19 April.

Overholt, W.A., Cuda, J.P., and Markle, L. 2012. Can novel weapons favor native plants? Allelopathic interactions between Morella cerifera (L.) and Schinus terebinthifolius Raddi. Florida Chapter of the Wildlife Society | Florida Exotic Pest Plant Council Joint 2012 Spring Conference, Ocala, Florida, 16-19 April.

Cuda, J.P. 2012. Classical biological control of Brazilian peppertree: Overview and new opportunities. Technical Advisory Committee Meeting, Florida Industrial and Phosphate Research Institute, Bartow, FL, 24 October.

Cuda, J.P., A. Mukherjee, C.A. Ellison, and W.A. Overholt. 2011. Results of native range surveys to identify natural enemies of Hygrophila polysperma. 35th Annual Meeting of the Florida Aquatic Plant Management Society, St. Augustine, Fl, 10-13 October.

Cuda, J.P., W.A. Overholt, J.Gillett-Kaufman, O. U. Onokpise, D.A. Williams, and B.P. Le Ru. 2011. Integrated management of cogongrass, Imperata cylindrica (Poaceae): Prospects for biological control. 38th Annual Natural Areas Conference, Tallahassee, FL, 1-4 November. IPM Award Nomination 24 James Cuda

Cuda, J.P., A. Mukherjee, and W.A. Overholt. 2011. Prospects for biological control of Hygrophila polysperma. 38th Annual Natural Areas Conference, Tallahassee, FL, 1-4 November. (Poster)

Cuda, J.P., J.L. Gillmore, J.C. Medal, B. Garcete-Barrett, and W.A. Overholt. 2011. Update on a promising biological control agent for Brazilian peppertree: .the stem boring weevil Apocnemidophorus pipitzi (Coleoptera;: Curculionidae). 26th Annual Symposium of the Florida Exotic Pest Plant Council, Maitland, FL, 17-20 May.

Hetrick, S., J. Cuda, W. Haller, D. Jones, A. Mukherjee, M. Netherland, and W. Overholt. 2011. Search for new management techniques for hydrilla and hygrophila. 22nd Annual Conference for the Florida Lake Management Society, St. Augustine, FL, 13-16 June.

Burckhardt, D., J.P. Cuda, V. Manrique, R. Diaz, W.A. Overholt, D.A. Willilams, L.R. Christ, and M.D. Vitorino. 2011. Calophya latiforceps, a new species of jumping plant lice (Hemiptera: Calophyidae) and natural enemy of Brazilian peppertree Schinus terebinthifolius (Anacardiaceae), in Brazil. 94th Annual Meeting of the Florida Entomological Society, Ft. Myers, FL, 24-27 July. (Poster)

Cuda, J.P. 2011. Update: Biological control of Florida’s arthropod pests and weeds. FLDACS Pesticide Review Council Meeting, Tallahasee, FL, 30 August.

Cuda, J.P., J.C. Medal, J.H. Pedrosa-Macedo, V. Manrique, P. Conant, and W.A. Overholt. 2010. Biology and host range of Episimus unguiculus (Lepidoptera: Tortricidae), a precedented biological control agent of Brazilian peppertree, Schinus terebinthifolius (Sapindales: Anacardiacae) for release in Florida. 84th Annual Meeting of the Southeastern Branch of the Entomological Society of America, Atlanta, GA, 7-10 March.

Cuda, J.P., J.C. Medal, J.H. Pedrosa-Macedo, V. Manrique, P. Conant, and W.A. Overholt. 2010. Biology and fundamental host range of Episimus unguiculus (Lepidoptera: Tortricidae), a new candidate for biological control of Brazilian peppertree, Schinus terebinthifolius (Anacardiacae) in Florida. 25th Annual Symposium of the Florida Exotic Pest Plant Council -Changes in Latitude, Crystal River, FL, 5-8 April. Invited.

Christ, L.R., J.P. Cuda, W.A. Overholt, and M.D. Vitorino. 2010. Biology, population growth, and feeding preferences of Calophya terebinthifolii (Hemiptera: Psyllidae), a candidate for biological control of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae). 25th Annual Symposium Florida Exotic Pest Plant Council-Changes in Latitude, Crystal River, FL, 5-8 April. (Poster)

Manrique, V. R. Diaz, W. A. Overholt, D. Williams, and J.P. Cuda. 2010. The effect of thrips herbivory on the performance of different genotypes of Brazilian peppertree in Florida. 25th IPM Award Nomination 25 James Cuda

Annual Symposium Florida Exotic Pest Plant Council-Changes in Latitude, Crystal River, FL, 5- 8 April. (Poster)

Cuda, J.P, A. Mukherjee, and W.A. Overholt. 2010. Prospects for classical biological control of the federal noxious weed Hygrophila. 50th Annual Meeting of the Aquatic Plant Management Society, Bonita Springs, FL, 11-14 July. Invited

Cuda, J.P. J.L. Gillmore, J.C. Medal, and B. Garcete-Barrett. 2010. Apocnemidophorus pipitzi (Coleoptera: Curculionidae), a new candidate for biological control of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae). 2010 Greater Everglades Ecosystem Restoration Planning, Policy and Science Meeting Naples, FL, 12-16 July. Invited

Cuda, J.P., J.L. Gillmore, J.C. Medal, and B. Garcete-Barrett. 2010. Biology and host range of the Brazilian peppertree stem boring weevil Apocnemidophorus pipitzi (Coleoptera: Curculionidae). 93rd Annual Meeting of the Florida Entomological Society, Jupiter Beach, FL, 25-28 July.

Overholt, W.A., D.A. Williams, R.C. Copeland, B. Nzigidahera, E. Nkubaye, F. Wanda, B. Gidudu and J.P. Cuda. Exploration for natural enemies of Hydrilla verticillata Royle (Hydrocharitaceae) in East/Central Africa and genetic characterization of worldwide populations. 2010 Biological Control for Nature Conference, Oct 3-7, 2010, Northampton MA. (Poster)

Cuda, J. P., A. Mukherjee, and W.A. Overholt. 2010. Update: biological control of Hygrophila. 34th Annual Meeting of the Florida Aquatic Plant Management Society, Daytona Beach, FL. 18-21 October. Invited

Cuda, J.P., J.L. Gillmore, J.C. Medal, and B. Garcete-Barrett. 2010. Update on the stem boring weevil Apocnemidophorus pipitzi, a promising biological control agent for Brazilian peppertree." 15th Annual Southwest Florida Invasive Species Workshop, Florida Gulf Coast University, 1 December. Invited.

Christ, L.R., J.P. Cuda, W.A. Overholt, M.D. Vitorino, and J.C. Medal. 2010. Biology, impact and feeding preferences of Calophya terebinthifolii (Hemiptera: Calophyidae), a candidate for biological control of Brazilian Peppertree, Schinus terebinthifolius (Sapindales: Anacardiaceae). 58th Annual Meeting of the Entomological Society of America, San Diego, CA, 12-15 December. (Poster)

Cuda, J.P., V. Manrique, W.A. Overholt, D.W. Williams, L.R. Christ, and G.S. Wheeler. 2010. Effect of host plant genotypes on the performance of Brazilian peppertree biological control agents. Biological Control for Nature Conference, October 3-7, Northampton, MA, Oral.

IPM Award Nomination 26 James Cuda

Cuda, J.P., A. Mukherjee, C.A. Ellison, and W.A. Overholt. 2009. Exploratory surveys for natural enemies of the invasive aquatic weed Hygrophila polysperma: Preliminary results. Inaugural International Congress on Biological Invasions, Fuzhou, China, 2-6 November, Oral.

Crow, W.T., J.P. Cuda, and B.R. Stevens. 2009. Nematicide potential of methionine on turfgrasess. Joint 48th Annual Meeting of the Society of Nematologists and 12th Biennial Meeting of the Soil Ecology Society, University of Vermont, Burlington, VT, 12-15 July, Oral.

Cuda, J.P., J.L. Gillmore, J.C. Medal, B. Garcete-Barrett, and W.A. Overholt. 2009. Biology and fundamental host range of the stem boring weevil Apocnemidophorus pipitzi, a new candidate for biological control of Brazilian peppertree, Schinus terebinthifolius. Annual Meeting of the Entomological Society of America, Indianapolis, IN, 13-16 December. (Poster)

Cuda, J.P. 2009. Biocontrol update for Florida. 33rd Annual Meeting of the Florida Aquatic Plant Management Society held in Daytona Beach, FL, 12-15 October.

Cuda, J.P., W.A. Overholt, V. Manrique, and S.M. Ewe. 2009. Synergistic effect of insect herbivory and plant parasitism on the performance of the invasive tree Schinus terebinthifolius (Anacardiaceae). 24th Annual Symposium of the Florida Exotic Pest Plant Council, Delray Beach, FL, 26-29 May, Oral.

Cuda, J.P., E. Hanlon, and W.A. Overholt. 2009. An IPM model for sustainable management of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae), in Florida. 24th Annual Symposium of the Florida Exotic Pest Plant Council, Delray Beach, FL, 26-29 May, Poster.

Cuda, J.P. 2009. Episimus unguiculus Clarke (Lepidoptera: Tortricidae), a candidate for biological control of Brazilian peppertree in Florida: Biology, host range, and impact studies. Fall Semester Seminar Series, McGuire Center for Lepidoptera and Biodiversity, 6 October, Oral.

Legaspi, J., J.P. Cuda, C.S. Gardner, G.L. Queeley, and N.C. Leppla. 2008. Demonstrating integrated pest management of hot peppers. Annual Meeting of the Caribbean Food Crops Society, Miami, FL, 13-17 July. (Poster)

Cuda, J.P. 2008. Novel approaches for risk assessment: Temporary releases of biocontrol agents using arrhenotoky or F1 sterility. Plant-Insect Ecosystems Section Symposium, 56th Annual Meeting of the Entomological Society of America, Reno, NV, November 16-19, Oral.

Cuda, J.P., J.C. Medal, J.H. Pedrosa-Macedo, R.W. Barreto, B.R. Garcete-Barrett, V. Manrique, and W.A. Overholt. 2008. New research activities on biological control of Brazilian peppertree. 15th Annual Conference of the Wildlife Society Miami, FL, 8-12 November, Oral.

(Presentation) Cuda, J.P., T. Stevens, R. Barreto, T. Schubert, and R. Charudattan. 2008. Septoria sp. (Sphaeropsidales): A new fungal pathogen for classical biological control of Schinus IPM Award Nomination 27 James Cuda terebinthifolius. Annual Meeting of the Weed Science Society of America, Chicago, IL, 4-7 February, Oral.

Cuda, J.P., J.L. Gillmore, J.C. Medal, and B. Garcete-Barrett. 2008. Apocnemidophorus pipitzi (Coleoptera: Curculionidae), a new candidate for biological control of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae). Annual Meeting of the Southeastern Branch of the Entomological Society of America, Jacksonville, FL, 2-4 March, Oral.

Cuda, J.P., J.C. Medal, W.A. Overholt, and J.H. Pedrosa-Macedo. 2008. Overview of biological control of Brazilian peppertree. Program Symposium, Recent and Current Events in the BioControl of Arthropods and Weeds in Florida. 91st Annual Meeting of the Florida Entomological Society, Jupiter Beach, FL, 13-16 July, Oral.

Cuda, J.P. 2008. Biological Control of Invasive Plants: A Florida Perspective. Joint Florida Institute of Phosphate Research and UF/IFAS Restoration Workshop: Native Plant Community Restoration and Management in Florida and the Southeast, Lakeland, FL, 2-3 April, Oral.

Cuda, J.P., A. Mukherjee, W.A. Overholt, and C.A. Ellison. 2008. Can insect defoliators be effective biocontrol agents of Hygrophila. 32nd Annual Meeting of the Florida Aquatic Plant Management Society, Daytona Beach, FL, 13-16 October. Oral.

Cuda, J.P., B.R. Coon, Y.M. Dao, and T.D. Center. 2008. Herbivores and hydrilla: Did chironomid herbivory contribute to the decline of hydrilla in Florida’s Crystal River watershed? 22nd Annual Meeting of the Florida Association of Benthologists, Crystal River, FL, 8-11 December, Oral.

Cuda, J.P. 2008. Novel approaches for risk assessment: Temporary releases of biocontrol agents using arrhenotoky or F1 sterility. Plant-Insect Ecosystems Section Symposium, 56th Annual Meeting of the Entomological Society of America, Reno, NV, November 16-19, Oral.

Cuda, J.P. 2008. Synopsis of biocontrol of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae). Entomology and Nematology Department Fall Seminar Series, 6 November, Oral.

Cuda, J.P., E.A. Hanlon, and W.A. Overholt. 2008. A model for sustainable management of Brazilian peppertree. Greater Everglades Ecosystem Restoration Meeting, Naples, FL, 28 July-1 August. (Poster)

Cuda, J.P. 2008. Current status of established arthropod and weed biological control agents of invasive pests. Everglades Invasive Species Summit, Florida International University, Miami, FL, 16-18 July, Oral.

Cuda, J.P. 2007. Biological control of invasive plants. The Delicate Balance of Nature Lecture Series, Key Largo, FL, 28 February. IPM Award Nomination 28 James Cuda

Cuda, J.P. 2007. Field testing the host range of a tortricid moth for biocontrol of Brazilian peppertree, Schinus terebinthifolius, in Florida: Is it possible? School of Natural Resources and Environment Minigrant Seminar Series, 20 February.

Cuda, J.P., L.S. Osborne, C.A. Jacoby, and K.A. Langeland. 2007. Establishment of the University of Florida Invasive Species Coordinating Council, TSTAR Workshop on Invasive Species, Miami, FL, 7-9 November, Oral.

Cuda, J.P., J.H. Frank, J.E. Maruniak, and J.C. Medal. 2007. Collaboration between Brazil and the USA in classical biological control: A Florida perspective - cases of success and lessons learned. X SICONBIOL, Brasilia, Brazil, 30 June-4 July, Oral.

Cuda, J.P., O.E. Moeri, J.C. Medal, W.A. Overholt, and V. Manrique. 2007. Novel approaches for risk assessment: Feasibility studies on temporary reversible releases of biocontrol agents. XII International Symposium on Biological Control of Weeds held in La Grande Motte, France, 22- 27 April, Oral.

Vitorino, M.D., and J.P. Cuda. 2007. Mass rearing of Calophya terebinthifolii (Hemiptera: Psyllidae) on Brazilian peppertree. XII International Symposium on Biological Control of Weeds held in La Grande Motte, France, 22-27 April. (Poster)

Vitorino, M.D., J.C. Medal, and J.P. Cuda. 2007. Specificity tests and biology of Gratiana graminea (Coleoptera: Chrysomelidae: Cassidinae), a potential biocontrol agent of Solanum viarum in Florida. XII International Symposium on Biological Control of Weeds held in La Grande Motte, France, 22-27 April. (Poster)

Cuda, J.P., J.C. Medal, and B. Garcete-Barrett. 2007. Exploratory surveys in Paraguay for new biocontrol agents of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae): Preliminary results. 47th Annual Meeting of the Weed Science Society of America, San Antonio, TX, 4-8 February, Oral.

Cuda, J.P. 2007. Biological control and vegetation management. Applied Management of Conservation Lands in Florida, Orlando, FL, 20-22 June, Oral.

(Cuda, J.P., J.C. Medal, and B. Garcete-Barrett. 2006. Laboratory biology of Apocnemidophorus spp. (Coleoptera: Curculionidae), new candidates for biological control of Brazilian peppertree, Schinus terebinthifolius (Anacardiaceae). Entomological Society of America Annual Meeting, December 10-14, 2006, Indianapolis, IN. (Poster)

Cuda, J.P., W.A. Overholt, and R. Copeland. 2006. Exploration for new natural enemies of the aquatic weed hydrilla in East Africa: Preliminary results. 46th Annual Meeting and 50th Anniversary Celebration of the Weed Science Society of America, New York, NY, February 1, Oral. IPM Award Nomination 29 James Cuda

Cuda, J.P. 2006. Update: Biological control of Brazilian peppertree and hydrilla. 11th Annual Southwest Florida Invasive Species Workshop, Rookery Bay Nature Center, Naples, FL, December 1, Oral.

Cuda, J.P. 2006. Brazilian peppertree defoliating sawfly Heteroperreyia hubrichi: Proposed field risk assessment study. U.S. Fish and Wildlife Service, South Florida Ecological Services Office, Vero Beach, FL, May 1, Oral.

(Cuda, J.P. 2006. Field host range and damage potential of the leaf galling psyllid Calophya terebinthifolii (Hemiptera: Psyllidae), a candidate for classical biological control of Brazilian peppertree in Florida. 80th Annual Meeting of the Southeastern Branch of the Entomological Society of America, Wilmington, NC, March 5, Oral.

Cuda, J.P. 2006. Update: Biological control of aquatic weeds. 30th Annual Meeting of the Florida Aquatic Plant Management Society, St. Petersburg, FL, November 2, Oral.

Cuda, J.P. 2006. Biological control of invasive weeds: Hydrilla as an example. Aquatic Weed Control Short Course, Coral Springs, FL, May 6, Oral.

Cuda, J.P. 2006. Integrated weed management plan for Brazilian peppertree (Schinus terebinthifolius) in Florida: An update. 21st Annual Symposium of the Florida Exotic Pest Plant Council, Gainesville, FL, April 1, Oral.

Leppla, N., and J.P. Cuda. 2006. Common myths about lovebugs, Florida folklore. 89th Annual Meeting of the Florida Entomological Society, Jupiter Beach, FL, June 1, Oral.

Overholt, W.A., J.P. Cuda, and R. Copeland. 2006. Could Africa be a source of Hydrilla natural enemies? 21st Annual Symposium of the Florida Exotic Pest Plant Council, Gainesville, FL, April 1, Oral.

Medal, J.C., and J.P. Cuda. 2006. A super beetle fighting the plant from hell: Tropical soda apple. 21st Annual Symposium of the Florida Exotic Pest Plant Council, Gainesville, FL, April 1, Oral.

Cuda, J.P. 2006. Biological control of Brazilian peppertree: Prospects for southwest Florida. Coccoloba Chapter of the Florida Native Plant Society, Pine Island, FL, March 9, Oral.

IPM Award Nomination 30 James Cuda

Awards and Other Recognition

Top 5 most downloaded articles published in the journal Biological Control during the first half of 2010 (Co-author).

U.S. Department of Interior - Partners in Conservation Award, member of the Everglades Cooperative Invasive Species Management Area, 18 October 2012 (Co-Recipient).

Technology Innovation Award for methionine pesticide technology, March 2012 (Co-Recipient).

Central District Extension Faculty Symposium 1st Place Poster Award, April 2012 (Co- Recipient).

2015 National Achievement Award/Innovative Program, Tropical Soda Apple Biological Control Extension Program, Association of Natural Resources Extension Professionals (Team Member)

Fulbright Scholar, 2014-2016.

College of Agricultural and Life Sciences Charles Steinmetz Hall Department of Entomology and Nematology Bldg. 970, Natural Area Drive PO Box 110620 Gainesville, FL 32611-0620 352-273-3901 352-392-0190 Fax Awards Committee 27 January 2017 SEB Award for Excellence in IPM

Re. James P. Cuda

Dear Committee:

To save your time, I have addressed each of the 7 criteria in order and provided brief evidence.

Criterion 1, recentness: Publications and presentations over the last five years suggest an ongoing research program, not belated accounts of a program that terminated several years ago.

Criterion 2, is it IPM? This study of compatibility of 3 control methods is an attempt to integrate the methods, and is IPM. Cuda, J.P., J. F. Shearer, E.N.I. Weeks, E. Kariuki, J. Baniszewski and M. Giurcanu. 2016. Compatibility of an insect, a fungus and a herbicide for IPM of dioecious hydrilla. Journal of Aquatic Plant Management 54: 20-25.

Criterion 3, quality of research. Acceptance of publications into 26 (by my count) refereed journals , as well as various conference proceedings, suggests quality of the reported research.

Criterion 4, originality of research. Classical biological control has sometimes been criticized as irreversible. However, the following publication shows that it CAN be reversible. Cuda, J.P. 2016. Novel approaches for reversible field releases of candidate weed biological control agents: Putting the genie back into the bottle, Chapter 7, pp. 137-152. In: J.F. Shroder and R. Sivanpillai (eds.), Biological and Environmental Hazards, Risks and Disasters. Elsevier, Inc., Amsterdam. http://dx.doi.org/10.1016/B978-0-12-394847-2.00010-3

Criterion 5, interpretation of research. I have not been able to fault any of the published papers for erroneous or incomplete interpretation.

Criterion 6, quality of publications. A multi-authored publication in the journal Biological Control was noted as one of the top 5 most downloaded articles published in that journal during the first half of 2010. Otherwise, if I read instructions correctly, all publications are to be taken into account. This criterion is highly related to no. 3.

Criterion 7, honors and awards. Award of a Fulbright Scholarship in 2015-2016 speaks to the views of the Fulbright award committee’s views. I support those views. Page 1 of 2

The Foundation for The Gator Nation An Equal Opportunity Institution

Page 2 of 2

Yours sincerely JH Frank Howard Frank Professor Emeritus (Biological Control)

College of Agricultural and Life Sciences Charles Steinmetz Hall Department of Entomology and Nematology Bldg. 970, Natural Area Drive PO Box 110620 Gainesville, FL 32611 352-392-1901 24 January 2017 Dr. Blair Siegfried, Chair Entomology & Nematology Department University of Florida Gainesville, FL 32611

Dear Blair: I am writing is support of Dr. Jim Cuda, who has been nominated for the Southeastern Branch Award for Excellence in Integrated Pest Management. Dr. Cuda is part of our weed biological control team, and has provided important leadership for 20 years. Florida seems to be an important gateway for invasive organisms, including non-native plants. Some have been deliberately introduced without adequate aforethought for impact, whereas others invade surreptitiously. In natural areas, weed suppression using herbicides is not economically feasible, nor desirable. Thus, cultural practices such as burning or encouragement of competition, or biological practices such as release of host-specific diseases and insects, are most desirable. Dr. Cuda has had great success in identifying and integrating such biotic factors into weed management systems. Dr. Cuda has been associated with many research projects, but the most important are: (1) suppression of Hydrilla, an aquatic weed that clogs Florida’s waterways, (2) suppression of Brazilian Pepper, a woody shrub that invades and dominates disturbed areas and pastures, and (3) suppression of Tropical Soda Apple, a spiny annual plant that invades pastures, making them unsuitable for cattle and people alike. Tropical Soda Apple is also not-so-affectionately known as “the plant from Hell”, which gives you a good idea of its importance. Probably Jim’s most spectacular success has involved Tropical Soda Apple. Jim and his team (Julio Medal and Dale Habeck) successfully identified a voracious leaf beetle, Gratiana boliviana, that not only defoliates Tropical Soda apple, but is host specific. They evaluated the beetle in Brazil, and found that it would not (contrary to the expectations of almost everyone) feed on other solaceous plants such as potato, tomato, and eggplant. After this screening, development of propagation techniques, and redistribution of the beetle, they attained suppression of Tropical Soda Apple within 2 years of release. This beetle has saved Florida’s ranchers up to $16 million per year, and of course the benefits continue to accrue, and the beetle is being sent to other nearby states that have been invaded by “the plant from Hell”. Dr. Cuda has made important contributions to integrated management of weeds, and the success of his effects warrants recognition. I hope the SEB will honor him with this award.

Sincerely,

John L. Capinera Emeritus Professor, Department of Entomology and Nematology

The Foundation for The Gator Nation An Equal Opportunity Institution Biocontrol Science and Technology

ISSN: 0958-3157 (Print) 1360-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/cbst20

Laboratory biology and field population dynamics of Trichopria columbiana (Hymenoptera: Diapriidae), an acquired parasitoid of two hydrilla biological control agents

B.R. Coon, N.E. Harms, J.P. Cuda & M.J. Grodowitz

To cite this article: B.R. Coon, N.E. Harms, J.P. Cuda & M.J. Grodowitz (2014) Laboratory biology and field population dynamics of Trichopria columbiana (Hymenoptera: Diapriidae), an acquired parasitoid of two hydrilla biological control agents, Biocontrol Science and Technology, 24:11, 1243-1264, DOI: 10.1080/09583157.2014.933311

To link to this article: http://dx.doi.org/10.1080/09583157.2014.933311

Accepted author version posted online: 30 Jun 2014. Published online: 30 Jun 2014.

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Download by: [University of Florida] Date: 25 January 2017, At: 12:47 Biocontrol Science and Technology, 2014 Vol. 24, No. 11, 1243–1264, http://dx.doi.org/10.1080/09583157.2014.933311

RESEARCH ARTICLE Laboratory biology and field population dynamics of Trichopria columbiana (Hymenoptera: Diapriidae), an acquired parasitoid of two hydrilla biological control agents B.R. Coona,b, N.E. Harmsc, J.P. Cudaa* and M.J. Grodowitzc

aDepartment of Entomology and Nematology, University of Florida, Gainesville, FL, USA; bPublic Health and Graduate School of Business and Management, Argosy University, Schaumburg, IL, USA; cU.S. Army Engineer Research and Development Center, Vicksburg, MS, USA (Received 6 December 2013; returned 14 January 2014; accepted 7 June 2014)

The biology and population dynamics of Trichopria columbiana, a native semi- aquatic parasitoid of ephydrid flies of the genus Hydrellia, were investigated in Florida and Texas, USA. Hydrellia pakistanae and Hydrellia balciunasi were introduced for classical biological control of the invasive aquatic weed Hydrilla verticillata in the 1980s and acquired T. columbiana post-release. Several life history parameters of T. columbiana were investigated in the laboratory, including fecundity, egg shape and size, number and location of egg(s) deposited per host, preferred host age, description and number of instars, mode of respiration and host-selection behaviour. Field studies included seasonal abundance of T. columbiana and the introduced Hydrellia spp., parasitism levels, overwintering stage and adult winter sex ratio. T. columbiana is a synovigenic solitary endoparasitoid that developed from egg to adult in 21.9 ± 0.2 days under laboratory conditions. Eggs of T. columbiana are hydropic, hymenopteriform in shape and possess a double membrane. Larvae have three instars; first instars have sclerotised mandibles, bifurcated abdominal appendages and are free floating in the host’s haemolymph. Second and third instars are grub-like and remain attached to the host’s tracheal system until pupation. Individual females produced on average 23.2 ± 0.6 eggs and survived 15.6 ± 1.8 days. Highest parasitism levels of early and intermediate stage pupae occurred when wasps were 8–9 days old. Field parasitism rates of the two introduced Hydrellia spp. averaged 19.1%. Keywords: Hydrellia pakistanae; Hydrellia balciunasi; Hydrilla verticillata; weed biocontrol; parasitoid accumulation; apparent competition

1. Introduction The submersed aquatic plant Hydrilla, Hydrilla verticillata (L.f.) Royle), is regarded as one of the worst invasive weeds worldwide (Holm, Doll, Holm, Pancho, & Herberger, 1997). After its introduction into the USA by the aquarium industry in the 1950s (Langeland, 1996), various control methods were developed and used to manage hydrilla infestations including biological control. Classical biological control studies were initiated in the 1970s (Buckingham, 1994). These efforts led to the release of four insects in the USA: a tuber and a stem-boring weevil of the genus

*Corresponding author. Email: [email protected]

This Article is a collaborative work. The contributions of N.E. Harms and M.J. Grodowitz were conducted as part of these persons’ official duties as employees of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105 no copyright protection is available for such works under U.S. law. B.R. Coon and J.P. Cuda waive their own assertion of copyright but not their status as co-Authors. 1244 B.R. Coon et al.

Bagous and two leaf-mining flies of the genus Hydrellia (Center, Cofrancesco, & Balciunas, 1990). Until recently, only the two leaf-mining flies Hydrellia pakistanae Deonier and Hydrellia balciunasi Bock (Diptera: Ephydridae) were confirmed as established (Balciunas, Grodowitz, Cofrancesco, & Shearer, 2002; Center et al., 1997; Grodowitz, Center, Cofrancesco, & Freedman, 1997). During the past decade, there has been a renewed interest in the biological control of hydrilla (Copeland, Nkubaye, Nzigidahera, Cuda, & Overholt, 2011; Copeland, Gidudu, et al., 2012; Copeland, Nkubaye, et al., 2012; Cuda, Coon, Dao, & Center, 2011; Overholt & Cuda 2005, Stratman et al., in press; Stratman, Overholt, Cuda, Netherland, & Wilson, 2013a, 2013b, 2013c; Wanda et al., 2011; Wheeler & Center, 2007; Zhang, Wheeler, Purcell, & Ding, 2010). Current efforts have been largely in response to the discovery in Florida of several hydrilla biotypes that have developed resistance to the herbicides fluridone (Michel et al., 2004) and, more recently, endothall (M.D. Netherland, pers. comm.). Furthermore, establish- ment of the Australian stem-mining weevil Bagous hydrillae O’Brien was recently confirmed in Louisiana, more than a decade after field releases were terminated (Center et al., 2013). Despite successful establishment and range expansion of three hydrilla biological control agents, population levels of the insects and associated plant damage have remained low (Bennett & Buckingham, 2000; Cuda et al., 2008; Forno & Julien, 2000; Grodowitz, Cofrancesco, Stewart, Madsen, & Morgan, 2003; Grodowitz et al., 2004). Although there is some evidence that recent hydrilla declines in Florida and Texas, USA, have been associated with local increases in Hydrellia fly populations (Grodowitz et al., 2004), several abiotic and biotic factors have been identified that could adversely affect their populations on a landscape scale (Cuda et al., 2008; Grodowitz et al., 2004). One of these is the acquisition of native parasitoids. Trichopria columbiana (Ashmead; Hymenoptera: Diapriidae; Figure 1) is a pupal endoparasitoid commonly associated with at least seven native Hydrellia spp. (Berg, 1950; Deonier, 1971, 1998; Grigarick, 1959; Hagen, 1956). Parasitism rates as high 90% have been reported in native Hydrellia populations (Deonier, 1971). This semi- aquatic wasp is widely distributed in North America (Bennett, 2008) and was first reported parasitising the puparia of the introduced H. pakistanae in Alabama (Cuda, Fox, & Habeck, 1997). Subsequent studies suggest this acquired parasitoid may have reduced the effectiveness of H. pakistanae (Doyle, Grodowitz, Smart, & Owens, 2002; Wheeler & Center, 2001) and perhaps prevented widespread establishment

Figure 1. (Colour online) Male (A) and female (B) T. columbiana. Males are readily distinguished by the threadlike, filiform, antennae as opposed to the shorter, clavate antennae in females. Biocontrol Science and Technology 1245 of the Australian leaf miner H. balciunasi in the USA (Grodowitz et al., 1997). T. columbiana has been recovered from established populations of H. pakistanae in Alabama (Grodowitz et al., 1997), Florida (Cuda et al., 1997; Wheeler & Center, 2001), and from both Hydrellia spp. in Texas (Doyle et al., 2002; Grodowitz, Nachtrieb, Harms, Swindle, & Snell, 2009). Deonier (1971) reared T. columbiana from two native Hydrellia spp. in Minnesota. In general, the biology and population dynamics of aquatic parasitoids in the genus Trichopria are not well understood. A few detailed studies have focused on terrestrial species (e.g., Huggert & Morgan, 1993; Morgan, Hogsette, & Patterson, 1990; Vaughn, 1985). However, except for Grigarick (1959) and O’Neill (1973), little biological information is available on semi-aquatic species. In order to assess the effect of T. columbiana on the two Hydrellia flies introduced for biological control of hydrilla, this paper presents the first in-depth study on the biology and ecology of this semi-aquatic parasitoid.

2. Materials and methods 2.1. Laboratory studies – Florida 2.1.1. Plant culture A stock culture of hydrilla (New Delhi strain) was propagated according to the procedures described by Goodson (1997). New Delhi strain was selected because Goodson (1997) found that both H. pakistanae and H. balciunasi developed equally well on this particular hydrilla strain. Briefly, hydrilla sprigs with intact apical meristems were cut into 5 cm lengths and planted in 150 ml plastic cups (three sprigs per cup) containing standard potting soil. The potting soil was supplemented with 6 gms of Osmocote® plant food (N:P:K–18:6:12) per litre of soil. A layer of clean sand (~1 cm) was placed on top of the soil in each cup to prevent leaching of excessive nutrients. Plastic cups with the hydrilla sprigs (n = 25) were placed in 37.9 litre rectangular aquaria filled with well water (pH ≈ 8.5). Each aquarium was provided with a bubble stone for aeration and covered with mosquito netting attached to a fitted wooden frame to exclude unwanted insects. The aquaria were located in a secured glasshouse at the University of Florida, Institute of Food and Agricultural Sciences Entomology and Nematology Department. The glasshouse was maintained at 27 ± 5°C and a 16:8 (L:D) photoperiod. Ambient lighting was supplemented with a combination of Gro-Lux® and standard fluorescent bulbs.

2.1.2. Rearing of Hydrellia flies H. pakistanae and H. balciunasi were reared according to the protocols established by Buckingham et al. (1989) and Buckingham and Okrah (1993). Because H. pakistanae is established in Florida, adults were collected periodically from the field and added to the lab colony to ensure genetic variability. Field samples of H. balciunasi were not readily available because they are established only in two areas of Texas (Grodowitz et al., 1997). Therefore, the source of flies used in this study was a colony maintained by the USDA, ARS, Biological Control Research Laboratory, Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, Florida. The fly colonies were isolated from each other to avoid contamination. Voucher specimens of the two Hydrellia spp. were preserved in 1246 B.R. Coon et al.

70% ethanol and deposited in the Florida State Collection of Arthropods, Gainesville, Florida.

2.1.3. Rearing of T. columbiana The colony of T. columbiana used in this study originated from a shipment of parasitised puparia of H. pakistanae obtained from the Tennessee Valley Authority (TVA), Muscle Shoals, Alabama, January 1995. Parasitoids were reared in the laboratory in a 3.8 litre glass jar fitted with the top of a BioQuip® mosquito breeder (12 cm diam. × 21 cm ht) containing a clear vinyl funnel. Each glass jar contained deionised water and 125 H. pakistanae puparia attached to hydrilla sprigs. Emerging parasitoids were collected when they would fly or crawl through the inverted funnel into the collecting chamber and then transferred to another jar containing hydrilla with newly attached H. pakistanae puparia. The parasitoid colony was housed in a Biotronette Mark III® Environmental Growth Chamber maintained at 27 ± 1°C under a 16-h light: 8-h dark photo regime with fluorescent lighting. Voucher specimens of T. columbiana were preserved in 70% ethanol and deposited in the Florida State Collection of Arthropods, Gainesville, Florida.

2.1.4. Life history of T. columbiana The objective of this study was to determine the number and location of egg(s) deposited per host and the duration of the egg, larval and pupal stages. One-day old unparasitised H. pakistanae puparia (n = 120) attached to a hydrilla whorl and placed in deionised water were exposed to newly emerged T. columbiana parasitoids

(n = 10). After 24 hours, the parasitoids were removed and transferred back into the colony. A cohort of H. pakistanae puparia (n = 10) was dissected and examined with the aid of a Zeiss® stereo microscope with 2.5× and 6.3× objectives daily thereafter to determine the life history of the immature stages of T. columbiana. A disposable scalpel was used to extract the parasitised puparium from the stem of the hydrilla. The cuticle of the parasitised pupa was then removed with micropins in modified Ringer’s solution. The exposed dipteran pupa was teased open for removal and measurement of the parasitoid life stages. Specimens were photographed with an Olympus OM2 35-mm camera. Parasitoid larvae were measured for length and width to determine the number of instars. The width was measured across the head. The globules enclosed in the exuviae of the parasitoid during the pupal stage were tested for Wolbachia (Jeyaprakash & Hoy, 2000). A subsample of the parasitoid instars was mounted on slides and photographed for descriptive purposes, and the others were preserved in 70% alcohol. In order to investigate the mode of respiration of the adult T. columbiana and the average time spent underwater during oviposition, newly emerged adults (n = 30) were exposed individually to five unparasitised H. pakistanae puparia attached to hydrilla submersed in deionised water. The length of time each parasitoid submersed to oviposit into each host puparium was recorded. To determine whether H. balciunasi is a suitable host for T. columbiana, one newly emerged wasp was placed in a parasitism chamber described previously with a cohort of 10 one-day old H. balciunasi puparia attached to hydrilla whorls. The exposed puparia of H. balciunasi were removed from the parasitism chamber daily Biocontrol Science and Technology 1247 and replaced with 10 fresh host puparia. The same female adult parasitoid was allowed to parasitise each cohort of 10 H. balciunasi puparia until she died. The host puparia exposed to T. columbiana were monitored for parasitoid emergence by placing them individually in plastic snap cap vials (12.9 ml) with small punctures in the lid for air exchange and half-filled with hydrilla colony water. The vials were held in the growth chamber at 27°C at 16-h light: 8-h dark photoperiod.

2.1.5. Ovigeny, generation time and rate of increase To determine ovigeny condition (pro- vs. synovigeny), a cohort of newly enclosed female parasitoids (n = 120 wasps) was randomly selected from the colony and placed in a BioQuip® Mosquito Breeder (12 cm diam. × 24 cm ht) containing 125 whorls of hydrilla and deionised water only. Ten parasitoids were removed daily for a period of 9 days and dissected to determine the egg load (number of mature eggs per host-deprived female). Female fecundity was determined by placing parasitoids (n = 120) into another BioQuip® Mosquito Breeder provisioned with 125 unparasitised H. pakistanae puparia attached to individual whorls of hydrilla and 100 ml of deionised water. On Day 1, 10 adult parasitoids were removed, frozen and dissected to establish a baseline. Each day thereafter for a period of 12 days, which coincided with approximately the life span of the adult wasp (see below), surviving adults were counted, and a subsample was removed, frozen and dissected. The number of eggs per female was recorded, and the mean number of eggs per adult was calculated. The longevity and parasitism level of individual T. columbiana females was determined in the following manner. Five newly eclosed wasps were transferred from the parasitoid colony into separate BioQuip® Mosquito Breeders . The containers were provided with 10 detached whorls of hydrilla, each with a newly formed H. pakistanae puparium. A new cohort of 10 whorls with attached puparia was exposed to each wasp daily until they died. Water (100 ml) obtained from jars with developing Hydrellia flies also was added to the containers and replaced daily. Hydrilla whorls with exposed puparia were then placed individually in plastic snap cap vials. The containers and vials with developing puparia were held in a Biotronette Mark III® Environmental Growth Chamber maintained at 18 to 24 ± 1°C under a 16-h light: 8-h dark photo regime with fluorescent lighting. Vials were examined daily for fly or parasitoid emergence. The cohort generation time, Tc, (Bengstron, 1969) and capacity for increase, rc (Laughlin, 1965), were calculated from data obtained on the survival (lx) and fecundity (mx) of the cohort of adult wasps reared on H. pakistanae. Survival of the immature stages was extrapolated from the maximum number of eggs deposited by a single female per host (three eggs) and a single larva survival rate (33%). For five adults monitored for their longevity and fecundity, the initial cohort size was estimated to be 15 eggs and immature development time of ~20 days (see Life History results).

2.1.6. Host-finding behaviour A choice test was conducted to detect what cues, if any, T. columbiana uses for locating a suitable host pupa for oviposition. A simple olfactometer was constructed using a glass Y-tube, with an outside diameter of 3 cm, having two arms and a stem 5.3 cm and 5.5 cm in length, respectively. In Experiment 1, the Y-tube was inverted 1248 B.R. Coon et al.

(stem up), and a snap cap vial (5 cm × 10 cm, 800 ml) half-filled with deionised water containing a whorl of artificial plastic hydrilla was attached to one side of the Y-tube with plastic tubing affixed to a hole punched in the lid. On the opposite side, was a vial containing a freshly cut hydrilla whorl with no damage or exposure to Hydrellia flies. Both vials also had separate aquarium pumps attached via plastic tubing, which operated at the lowest output circulating air through the water in each vial. A newly emerged parasitoid (n = 25) was placed in a small funnel connected to stem of the ‘Y’ tube and was allowed 2 minutes to move down one of the olfactometer arms. The experiment was repeated with a new group of parasitoids except that the plastic hydrilla was removed and replaced with a piece of hydrilla damaged by a larva of H. pakistanae (Experiment 2). In the final test (Experiment 3), the undamaged hydrilla whorl was removed and replaced with a hydrilla whorl with attached H. pakistanae puparia.

2.2. Laboratory studies – Texas 2.2.1. Collection of Hydrellia flies and T. Columbiana H. pakistanae and H. balciunasi were obtained from adventive populations associated with hydrilla in earthen ponds (~0.2–0.8 surface ha, averaging 1 m deep) located at the Lewisville Aquatic Ecosystem Research Facility (LAERF), Lewisville, Texas (33° 04′45″ N, 96° 57′ 30″ W). T. columbiana was collected from the same ponds used for mass-rearing Hydrellia spp. at the LAERF as follows. Hydrellia spp. pupal collections coincided with hydrilla stem collections. The pupae were collected by sorting through hydrilla and excising stem areas containing a pupa. Each 8- to10-cm section containing a single pupa was placed in a plastic snap cap vial (55.4 ml) with a ventilated lid and incubated in a growth chamber at 25°C with a photoperiod of 16 h light:8 h dark (Hot Pack Corp., Philadelphia, PA). Vials contained sufficient water to cover stems, yet allowed room for an emerged adult to perch. Water was added as needed to maintain levels. All vials were checked daily for emergence of either an adult Hydrellia or T. columbiana.

2.2.2. Host stage oviposition preference H. pakistanae larvae were collected from a laboratory colony. Laboratory rearing followed methods outlined in Freedman, Grodowitz, Cofrancesco, and Bare (2001). Wasps were given the choice of two H. pakistanae life stages and observed through a stereomicroscope in a Petri-dish (140 mm diam.) for 1 h. Life stages of H. pakistanae were designated as first, second or third instars and early, intermediate or late pupae. Immatures were not extracted or detached from hydrilla during observations. The oviposition preference experiment had an average of nine replicates per life stage. Oviposition was assumed if the wasp was observed piercing the larval or pupal cuticle. Percent oviposition was calculated for each immature life stage as (number of parasitised/total number offered) × 100. Parasitised immatures were placed into a snap cap vial for incubation, and wasp emergence was recorded. Incubation procedures followed those described in the % parasitism section (see Texas field studies below). Percent survival was subsequently calculated as (number of adult wasps emerged/number of parasitised immatures) × 100. Biocontrol Science and Technology 1249

2.2.3. Parasitoid behaviour Average time (%) spent in each of four behavioural categories (searching, stem examination, ovipositing and grooming/resting) was recorded concurrently for all wasps observed and for wasps that oviposited in the aforementioned host stage preference study. A stopwatch was used during the hour of observation to record the intervals of each behavioural event.

2.3. Field studies – Florida 2.3.1. Seasonal abundance of H. pakistanae and T. columbiana Sampling of both insects was conducted in a series of 10 rectangular ponds (~24 m × 6 m) planted with hydrilla in April 1994. The 10 ponds, five in each of two rows, were located at the UF/IFAS Center for Aquatic and Invasive Plants (29° 43′ 14.8″ N, 82° 24′ 58.4″ W). A modified 0.25 m2 PVC quadrat was used to collect the insects and quantify density. The quadrat was suspended from the end of a telescopic aluminium pole used for cleaning swimming pools with two large plastic cable ties (~50 cm length). The cable ties were attached to opposite corners of the frame with large binder clips and joined at the centre with an eyebolt. The open area delineated by the floating quadrat was fitted with a plastic grid (egg crate drop ceiling, Home Depot®) cut and affixed to the frame with tie wraps. The grid (cell size 11 × 11 mm) was covered with a piece of mosquito netting secured with adhesive spray. Insects were sampled by gradually removing the netting from one corner and collecting the flies and parasitoids from the grid with a battery-powered vacuum device (DC Insect Vac, BioQuip®, Rancho Dominguez, CA). Three samples were collected from each of the 10 ponds (n = 30). Sampling points within each pond were determined by coordinates generated by rolling a 28-sided die. Each pair of numbers produced corresponded to the number of paces along the length and width of each pond. Samples were collected biweekly from 6 July 1995 to 17 January 1997 to ensure the sampling interval encompassed the minimum generation time of 18 days reported for H. pakistanae (Buckingham, 1994). Parasitoids collected in field samples were preserved in 80% ethanol, and submitted to Dr. Lubomir Masner, Agriculture and Agri-Food Canada, for identification. Mat surface temperatures were recorded with a hand-held temperature probe (Atkins® Thermocouple Thermometer Model 39658- T, Atkins Technical Inc., Gainesville, FL)

2.4. Field studies – Texas 2.4.1. Seasonal abundance and % parasitism of Hydrellia spp In 1999 and 2001, hydrilla stems were randomly collected from rearing ponds at the LAERF, usually every 2 weeks during the months of August through December (1999) and June through December (2001). Length and fresh weight were recorded, and each stem was examined under a stereomicroscope. Immatures per kilogramme of hydrilla were calculated for each sample period. To determine the rate of parasitism of Hydrellia spp. by T. columbiana, host puparia were collected during the same time period and with the same procedures as described above for the laboratory studies. Puparia were collected by sorting through hydrilla and excising stem areas containing a puparium. Each 8–10 cm stem section containing a single puparium was placed in a 55.4 ml plastic snap cap vial with a 1250 B.R. Coon et al. ventilated lid and incubated in a growth chamber at 25°C with a photoperiod of 16 h light:8 h dark (Hot Pack Corp., Philadelphia, PA). All vials were checked daily and emergence of either an adult Hydrellia or T. columbiana was recorded. Percentage of parasitism of Hydrellia spp. [(total T. columbiana emergence/total pupae) × 100], was calculated for each pond per sampling date, and ponds were averaged together for each sampling date and month.

2.4.2. Parasitoid overwintering biology and sex ratio A study was undertaken during the winter months of 2007–2008 in order to investigate the overwintering biology of T. columbiana. Data were collected from two H. pakistanae rearing ponds at the LAERF (between the months of November 2007 and July 2008). In June and July 2008, only one pond was sampled due to unexpected draining of the other pond. Hydrilla was collected from the study ponds monthly, November 2007 until June 2008. Five plant samples (~5 L plant material) were randomly taken from each pond, weighed and placed in Berlese funnels for one week or until dry. Extracted insects were preserved in 70% ethanol for later identification. To sample adult T. columbiana, pond-edge debris such as leaf-litter and cut grass was collected from the ponds’ edges monthly, November 2007 until March 2008. Five large handfuls of debris were collected randomly, weighed and placed in Berlese funnels (60-watt bulbs) to extract adult T. columbiana. Debris samples were allowed to remain in Berlese funnels for at least seven days, until dried. Collections were discontinued in March because new growth along the ponds’ edges made sampling

difficult and debris became minimal after this time. Because T. columbiana has been previously observed to display parthenogenesis in some areas of its distribution (L. Masner, pers. comm.), with southern populations consisting almost entirely of females (unpublished data), all collected specimens were sexed to determine if overwintering populations maintained the female-biased sex ratio.

2.4.3. Data analysis Unless otherwise indicated, data are reported as means ± SE. A two-sample t-test was used to determine if there was a difference in the size of eggs produced by females in the egg production experiment. For the parasitoid respiration study, Statistix® for Windows 97 was used to calculate the average length of time the adult parasitoid was submersed underwater. A Pearson’s correlation was used to analyse the relationship between % oviposition and % survival of the parasitoid in different life stages of H. pakistanae.

3. Results 3.1. Life history of T. columbiana This semi-aquatic parasitoid swims underwater to search for a submersed puparium of Hydrellia spp. in which to oviposit. After locating a suitable host, the female inserts her ovipositor into the thorax, which is contiguous with the cuticle of the puparium. Pupal dissections of both Hydrellia spp. revealed T. columbiana deposits a maximum of three eggs per host. Eggs are deposited in the host haemolymph, and Biocontrol Science and Technology 1251 embryogenesis is completed in 1 to 3 days. In this study, individual females (n = 50) produced on average 23.2 ± 0.6 eggs (14–32 eggs). The length and width of pre- ovipositioned eggs dissected from females (n = 1162) were 0.19 ± 0.01 mm and 0.06 ± 0.01, respectively, whereas 72 h post-ovipositioned eggs dissected from H. pakistanae pupae (n = 32) were significantly larger, measuring 0.57 ± 0.03 mm by 0.28 ± 0.002 mm, respectively (length, F = 16.77; width, F = 13.66, p < 0.002). Pre-ovipositioned chorionated eggs dissected from the ovaries of T. columbiana were hymenopteriform in shape (Clausen, 1940; Iwata, 1959). The chorion is smooth, thin and transparent, making the developing embryo visible (Figure 2A). The eggs possess a double membrane that allows them to increase in size once oviposited in the host’s haemolymph (DeBach, 1964). Based on measurements of the width of the head, larvae of T. columbiana appear to have three instars (Figure 3). The first instar is segmented and has large sclerotised mandibles; the last abdominal segment is bifurcated (two lobes) with several teeth on each lobe (Figure 2B). The length of the first instar (n = 127) was 0.49 ± 0.01 mm (0.37–0.62 mm) and the width was 0.14 ± 0.01 mm (0.06–0.18 mm). The mode of respiration for the first instar is unknown, but diffusion of oxygen via the haemolymph cannot be ruled as this is the only free-floating instar. Only a single larva survived the first instar, which was completed in 1–3 days. Apparently, the surviving larva uses its mandibles to eliminate siblings thereby avoiding competition for resources. The second and third instars are grub-like in appearance with 11 to 13 segments and possess indistinct mouthparts (Figure 2C). They were attached to the longitud- inal tracheal system of the host, where they presumably obtain air. The length and

Figure 2. (Colour online) Immature stages of T. columbiana. (A) Hymenopteriform egg after 72 hours post-oviposition, (B) Mandibulate first instar floating free in host haemolymph, (C) Grub-like second instar and (D) pupa. 1252 B.R. Coon et al.

35

30

25

20

Frequency 15

10

5

0 0.06 0.09 0.14 0.16 0.19 0.23 0.28 0.31 0.35 0.37 0.40 0.45 0.49 0.51 0.57 0.62 0.66 0.71 Width of head (mm)

Figure 3. Frequency distribution of instars of T. columbiana based on width of head region. width of the second instar (n = 184) were 0.92 ± 0.01 mm (0.63–1.04 mm) and 0.31 ± 0.01 mm (0.19–0.38 mm), respectively. The third instar length (n = 172) was 1.50 ± 0.01 mm (1.06–1.69 mm) and the width was 0.52 ± 0.01 (0.40–0.71 mm). Except for the size differences, the external appearance of the second and third instars was similar. The estimated stadial lengths for the second and third instars were 2–5 and 5–8 days, respectively. In total, the larval stage is completed in 12.2 ± 1.79 days (8– 16 days). The pupal stage (Figure 2D) lasted between 5 and 7 days and was enclosed in a thin transparent sac, presumably the last larval exuvia. Enclosed inside the sac around the parasitoid pupa were small globules, presumably faecal material expelled by the last instar before pupation. These globules consistently appeared only in the pupal stage. After the parasitoid completes its life stages inside the puparium, the newly formed adult exits the host, which is below the water surface. The adult parasitoid emerges from the host by cutting a hole in the ventral side of the puparium and exits with an air bubble attached to the setae on her abdomen. The air bubble presumably was acquired from the internal environment of the host puparium. This finding is consistent with adult eclosion by H. pakistanae and H. balciunasi. Hydrellia adults exit the puparium in the same manner but ascend to the surface enclosed in an air bubble obtained from the puparium (Balciunas et al., 2002). Under laboratory conditions, total development time from the egg to emerged adult was 21.9 ± 0.2 days (14–26 days).

3.2. Ovigeny, generation time and rate of increase T. columbiana was determined to be a synovigenic parasitoid because the egg load of host-deprived females increased over time (Jervis, Heimpel, Ferns, Harvey, & Kidd, 2001). When deprived of hosts for a period of 9 days, the number of mature eggs per female increased from 22.0 ± 2.1 to 25.1 ± 1.3. The ovigeny index for T. columbiana was 0.66, which also indicates synovigeny (Jervis et al., 2001). A laboratory survivorship curve and an adult fecundity table based on stadial lengths estimated for the immatures, and the reproductive performance of five females was constructed to calculate basic population statistics for T. columbiana (Southwood, 1978). The cohort generation (Tc) was ≈ 27.5 days (Figure 4). The net reproductive rate (Ro) was 6.4, which indicates the population is increasing. The Biocontrol Science and Technology 1253

100 5

90 mx Ix 80 4

70 Wasps / cohort (mx)

60 3

50

40 2 Survival (lx) Survival 30

20 1

10

0 0 1 357911131517192123 25 27 29 31 33 35 37 39 Age (days)

Figure 4. Survival and age-specific fecundity of five T. columbiana adults in a growth chamber at 26° ± 1°C and a 16:8 (L:D) photoperiod. Survival of immature stages was extrapolated from host dissections and a 33% survival rate of 15 larvae. capacity for increase (rc) was calculated to be 0.067. The number of times the population would increase per day (λ) was 1.07, and the time required for the population to double under the specified laboratory conditions was 10.2 days.

3.3. Parasitoid behaviour The adult inserts her antennae into the water before entering, suggesting that this parasitoid perceives some type of chemical cue from the larval-damaged plant, host or combination of both. Once under the water, she traps an air bubble under her wings and moves the air bubble to the tip end of her abdomen using her hind legs. Although capable of swimming underwater using a ‘dog paddle’ motion, she usually walks down the stem of the hydrilla, drumming the stem with her antennae to detect Hydrellia-damaged leaves. In general, females can spend up to 20 min submersed underwater searching for a suitable host(s) in which to oviposit. When a host puparium was located, she would use the tip of her abdomen to detect the position of the host pupa. The parasitoid would then insert her ovipositor between the segments where the host thorax was located and deposit up to three eggs. The oviposition process lasted approximately 9 min. If a host was located immediately, several hosts were attacked before the female had to resurface to replenish the air bubble under her wings. In the Y-tube olfactometer tests (Experiment 1), none of the parasitoids chose the vial containing water with the plastic hydrilla whorl or the undamaged hydrilla whorl. In Experiment 2, however, 17 of 25 parasitoids (68%) chose the side with the hydrilla whorl damaged by the H. pakistanae larva over the undamaged hydrilla. Finally, in Experiment 3, 24 of the 25 parasitoids tested (96%) selected the hydrilla with the puparia over the larva-damaged hydrilla within the allotted 2-min interval. Four general behavioural categories were observed in the laboratory: searching, stem examination, oviposition and grooming/resting (Figure 5). When searching for a prospective host, wasps apparently drum their antennae against hydrilla leaves and 1254 B.R. Coon et al.

Searching

% of time spent Oviposition

Stem examination

Grooming/resting

All data combined Ovipositing only

Figure 5. Behaviour of T. columbiana. A. Average time (%) in each of the four behavioural categories for all wasps combined and for ovipositing wasps only. Over twice as much time was spent depositing eggs by the ovipositing wasps. stems while walking on hydrilla above and below the water’s surface. Wasps examined stems with their ovipositor and/or mouthparts while remaining stationary. To oviposit, wasps pierced the immature host with its ovipositor in a stabbing motion. Grooming and resting were both accomplished while stationary and were often alternated. Wasps typically cleaned their antennae and legs. These four behaviours occurred in no specific order, but oviposition often was followed by a grooming session. On average, wasps spent the greatest percentage of time searching for prey to parasitise (58%) and the least amount of time examining stems (5%; Figure 5). Grooming/resting also was a substantial component of the wasp’s behaviour (27%), yet T. columbiana only allocated 10% of time on average to ovipositing (Figure 5). Of the 33 wasps observed, only 15 (45%) successfully oviposited. Due to the large number of wasps that did not oviposit during this behavioural study, time spent ovipositing could have been underestimated. For this reason, the 15 wasps that oviposited were analysed as a subgroup (known as ovipositing wasps, Figure 5) and average behavioural times were compared to the entire sample of wasps. Amount of time spent in each behavioural category was similar for both groups except for oviposition time (Figure 5). Ovipositing wasps spent more time, on average, searching for prey to parasitise (53%) and the least time examining stems (7%), whereas grooming/resting occupied 19% of the time (Figure 5). On average, 21% of time was spent ovipositing (Figure 5), which was a two-fold increase over the entire sample of wasps. By analysing the ovipositing wasps separately, oviposition was shown to be a more substantial component of the parasitoid’s behaviour than originally observed. Biocontrol Science and Technology 1255

3.4. Host stage oviposition preference Early and intermediate pupae were chosen for oviposition 40% and 60% of the time, respectively. Other stages ranged in % oviposition from 0% (first instar) to approximately 35% (third instar). Mature larvae and young pupae stages yielded the greatest % survival of immature T. columbiana to adulthood (Figure 6). First instars were not selected by T. columbiana as suitable hosts (Figure 6). A significant curvilinear relationship was found between % oviposition and % survival (p < 0.05, r2 = 0.79).

3.5. Seasonal abundance of H. pakistanae and T. columbiana The temporal distributions of H. pakistanae and T. columbiana in Florida and Texas are shown in Figures 7 and 8, respectively. In Florida, maximum densities of H. pakistanae were recorded in November 1995 (841.2 insects/m2) and November 1996 (703.7 insects/m2; Figure 7). Parasitoid activity was highest during the cooler months of the year (October–January). Population peaks for T. columbiana occurred in February 1996 (125 insects/m2) and also January 1997 (200/m2)(Figure 7). Based on these sample densities, the average maximum field parasitism rate for T. columbiana observed in Florida was 21.7%. In Texas, the average Hydrellia spp. immatures collected per kilogramme of hydrilla at the LAERF followed a similar seasonal trend both years of the study (Figure 8). Hydrellia spp. populations increased 1.2-fold from August to October in 1999 and 2.4-fold in 2001 (Figure 8). However, average Hydrellia spp. immatures per

kilogramme decreased 52% from October to December in 1999 and 66% during the same interval in 2001 (Figure 8).

Figure 6. Percent oviposition and % survival to adulthood of T. columbiana in the different life stages of H. pakistanae. Pearson’s correlation analysis between % oviposition and % survival of T. columbiana in life stages of H. pakistanae was significant (p < 0.05, r2 = 0.79). 1256 B.R. Coon et al.

Figure 7. Seasonal abundance of adult T. columbiana in hydrilla ponds located at the UF/ IFAS Center for Aquatic and Invasive Plants, Gainesville, FL, July 1995–January 1997.

Mean % parasitism by T. columbiana followed a distinct seasonal trend both years of the study (Figure 8). In 2001, a putatively density-dependent numerical response between wasp and fly populations was observed. In 1999, parasitism peaked in September and December at 12.7 and 12.0%, respectively. In 2001, %

parasitism increased 47-fold from August to a peak in December of nearly 30% parasitism. From 1999 to 2001, Hydrellia spp. numbers increased despite a consecutive increase in parasitism levels by T. columbiana. In 1999, Hydrellia spp. populations peaked at 3145 immatures per kilogramme (Figure 8). In 2001, a 1.8-fold increase from the peak of 1999 was detected. Hydrellia spp. populations reached 5693 immatures per kilogramme in 2001 (Figure 8). From 1999 to 2001, T. columbiana parasitism increased 2.3-fold from a peak of 12.7% parasitism in 1999 to nearly 29% parasitism in 2001 (Figure 8).

3.6. Parasitoid overwintering biology and sex ratio Adult T. columbiana were found along the edge of ponds at the LAERF from mid- November to mid-March (Figure 9), reaching densities in one sample as high as 5619 wasps per kilogramme of pond-edge debris. A significant trend was found in the decline of T. columbiana numbers in pond-edge debris from December to March (y = –0.99x + 300,246.9, df = 1, 2, F = 154.64, R2 = 0.99, p = 0.006). Berlese extraction of fresh hydrilla resulted in the collection of adult wasps throughout the entire study, although there was a decline after December (Figure 10). This is likely a combination of adults foraging in the hydrilla plant material at the time of sampling as well as newly emerging adults caught by the extraction method. The number of wasps in hydrilla began to increase again in May and in June, which corresponds to warmer temperatures, increased daylength and an increased availab- ility of host insects (Harms & Grodowitz, 2011). Biocontrol Science and Technology 1257

Figure 8. Average Hydrellia spp. immatures/kg of hydrilla and average % parasitism by T. columbiana at the LAERF, Lewisville, TX, August–December 1999 and June–December 2001.

In total, 15,377 specimens of T. columbiana were collected and sexed, of which 14,776 were collected in the current study, and 601 were reared from host pupae or represented past collections. Only four males were found during the current study, 1258 B.R. Coon et al.

Figure 9. Mean (±SE) T. columbiana adults per kilogramme of pond-edge debris recovered by Berlese extraction, LAERF, Lewisville, TX, November 2007–March 2008.

Figure 10. Mean (±SE) T. columbiana adults per kilogramme of hydrilla plant material recovered through Berlese extraction, LAERF, Lewisville, TX, November 2007–July 2008. giving a winter sex ratio of 1:3694 (male: female). In addition, only one male was identified from past rearing/collections, giving an estimated sex ratio of 1:600.

4. Discussion T. columbiana is a synovigenic endoparasitic wasp native to North America and has been reported to parasitise puparia of at least six indigneous Hydrellia spp. (Berg, 1950; Deonier, 1971). Our study confirmed T. columbiana also is an acquired parasitoid of the introduced hydrilla biological control agents H. pakistanae and Biocontrol Science and Technology 1259

H. balciunasi. Females dive beneath the water surface to search for and parasitise their host. Adults can remain submerged for up to 24 hours (Deonier, 1971). After ovipositing up to three eggs in the host puparium, only a single larva develops to the pupal stage within the submersed host. Upon eclosion, the adult wasp rises to the water surface enclosed in an air bubble. Life cycle from egg to adult is completed in 14–24 days at 26°C. The development time is similar to that reported for other Trichopria spp. (O’Neill, 1973; Morgan et al., 1990; Vaughn, 1985). However, the solitary life history strategy exhibited by T. columbiana in this study, in Hydrellia griseola (Fallen; Grigarick, 1959) and also by T. painteri Huggert and Morgan (Huggert & Morgan, 1993), does not support the gregarious habit assumed by Hagen (1956) for the entire genus and later documented in other Trichopria spp. (Morgan et al., 1990;O’Neill, 1973; Vaughn, 1985). Eggs of T. columbiana are hymenopteriform in shape and have a smooth, thin, transparent chorion that allows the developing embryos to be visible within the egg. The eggs also have a flexible membrane beneath the chorion that is presumably the vitelline membrane. It has been suggested this ‘double membrane’ is important for nutrient uptake (Flanders, 1950) and is characteristic of hydropic eggs (DeBach, 1964). Henneguy (1892) was the first to observe hydropic eggs and found these types of eggs are poor in yolk and need to be in an environment where there are abundant nutrients. Other researchers also reported that many eggs of hymenopterous parasitoids are hydropic and have a double membrane, which allows the diffusion of nutrients from the host (DeBach, 1964; Iwata, 1959). Flanders (1950) reported that parasitoids producing hydropic eggs generally deposit the eggs inside their host

so that the resources are available to the developing embryo. Although a protein analysis of the yolk was not performed on the eggs of T. columbiana, they probably are of the hydropic type for several reasons. First, the eggs of T. columbiana possess a double membrane. Second, the parasitoid eggs dissected from the host were larger than the eggs dissected out of the adult T. columbiana, which suggests nutrient uptake from the haemolymph had occurred. Third, the relatively high ovigeny index for this parasitoid is consistent with producers of hydropic eggs (Jervis et al., 2001). According to Bennett (2008), first instars of Trichopria larvae are characterised by having small, indistinct mouthparts. However, first instars of T. columbiana dissected out of the host in this study possessed large sclerotised mandibles. Up to three first instars were discovered free floating in the host haemolymph of the parasitised host, but only one first instar survived. Because they have sclerotised mandibles, first instars of this solitary parasitoid presumably seek out conspecifics and/or competitors and destroy them with their mandibles before losing them in subsequent instars (Fisher 1961; Salt 1961). T. columbiana apparently overwinters primarily in the adult life stage, in organic debris along the edge of water bodies. The wasps appear to rest in shoreline vegetation and occasionally venture out onto the water surface, searching for a suitable host when temperatures become warmer. Because hydrilla populations decline and suitable host life stages are limited during winter months, a dramatic decline in parasitism of H. pakistanae was observed. It is possible, although not examined in the current study, that native hosts, such as H. bilobifera Cresson and H. discursa Deonier, may be used as winter hosts. How abundant either of these native Hydrellia spp. are during winter months was not examined in this study, but 1260 B.R. Coon et al. their presence may allow T. columbiana to remain active year-round if environmental conditions are favourable. In this study, field parasitism levels of H. pakistanae in Florida and of the two Hydrellia spp. in Texas were over 20%. Based on empirical evidence, Hawkins and Cornell (1994) suggest that parasitism levels below ~35% would not lead to successful host suppression. However, Grodowitz observed field parasitism levels as high as 90% for H. pakistanae (unpublished data; Doyle et al., 2002). Under these circumstances, T. columbiana could be negatively affecting populations of the two introduced Hydrellia flies at some release sites where parasitism exceeds the 35% threshold. Although no trend was obvious in 1999, the 2001 data combined with wasp developmental time indicated a possible density-dependent relationship between the parasitoid and its host. Because T. columbiana develops from egg to adult in approximately 3–4 weeks, peaks in parasitism should follow peaks in Hydrellia spp. immature numbers by 3–4 weeks. For example, from October to November 2001, average % parasitism sharply increased 3.1-fold, corresponding to October peaks of Hydrellia spp. immatures per kilogramme. Following spikes in parasitism, immature numbers decreased 62% from October to November and continued to decrease into December. Because fly populations decreased at the onset of winter months when other factors (photoperiod, temperature, etc.) also could have affected fly densities, parasitoid-induced decreases could not be distinguished from seasonal declines. Despite the extreme female-biased sex ratio observed during the course of our studies, there is some evidence that in the northern range of T. columbiana, the sex ratio is nearly 1:1 (L. Masner, person. comm.). The reasons for this geographic parthenogenesis are unknown, although infection by Wolbachia is a possibility (Jeyaprakash & Hoy, 2000). The proteobacterium Wolbachia, which has been reported from a congener of T. columbiana (Hunter, 1999), was isolated from H. pakistanae and its acquired parasitoid. Regardless, it appears that male T. columbiana are rare in nature, at least in Florida and in Texas. A basic premise of biological control research is that parasitoids can exert a strong impact on insect populations. Consequently, in weed biological control programmes, it is essential that host-specific herbivorous insects are introduced without their parasitoid complex; otherwise, these co-evolved natural enemies would reduce the efficacy of the weed biological control agents. However, new association parasitoids cannot be excluded from the area of introduction (Doyle et al., 2002; Hill and & Hulley, 1995; Kula, Boughton,. J., & Pemberton, 2010; McFadyen & Jacob, 2004), and their acquisition can interfere with the establishment or impact of insects introduced for weed biological control (Goeden & Louda, 1976; McFadyen & Jacob, 2004; Paynter et al., 2010). In fact, Buckingham and Okrah (1993) concluded that parasitism of H. pakistanae and H. balciunasi by parasitoids of native Hydrellia spp. could be more of a problem than interspecific competition between the two introduced biological control agents, and they suggested that parasitism should be carefully monitored. In hindsight, attack of the two introduced Hydrellia spp. by T. columbiana, or another specialist parasitoid of native Hydrellia spp., was not unexpected because they were not released in an ‘enemy-free space’ (Lawton, 1985). Clearly, weed biocontrol practitioners should carefully consider the potential for acquiring novel parasitoids when selecting agents in order to avoid reducing biocontrol agent effectiveness and apparent competition with native ecological analogues of the agent (McFadyen & Jacob, 2004; Paynter et al., 2010). Biocontrol Science and Technology 1261

Acknowledgements We thank the following individuals for their technical contributions to this research, in particular, Jerry Butler, Judy Gillmore, Pam Howell, Pauline Lawrence, Michelle Leonard, Karen McKenzie, Julie Nachtrieb, James Nation, Linda Nelson, Christi Snell and Robin Swindle. The authors also thank Lubomir Masner for identification of T. columbiana as well as for comments on the ecology of the wasp, and Howard Frank and Bill Overholt for reviewing an earlier draft of this manuscript.

Funding This work was funded in part by a US Department of Agriculture, Agricultural Research Service/Institute of Food and Agricultural Sciences, University of Florida Cooperative Agreement [No. 58-6629-4-008]; the US Army Engineer Aquatic Plant Control Research Program.

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Roley, S. S. and R. M. Newman. 2006. Developmental performance of the Strefeler, M. S., E. Darmo, R. L. Becker and E. J. Katovich. 1996. Isozyme milfoil weevil, Euhrychiopsis lecontei (Coleoptera: Curculionidae), on characterization of genetic diversity in Minnesota populations of purple northern watermilfoil, eurasian watermilfoil, and hybrid (northern x loosestrife, Lythrum salicaria (Lythraceae). Am. J. Bot. 83:265-273. eurasian) watermilfoil. Environ. Entomol. 35:121-126. Takhtajan, A. L. 1969. Flowering plants. Smithsonian Institute Press, Wash- Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, ington DC. 510 pp. S. Baughman, R. J. Cabin, J. E. Cohen, N. C. Ellstrand, D. E. McCauley, P. Tranel, P. J. and T. R. Wright. 2002. Resistance of weeds to ALS-inhibiting O’Neil, I. M. Parker, J. N. Thompson and S. G. Weller. 2001. The popula- herbicides: what have we learned? Weed Sci. 50:700-712. tion biology of invasive species. Ann. Rev. Ecol. Syst. 32:305-332. Urbanska, K. M., H. Hurka, E. Landolt, B. Neuffer and K. Mummenhoff. Saltonstall, K. 2002. Cryptic invasion by a non-native genotype of the com- 1997. Plant Syst. Evol. 204:233-256. mon reed, Phragmites australis, into North America. Proc. Natl. Acad. Sci. Vilà, M, E. Weber and C. M. D’Antonio. 2000. Conservation implications of USA 99:2445-2449. invasion by plant hybridization. Biol. Invasions 2:207-217. Saltonstall, K. 2003. A rapid method for identifying the origin of North Amer- Williams, D. A., W. A. Overholt, J. P. Cuda and C. R. Hughes. 2005. Chloro- ican Phragmites populations using RFLP analysis. Wetlands 23:1043-1047. plast and microsatellite DNA diversities reveal the introduction history Simberloff, D. 2003. Eradication—preventing invasion at the outset. Weed of Brazilian peppertree (Schinus terebinthifolius) in Florida. Mol. Ecol. Sci. 51:247-253. 14:3643-3656.

J. Aquat. Plant Manage. 46: 15-32 Recent Advances in Biological Control of Submersed Aquatic Weeds

J. P. CUDA1, R. CHARUDATTAN2, M. J. GRODOWITZ3, R. M. NEWMAN4, J. F. SHEARER3, M. L. TAMAYO5 AND B. VILLEGAS6

ABSTRACT ners in the field. It also covers the types of natural enemies commonly used as biological control agents and the various The submersed aquatic plants hydrilla (Hydrilla verticillata abiotic, biotic, and technical factors that have contributed to [L.f.] Royle), Eurasian watermilfoil (Myriophyllum spicatum project successes and failures. Finally, priority areas are iden- L.),and Brazilian egeria (Egeria densa L.) are three of the tified where more resources are needed for research and worst invasive aquatic weed problems in the U.S., with mil- outreach programs to increase the effectiveness and accep- lions of dollars spent annually to control large infestations in tance of biological control technology for managing sub- all types of waterbodies. Historically, various control technol- mersed aquatic weeds in the future. ogies have been used to manage infestations of these sub- Key words: Brazilian egeria, hydrilla, Eurasian watermilfoil, mersed species, including biological control. During the past limiting factors, natural enemies. five years, there has been renewed interest in biological con- trol of submersed aquatic weeds nationally, primarily in re- INTRODUCTION sponse to the discovery in Florida of several hydrilla biotypes that have developed resistance to the herbicide fluridone. “One of the success stories revealed in the catalogue [by This paper summarizes the current status of biological con- Julien and Griffiths] is the biological control of several major trol activities in North America during the past 10-15 years. It water weeds; yet 40 years ago they were regarded as unprom- includes a preferred definition of biological control and de- ising targets.”—in Forward by D. F. Waterhouse, Julien and scribes the different approaches currently used by practitio- Griffiths (1998: vi). This review is not intended to be a comprehensive treat- ment of biological control methods for all aquatic weeds. In- 1Entomology and Nematology Department, Institute of Food and Agri- stead, it will: (1) focus on the use of arthropods (mainly cultural Sciences, P.O. Box 110620, University of Florida, Gainesville, FL 32611-0620, e-mail: jcuda@ufl.edu. insects), fish, and pathogens, both introduced and natural- 2Plant Pathology Department, Institute of Food and Agricultural Sci- ized, for biological control of submersed aquatic weeds; (2) ences, P.O. Box 110680, University of Florida, Gainesville, FL 32611-0680. examine the factors contributing to the repeated and often 3U.S. Army Engineer Research and Development Center, CEERD-EE-A, predictable control of certain aquatic weeds as well as identi- 3909 Halls Ferry Road, Vicksburg, MS 39280. fy possible reasons for failure; and (3) discuss biological con- 4Fisheries, Wildlife and Conservation Biology, 120 Hodson Hall, 1980 Folwell Ave., University of Minnesota, St. Paul, MN 55108-6124. trol research and outreach priorities for the most invasive 5Washington Cooperative Fish and Wildlife Research Unit, School of submersed aquatic plant species. Aquatic and Fishery Sciences, Box 355020, University of Washington, Seat- For general information on the theoretical and practical tle, WA 98195-5020. aspects of weed biological control, consult recently pub- 6California Department of Food and Agriculture, Biological Control Pro- gram, 3288 Meadowview Rd., Sacramento, CA 95832. Received for publica- lished references (Harris 1991, Harley and Forno 1992, Cen- tion June 21, 2006 and in revised form February 21, 2007. ter et al. 1997a, Deloach 1997, Julien and White 1997,

J. Aquat. Plant Manage. 46: 2008. 15

McFadyen 1998, Bellows and Headrick 1999, Goeden and lease a natural enemy into the U.S. for classical biological Andres 1999, Isaacson and Charudattan 1999, Rosskopf et al. control of an invasive aquatic plant, the potential agent must 1999, Van Driesche et al. 2002, Randall and Tu 2003, undergo rigorous testing in quarantine to ensure it will not Coombs et al. 2004, Cuda 2004). In addition, the following harm nontarget species. The candidate agent is exposed to a reviews, devoted exclusively to aquatic weed biological con- series of carefully chosen test plants in no-choice and multi- trol, are highly recommended (Charudattan 1990, 2001, ple-choice replicated trials to determine if the natural enemy Harley and Forno 1990, Pieterse 1990, Buckingham 1994, is safe to release. The U.S. Department of Agriculture Animal Center 1994, Barreto and Evans 1996, Madsen 1997, Cof- and Plant Health Inspection Service, Plant Protection Quar- rancesco 1998, Center et al. 1990, 2002, Barreto et al. 2000, antine unit (APHIS PPQ) controls the release approval pro- Balciunas et al. 2002, Johnson and Blossey 2002). cess (Buckingham 1994, Scoles et al. 2005). A voluntary multi- agency Technical Advisory Group reviews information provid- DEFINITION AND SCOPE OF ed by the requesting scientist prior to making a recommenda- BIOLOGICAL WEED CONTROL tion to APHIS PPQ concerning the release of an agent (Buckingham 1994, Scoles et al. 2005). Defining biological control in the context of other pest The augmentation approach involves the mass rearing management practices is like differentiating between a wet- and periodic releases of resident or naturalized aquatic weed land and an aquatic weed. The transition from one to the biological control agents to increase their effectiveness. This other is often difficult to distinguish. Recent advances in the approach is used primarily with pathogens, but it also can ex- field of biological control, particularly in the area of biotech- tend to other types of natural enemies, such as insects (Gro- nology (Nordlund 1996), and changes in public policy in the dowitz 1998, Jester et al. 2000, Hairston and Johnson 2001) last two decades have generated a new definition of biologi- and fish (Cassani 1996, Sutton and Vandiver 1998). For in- cal control, which states “. . . the use of natural or modified stance, augmentative releases of native or naturalized insects organisms, genes, or gene products to reduce the effects of have been proposed for biological control of water hyacinth undesirable organisms (pests), and to favor desirable organ- (Eichhornia crassipes [Mart.]) Solms; Center and Hill 2007), isms such as crops, trees, animals, and beneficial insects and hydrilla (Hydrilla verticillata [L.f.] Royle; Cuda et al. 2002, microorganisms” . . . (Anonymous 1987). Regrettably, this Wheeler and Center 2007), and Eurasian watermilfoil (Myrio- expanded all-inclusive definition of biological control advo- phyllum spicatum L.; Jester et al. 2000). cated by the U.S. Committee on Science, Engineering, and The conservation approach involves identifying and ma- Public Policy (COSEPUP) fails to capture the “natural ene- nipulating factors to enhance the abundance of potentially my” component that is the foundation of the discipline of bi- effective native or introduced natural enemies of aquatic ological control. weeds. Although conservation strategies have rarely been ex- Although there are numerous definitions of biological ploited (Harris 1993), their importance in the biological control, we follow the definition proposed by DeLoach control of aquatic weeds is now gaining recognition (MacRae (1997) because it preserves the natural enemy aspect that et al. 1990, Creed and Sheldon 1995, Sheldon and O’Bryan sets biological control apart from other methods of weed 1996, Newman et al. 1998, Creed 2000, Tamayo et al. 2000, control. He defines biological control of weeds as “. . . the 2004, Newman 2004). planned use of undomesticated organisms (usually insects or The “new association” approach is a variation of classical plant pathogens) to reduce the vigor, reproductive capacity, biological control first proposed by Pimental (1963) and lat- or density of weeds . . . it excludes cultural controls (grazing er by Hokkanen and Pimental (1984). They contend that management, crop rotation, etc.) and natural control (the natural enemies from closely related plant species growing in action of organisms without human intervention) . . .” The similar climates but different geographical areas from the intentional use of grass carp for aquatic weed control is con- target plant are potentially more damaging than co-evolved sistent with this definition. However, the application of bio- natural enemies. The target weed is more likely to be dam- technology to genetically modify organisms as well as aged by the new associate because presumably it lacks the ap- cultural practices like hand weeding, plant competition, al- propriate defense mechanisms to resist attack. This lelopathy, and other management practices that alter the bi- approach, more recently referred to as “neoclassical biologi- otic balance of the soil are not included in this definition. cal control” (Lockwood 1993), differs from classical biologi- Three different approaches are currently used in the bio- cal control in that the natural enemies have not played a logical control of aquatic weeds: classical (importation), non- major role in the evolutionary history of the host plant, and classical (augmentation), and conservation (habitat manipu- are therefore considered new associates (Hokkanen and Pi- lation) (Julien and White 1997, McFadyen 1998, Goeden and mentel 1984). The neoclassical approach for selecting plant- Andres 1999). The classical approach is by far the most com- feeding insects as biological control agents has been actively mon method and typically involves the planned introduction supported by some practitioners of biological weed control of natural enemies from their native range to control a non- (Dennill and Moran 1989, DeLoach 1995), and vigorously native invasive species. Researchers travel to the native range criticized by others (Goeden and Kok 1986). Because organ- of the invasive plants to find natural enemies of the unwanted isms used in neoclassical biological control are not by defini- species. These organisms are tested initially for efficacy and tion entirely host specific, they also represent a threat to host-specificity in their native range; successful candidates are nontarget congeners of the weed. Therefore, this approach then imported under permit into approved containment lab- is appropriate only in those cases where the target weed has oratories for final host range testing. Before scientists can re- few or no native relatives in the area of introduction.

16 J. Aquat. Plant Manage. 46: 2008.

In a broad sense, the term neoclassical biological control and that insects would not be effective in controlling aquatic could be applied to those cases where a native organism de- weeds (Wilson 1964). Some authors still support this notion. velops a new association with a non-native weed species. For For instance, Jolivet (1998) stated that, “Insects rarely eat example, a number of studies have demonstrated the native aquatic plants, more often eating subaquatic ones . . . It milfoil weevil (Eurychiopsis lecontei [Dietz]) is an important bi- seems that the variety of deterrents . . . reduces the chances ological control agent of the non-native Eurasian watermil- for specialization. This is one of the reasons why it is so diffi- foil in the U.S. and Canada (Creed et al. 1992, Creed and cult to find specific herbivores to use to control introduced Sheldon 1993, 1994, 1995, Sheldon and Creed 1995, Shel- aquatic weeds . . .” However, studies conducted over the past don 1997, Creed 1998, Engel and Crosson 2000, Jester et al. several decades indicate many instances of successful control 2000, Newman and Biesboer 2000, Cofrancesco et al. 2004, of aquatic weeds worldwide by insects (Andres and Bennett Newman 2004). The milfoil weevil is native to North America 1975, Julien and Griffiths 1998, McFadyen 1998, Hill 1999). and attacks milfoils (Myriophyllum spp. Haloragaceae). Re- Furthermore, the extensive recent literature on specialized cent studies have shown that weevils reared on Eurasian wa- insects that mine and feed on the living tissues of the sub- termilfoil not only develop faster and survive better on the mersed macrophytes hydrilla and Eurasian watermilfoil introduced milfoil (Newman et al. 1997, Roley and Newman clearly demonstrates the biological control potential of 2006), but also will preferentially attack the non-native spe- aquatic insects (MacRae et al. 1990, Balciunas and Purcell cies over its natural host plant northern water milfoil (M. si- 1991, Newman 1991, Buckingham and Okrah 1993, Kangas- biricum Komarov) (Solarz and Newman 1996, 2001). niemi et al. 1993, MacRae and Ring 1993, Creed and Shel- don 1994, Allen and Center 1996, Balciunas and Burrows TYPES OF BIOLOGICAL CONTROL AGENTS 1996, Buckingham and Bennett 1996, Grodowitz et al. 1997, 2004, Wheeler and Center 1997, 2001, 2007, Buckingham Three major groups of organisms are commonly used in 1998, Johnson et al. 1998, Cuda et al. 1999, 2002, Bennett biological control of aquatic weeds: arthropods (insects and and Buckingham 2000, Epler et al. 2000, Johnson et al. 2000, mites), fish (primarily grass carp), and pathogens (fungi and Newman 2004). bacteria). Fish Arthropods One of the most controversial biological control agents In an early review article on biological control of weeds, currently used to control hydrilla and other submersed Wilson (1964) stated that . . . “no insects have yet been used aquatic weeds is the grass carp (Ctenopharyngodon idella Val.) for the biological control of aquatic weeds, . . . it may be that (Chilton and Muoenke 1992, Cassani 1996, Elder and Mur- in the fresh-water environment the relatively small numbers phy 1997, Killgore et al. 1998). Native to cold and warm water of species of plants and phytophagous insects, and perhaps regions of China and Russia (Sutton and Vandiver 1998), the the domination of this environment by fish, have caused in fish is highly adaptable to a wide range of temperature ex- aquatic phytophagous insects a level of host specialization tremes and has been introduced into many countries world- much lower than that occurs in the species-rich terrestrial wide for aquatic weed control (Julien and Griffiths 1998). environment.” A very different viewpoint is presented in lat- Although interest in expanding the use of grass carp for con- er review articles on the same topic (Andres and Bennett trolling hydrilla has increased following the recent discovery 1975, McFadyen 1998). Biological control of aquatic weeds of herbicide resistance in some Florida hydrilla populations with insects has been remarkably successful since it was first (Michel et al. 2004, Netherland et al. 2005), the widespread attempted in the U.S. against alligatorweed (Alternanthera use of grass carp for aquatic weed control has been ques- philoxeroides [Mart.] Griseb.) in 1964 (Hawkes et al. 1967), tioned because of concerns about its negative impact on wa- which was coincidentally the same year Wilson’s article was ter quality and nontarget species (McKnight and Hepp published. Complete or substantial biological control of the 1995). Unlike host-specific arthropods and pathogens typical- floating macrophytes water hyacinth, water lettuce (Pistia ly used in biological weed control programs, grass carp are stratiotes L.), salvinia (Salvinia molesta D.S. Mitch.), and red nonselective grazers that can potentially alter entire freshwa- water fern (Azolla filiculoides Lamarck) by insects has been ter ecosystems and may be unsuitable for biological control achieved in most countries where it has been attempted of aquatic weeds in some water bodies (Bain 1993, Kirk and (Julien and Griffiths 1998, Hill 1999). Although biological Socha 2003, Kirkagac and Demir 2004). Consequently, this control of many introduced weeds is not always effective fish may be regarded as unsuitable for biological control of (Crawley 1989), the success rate for the control of aquatic aquatic weeds in some water bodies and is illegal to release in weeds is much higher. A cursory examination of the various some states (e.g., Minn., Vt., Wisc.) (Getsinger et al. 2004). projects (e.g., see Julien and Griffiths 1998, McFadyen 1998, Consumption of aquatic plants by grass carp depends on a Hill 1999) suggests this high success rate may be associated variety of factors (Pine and Anderson 1991, Sutton and Van- with the growth form of the weeds, the insect taxa used as bi- diver 1998). Generally, grass carp tend to feed in relatively ological control agents, susceptibility to disease-causing shallow areas and near the surface of a water body, prefer- pathogens, fluid nature of the aquatic environment, or some ring to graze on the soft tips of tender submersed aquatic combination of these elements. plants. In Florida, large fish preferentially consume hydrilla Historically, it was thought that herbivory on aquatic mac- over other non-native species such as Brazilian egeria (Egeria rophytes was uncommon and unimportant (Lodge 1991) densa Planch.), hygrophila (Hygrophila polysperma [Roxb.] T.),

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and Eurasian watermilfoil (Sutton and Vandiver 1998). How- out in the 1990s in Asia and Europe (Harvey and Varley ever, small fish exhibit a clear preference for native musk- 1996, Harvey and Evans 1997, Shearer 1997). Although the grasses (Chara spp.) over hydrilla where the plants occur to- biological control potential of several promising isolates gether (Sutton and Vandiver 1998). from these surveys has been evaluated (Harvey and Varley Because of fears about grass carp’s potential for reproduc- 1996, Shearer 1999a), further studies are needed to demon- ing and possible negative impacts on native fisheries, early strate the safety and efficacy of these pathogens. research focused on developing a nonreproductive fish (Cas- Pathogens indigenous to a region and those that cause sani 1996, Sutton and Vandiver 1998). Sterile fish are now endemic diseases are ideal candidates for development as produced routinely by shocking fertilized eggs with hot or nonclassical (augmentative or inundative) biological control cold water, or with pressure. Eggs that are shocked retain an agents. Generally, inundative biological control agents are in- extra set of chromosomes (triploids) that causes sterility. To dustrially developed and registered as bioherbicides by gov- ensure that all stocked fish are incapable of reproducing, ernmental agencies such as the Environmental Protection each fish is screened by scanning the blood cell nuclei with a Agency (EPA). These pathogens must have high levels of vir- Coulter Counter™ (Cassani 1996). This instrument is used ulence to be capable of inflicting acceptable damage. Host to measure the diameter of the nuclei, which is larger in trip- specificity is not a major concern with these pathogens be- loid fish. cause their effectiveness is contingent on inundative applica- Grass carp are difficult to remove from a body of water af- tion, and in the absence of such applications, the pathogens ter they have been introduced. Consequently, rigid barriers cease to spread or do not cause a prolonged or escalating ep- capable of confining the fish while maintaining unrestricted idemic. Bioherbicide pathogens should be easily cultured to movement of water are usually installed on culverts or canals produce infective propagules, and the propagules should to prevent grass carp from escaping into other areas. Because have good viability and shelf life. They also should be capable the life span of grass carp can be 20 or more years, appropri- of causing infection and disease cycles over a range of envi- ate methods of removal must be considered prior to stocking ronmental conditions. Pathogens that have high levels of ge- the fish (Sutton and Vandiver 1998). Draining the water netic stability are desirable for the sake of long-term safety. body or using a fish toxicant like rotenone are normally used Currently, there are no registered bioherbicides to control to remove grass carp (Sutton and Vandiver 1998), but these any submersed aquatic weed, but several promising candi- methods are nonselective and can be ecologically disruptive. dates have been the subject of numerous investigations. The development of Grass Carp Management Baits (GCMB; Typically, a variety of microorganisms, including common a floating alfalfa-based pellet laced with rotenone) may help plant-associated saprophytes, plant parasites, and general to alleviate some of these problems (Mallison et al. 1994). Al- members of the microbial community, reside on submerged though GCMBs can selectively remove up to 80% of the grass plants such as hydrilla. For instance, in one Florida study by carp from a water body with minimal effects on nontarget Shabana and Charudattan (1996), 458 different microorgan- fish species (Mallison et al. 1994), the sudden appearance of isms (211 bacteria, 202 fungi, 44 actinomycetes, and 1 cyano- dead grass carp on the surface of a water body could create a bacterium) were recovered from 48 samples taken from the public relations problem. The public perception of fish kills ponds. Another 287 pathogens (132 bacteria, 154 fungal iso- is generally negative, regardless if they are intentional or a lates, and 1 cyanobacterium) were recovered in 25 samples natural occurrence. collected from the two lakes. Fungi belonging to several plant pathogenic genera, including Botryosporium, Cercosporid- Pathogens ium, Chaetophoma, Diplodia, and Pyrenochaeta, were found mainly on hydrilla and in soil samples. The frequency and di- Generally, pathogens with a capacity for rapid secondary versity of the microorganisms isolated confirmed the occur- reproduction (i.e., having the potential to cause secondary rence of a rich microbial flora associated with hydrilla infections and disease spread) and capable of causing high (Shabana and Charudattan 1996, Shabana et al. 2004); this levels of damage to the weed’s vegetative or reproductive condition should be typical in any body of water infested parts are most suitable as classical biological control agents. with a submersed aquatic weed. Several factors contribute to the effectiveness of these patho- Despite this rich microbial biodiversity, no practical mi- gens, including host-pathogen disjunction (i.e., lack of host- crobial herbicide has been developed thus far for hydrilla. pathogen homeostasis), presence of a target weed popula- One reason is the lack of understanding of the epidemiolog- tion that is predominantly or wholly susceptible (i.e., lacking ical principles involved in underwater diseases. For instance, in genetic diversity), and high levels of virulence and accept- little information exists on the mode of inoculum dispersal, able levels of host specificity of the pathogen. Presence of settlement, and early infection processes in underwater pa- dense weed populations and environmental conditions con- thosystems. Recent studies have attempted to address this de- ducive for epidemic build-up also are required. Currently, ficiency. Smither-Kopperl et al. (1998, 1999a) studied the only one pathogen has been deployed as a classical biological epidemiology of disease caused by an isolate of Fusarium cul- control agent of an aquatic weed anywhere in the world. This morum (Wm.G.Sm.) Sacc. originally obtained from water sol- fungal agent, Cercospora piaropi Tharp (= C. rodmanii Conway; diers (Stratiotes aloides L.) but shown to be pathogenic to Tessman et al. 2000), was imported into South Africa from hydrilla (Charudattan and McKinney 1978). The process of Florida and released against water hyacinth (Morris et al. deposition and attachment of spores in the hydrilla—F. cul- 1999). Surveys for pathogens of hydrilla and Eurasian water- morum pathosystem is quite complex (Smither-Kopperl et al. milfoil with classical biological control potential were carried 1999a) and must be understood in relation to spore dispersal

18 J. Aquat. Plant Manage. 46: 2008.

in water. Smither-Kopperl et al. (1998) investigated the dis- EcoScience Corporation, Worcester, Massachusetts, devel- persal of spores of F. culmorum in still and moving aquatic sys- oped a prototype formulation of M. terrestris, named Aqua- tems. They found that the physical components of dispersal Fyte™, for potential registration and commercial use. Verma of F. culmorum spores in a still aquatic system were defined by and Charudattan (1993) tested the prototype formulation rapid lateral dispersal and sinking due to gravity. In moving on several aquatic and terrestrial plants and found a number water, the dynamics of water movement were superimposed of species (including hydrilla) to be susceptible to infection over the other two factors, which complicated the movement with the Gunner (1983) isolate applied as Aqua-Fyte™. dynamics of the spores. Aqua-Fyte™ was effective in controlling milfoil in growth Another pathogen with bioherbicide potential is Plectospo- chamber studies when water temperatures were between 20 rium tabacinum (van Beyma) Palm et al., the anamorph of and 28 C, the optimum disease-inducing range for this fun- Plectosphaerella cucumerina (Lindfors) Gams. This pathogen gus. Successful tests in laboratory, pool, and pond experi- was isolated in 1996 from naturally diseased hydrilla shoots ments gave impetus for further evaluations on a field (Smither-Kopperl et al. 1999b). In the laboratory, P. tabaci- population of Eurasian watermilfoil in Guntersville, Ala- num was pathogenic to hydrilla shoots maintained in aque- bama, Lewisville, Texas (Shearer 1994). However, the myco- ous solutions in test tubes. Koch’s postulates (establishing a herbicide was ineffective in reducing aboveground biomass causal relationship between a causative microbe and a dis- of milfoil under natural conditions at these sites (Smith and ease) were fulfilled in several replicated experiments. Infect- Winfield 1991). A reevaluation of the formulation was ed shoots became slightly chlorotic within 24 h and the deemed necessary to understand the reduced levels of effica- leaves became flaccid. There was also an increase in disease cy between laboratory and field trials. Using naturally infect- severity as inoculum concentration increased from 105 to 107 ed plant material from Florida, Shearer (2002) recently conidia ml-1 and the disease developed over a range of tem- examined how M. terrestris could become pathogenic when peratures from 15 to 30 C. This fungus clearly has potential milfoil is stressed. as a biological control agent for hydrilla. Morris et al. (1999) recorded the occurrence of a bacteri- An isolate of Mycoleptodiscus terrestris (Gerdemann) Osta- al disease of parrotfeather (Myriophyllum aquaticum [Vell.] zeski, first reported as a pathogen of Eurasian watermilfoil by Verdc.), in South Africa. Diseased aerial shoots of parrot- Gunner (1983), is capable of causing disease in hydrilla (Joye feather plants were found in most areas infested with this and Paul 1992, Verma and Charudattan 1993). Prototype for- weed in that country. The disease was characterized by the mulations of M. terrestris tested against dioecious hydrilla in wilting of scattered, individual aerial shoots from the tip laboratory and field studies showed that hydrilla was suscepti- downward for about 10 cm accompanied by a greying color. ble to infection by the fungus (Joye 1990, Joye and Cof- Microscopic examination revealed that the xylem vessels of rancesco 1991). Incorporating the fungus into a patented the stems and leaves were filled with bacterial cells. The caus- biocarrier, Biocar™ 405, produced more recent formula- al bacterium was isolated in pure culture and identified as a tions. Initial test-tube studies demonstrated that both granu- strain of Xanthomonas campestris (Pammel) Dawson. Although lar and caplet formulations induced severe disease or death natural infections seldom caused more than 1% of the aerial of excised hydrilla shoots 2 weeks after inoculation. Low, me- shoots to be affected, an inundative application of the bacte- dium, and high dosage rates of the granular formulation ap- rium at 108 colony-forming units per ml produced 100% plied to rooted hydrilla in 12-L columns reduced shoot shoot infection when the plants were sprayed in the morning biomass at 4 weeks after application by 87.7, 94.8, and 99.2% when guttation droplets were still present on the leaves respectively compared to untreated controls (Shearer 1998). (Morris et al. 1999). Although all aerial parts of the plant In microcosm studies, a granular formulation reduced shoot were dead, about 6 weeks later new shoots appeared from biomass of hydrilla grown in 1700-L tanks by 97.5% at 4 the submersed stems and the plants recovered. Microscopic weeks after application (Shearer 1996, 1998). However, ini- examination revealed that the bacterium did not invade the tial field trials of M. terrestris formulated with Biocar™ 405 older underwater stems. Because of this inability to kill sub- failed because the company changed the ingredients in the mersed biomass and the ability of the plant to replace killed carrier that inadvertently killed the fungus (Shearer 1999b). shoots, the bacterium was not considered an effective bioher- Further development and registration of a Texas isolate of bicidal agent (Morris et al. 1999). However, it may prove to M. terrestris as a bioherbicide are anticipated now that some be more effective if used in combination with an approved hydrilla populations in Florida have become resistant to flu- herbicide (see Integration of control tactics). ridone (Michel et al. 2004). Current research is focusing on Brazilian egeria and its congener Egeria najas Planc. are two better ways of processing the fungus for commercialization submersed species native to Brazil that have become serious (J. F. Shearer, pers. comm.). weeds in hydroelectric reservoirs in the southern part of this In the late 1970s, M. terrestris was isolated from Eurasian South American country. Because the use of chemical herbi- watermilfoil plants collected in Massachusetts (Gunner cides not only is impractical but prohibited in these reservoirs, 1983). Preliminary greenhouse and laboratory studies estab- biological control studies were initiated by Nachtigal and Pitel- lished the effectiveness of the fungus in reducing milfoil bio- li (2000). This research resulted in the discovery of a Fusarium mass (Stack 1990, Gunner et al. 1991, Smith and Winfield sp. (tentatively identified as F. graminearum; R. A. Pitelli, pers. 1991). A small-scale field trial using fungal mycelia in Stock- comm.) from naturally diseased shoots of the two egeria spe- bridge Bowl, Massachusetts, supported the laboratory find- cies. Pathogenicity studies proved that this fungus caused a dis- ings by inducing a 16-fold reduction in shoot biomass in ease characterized by stem necrosis and foliar chlorosis that treated versus untreated plots (Gunner 1987). intensified progressively until a complete breakdown of the

J. Aquat. Plant Manage. 46: 2008. 19

plant tissues occurred. Propagation of the fungus on sterilized Reproduction in these weeds is primarily by rapid vegetative rice grains was the most suitable method for inoculum pro- growth (Hoyer et al. 1996). Crawley (1989, 1990) suggests duction. The rice-grown inoculum was highly efficacious in that high genetic uniformity usually associated with vegetative killing egeria plants at the rate of 0.5 g/L, and it could be reproduction is a necessary prerequisite for successful biolog- stored for more than 8 months at 4 C. The specificity of the ical control, although its importance has been questioned fungus was tested on 14 cultivated species and 11 aquatic (Chaboudez and Sheppard 1995). (3) These weeds are highly plants, but only hydrilla and the two egeria species developed susceptible to secondary infection. Aquatic plants that have symptoms. The biological control potential of this fungus sustained damage by insects or disease will rot and disinte- needs to be investigated further (Nachtigal and Pitelli 2000). grate very rapidly (Buckingham 1994). (4) Beetles, especially weevils, have been responsible for most of the control. Nu- FACTORS CONTRIBUTING TO PROJECT merous successes in weed biological control have been associ- SUCCESSES AND FAILURES ated with this group of insects (Crawley 1989, 1990, O’Brien 1995). These agents also tend to remain above the water, Defining success in biological control of weeds is usually which may reduce fish predation pressure (Newman 2004). subjective and highly variable. A project may be considered Classical biological control programs targeting submersed successful in an ecological sense when a weed biological con- aquatic weeds such as hydrilla have been less predictable trol agent establishes and negatively changes the weed’s equi- (Bennett and Buckingam 2000, Forno and Julien 2000; see librium density (Crawley 1990, Grodowitz et al. 2004). also Cuda et al. 1999, 2000, 2002, Doyle et al. 2002, Grodow- However, the type of damage inflicted by the biological con- itz et al. 2003, 2004, Owens et al. 2006). Success or failure trol agent may not cause the desired level of economic con- can be attributed to a variety of factors that may be grouped trol (Ehler and Andres 1983). Forno and Julien (2000) also into three general categories: physical, biological, and tech- make a clear distinction between biological success and im- nical. These factors, working alone or in combination, can pact success. Biocontrol agents can be biologically successful affect the population dynamics of the biological control in establishing and sustaining high population densities on agents as well as the weeds. the target weed but may not provide the desired level of con- trol or impact on the weed. ABIOTIC FACTORS Julien (1997) argues that the use of short descriptive terms to define success, such as complete, substantial, or neg- Climate and Weather ligible, as proposed by Hoffman (1995), oversimplifies reality because variations in time and space are not taken into ac- Several independent studies have shown that approxi- count. For example, in those countries where alligatorweed mately one-half of the failures in weed as well as insect bio- has been introduced, biological control can range from com- logical control programs is climate and weather related plete to negligible depending on the season, geographic (Stiling 1993, Cullen 1995). Climate matching should be an area, and habitat. However, Hoffman’s system is more mean- important consideration when planning releases of biologi- ingful from an operational perspective because it equates the cal control agents (Buckingham 1994, McFadyen 1998, New- degree of biological control with the extent to which other man et al. 1998), but its importance to aquatic weed control measures (e.g., harvesters, aquatic herbicides) must biological control is not well understood (Buckingham be used. The advantage of this approach is that it describes 1994). Climate matching may be less important in aquatic success in practical terms that are more readily understood systems because the thermal capacity of water dampens tem- by aquatic plant managers and bureaucrats. For example, bi- perature fluctuations, and relative humidity in aquatic sys- ological control is defined by Hoffmann (1995) as complete tems is more or less constant. when no other control method is required, substantial when However, physical factors may have an effect in some other methods such as herbicides are still required but at re- aquatic systems. For example, dense mats of hydrilla increase duced level, and negligible when other control methods are the surface water temperature considerably, creating unfa- necessary for managing the weed problem. Measuring bio- vorable conditions for a biocontrol agent. For instance, the logical control success in economic terms (e.g., reduced her- Asian hydrilla leaf-mining fly (Hydrellia pakistanae Deonier) bicide applications) has an additional benefit. Funding was introduced as a biological control agent of hydrilla in agencies are more inclined to continue supporting biologi- 1987, and establishment has been confirmed at most loca- cal control when they can see a return on their investment. tions in the southeastern U.S. where hydrilla occurs (Center The most recent comprehensive listing of aquatic weed bi- et al. 1997b, Balciunas et al. 2002). However, laboratory stud- ological control programs worldwide was published by Julien ies by Buckingham and Okrah (1993) showed that a constant and Griffiths (1998). Alligatorweed, water hyacinth, water let- water temperature of only 36 C prevented adult emergence. tuce, salvinia, and red water fern have been predictably con- Water temperatures in excess of 40 C for extended periods trolled using the classical approach (McFayden 1998, 2000, are not uncommon in hydrilla canopies during the summer Hill 1999). An interesting pattern emerges when the weed months in Florida. Growth chamber studies simulating mid- and natural enemy attributes associated with these successes summer hydrilla mat surface temperatures in Florida support are examined. (1) All the aforementioned weeds are free- the high temperature mortality hypothesis (Cuda et al. floating, or produce floating mats in the case of alligator- 1997). Therefore, it appears that high temperatures occur- weed. This plant growth form is strongly affected by wave ac- ring in the hydrilla mats in the summertime are probably det- tion and currents that are inherent in aquatic systems. (2) rimental to H. pakistanae. Regression analysis of the density of

20 J. Aquat. Plant Manage. 46: 2008.

H. pakistanae larvae with the maximum surface water temper- the number of sites available for larval development of the ature showed a significant negative correlation (Cuda et al. hydrilla stem tip midge (Cricotopus lebetis Sublette) (J. P. Cu- 1997). Further evidence for the high temperature-mortality da, pers. observ.). hypothesis is supported by the seasonal abundance of H. paki- Applying pesticides to control biting flies and aquatic stanae in north Florida. Similar patterns of low density in mid- weeds also may affect the density and performance of certain summer and progressively higher densities later in the season biological control agents. In Florida, for example, insecti- when water temperatures are cooler were observed in experi- cides used for controlling mosquitoes are routinely applied mental ponds and lakes with established populations of H. to areas in close proximity to water bodies with established pakistanae (Cuda et al. 1997, Wheeler and Center 2001). populations of aquatic weed biological control agents (J. P. Heavy rainfall and cold weather also have been observed to Cuda, pers. observ.). Drift from the aerial application of mos- cause high mortality of leaf-mining flies (D. L. Deonier, pers. quito adulticides and larvicides is unavoidable due to the comm. in Buckingham 1994; Wheeler and Center 2001). density of these waterbodies in peninsular Florida. In a re- Yet some aquatic insects have been able to adapt to ex- cent laboratory study, the Asian hydrilla leaf-mining fly H. pa- treme weather conditions. For instance, overwintering tem- kistanae was found to be highly susceptible to aerial peratures do not appear to be a limiting factor for the milfoil application of malathion at rates typically used for control- weevil in Minnesota (Newman et al. 2001), which is not sur- ling adult mosquitoes in Florida (N. Tietze, unpubl. data). prising because the weevil is native to northern North Ameri- The mosquito larvicides temephos and methoprene also are ca. High summer water temperatures (>35 C) probably extremely toxic to the larvae of H. pakistanae (J. P. Cuda, un- precludes establishment of the weevil in southern lakes published data). This discovery led to the use of these insec- (Newman 2004). ticides in manipulative laboratory and field studies where the effect of this classical biological control agent on hydrilla was Habitat Conditions evaluated experimentally by using these pesticides to chemi- cally exclude the insect from control tanks or ponds (Cuda Lack of fluctuating water levels and drought conditions et al. 1997). Likewise, the commercially available microbial can affect the establishment or survival of some insect biolog- mosquito larvicide Bacillus sphaericus is toxic to the Indian ical control agents of aquatic and semi-aquatic weeds. For in- moth Parapoynx diminutalis (Snellen), an adventive natural stance, larvae of the Indian weevil (Bagous affinis Hustache) enemy of hydrilla (Haag and Buckingham 1991). and the Australian weevil (B. hydrillae O’Brien) severely dam- Biological control agents are more likely to come into di- age the tubers and stems, respectively, of hydrilla in its native rect contact with herbicides used for aquatic plant control. In range (Balciunas and Purcell 1991, Buckingham 1994). How- most cases, aquatic herbicides, when applied at recommend- ever, these insects failed to become permanently established ed field rates, are regarded as harmless to fish and arthropods following their release in the United States because they used as biological control agents. Under laboratory condi- were unable to complete their development entirely on sub- tions, larval mortality of the Asian leaf-mining fly H. pakistanae mersed hydrilla (Buckingham 1994, Godfrey et al. 1994). was attributed to loss of habitat rather than to direct toxicity Three additional Bagous weevils from Thailand that failed to following exposure to the herbicides endothall, fluridone and complete their life cycles on submersed hydrilla in quaran- diquat (Haag and Buckingham 1991). The hydrilla tuber wee- tine studies were dropped from further consideration (Ben- vil (B. affinis) also was not adversely affected by direct contact nett and Buckingham 1999, 2000). with these same herbicides (Haag and Buckingham 1991). For insects that overwinter on the shoreline, both shore- However, herbicides also can have a negative effect on biolog- line and in-lake habitat can be important. Milfoil weevil densi- ical control agent populations by removing too much of their ties have been correlated with shoreline development (Jester food supply. For example, reduced feeding activity was ob- et al. 2000) and plant cover (Tamayo 2003); the weevils need served in the grass carp after hydrilla was treated with diquat dry sites with good duff to overwinter successfully (Newman et or fluridone, suggesting the plant’s food quality or palatability al. 2001). Within a lake, weevil densities appear highest in was altered by exposure to the herbicides (Kracko and Noble large beds of watermilfoil in shallower water (Jester et al. 1993). Center (1994) showed repeated herbicide treatments 2000, Johnson et al. 2000, Tamayo et al. 2000). Plants in deep- that eliminate or reduce the host plant can eliminate weed er water may be harder to find by adult weevils, are subjected biocontrol agents (see also Newman et al. 1998). Although a to wave action that may displace the insects, and also are more combination of herbicides and natural enemies often is sug- accessible to fish that feed on the insects (Newman 2004). gested as an integrated approach for managing aquatic weeds, more research is needed on a case-by-case basis to determine Other Control Practices the compatibility of these two methods.

Depending on the circumstances, mechanical harvesting BIOTIC FACTORS operations may disrupt or enhance the effectiveness of aquatic weed biological control agents. For example, the Host Quality density of the weevil E. lecontei was reduced significantly after its host plant Eurasian watermilfoil was subjected to harvest- Texture and nutrient content of aquatic plants are two of ing (Sheldon and O’Bryan 1996). Conversely, there is anec- the more critically studied aspects of host plant quality be- dotal evidence indicating that new shoot growth stimulated cause they directly affect palatability of the plants and con- by harvesting hydrilla in Crystal River, Florida, may increase sumption by the natural enemies. Variations observed in the

J. Aquat. Plant Manage. 46: 2008. 21

texture of aquatic plants can be due to interspecific, intraspe- (Goodson 1997). Conversely, H. balciunasi Bock, a related spe- cific, or induced differences. For instance, the grass carp will cies native to Australia (Deonier 1993), failed to become wide- preferentially consume hydrilla over other submersed aquat- ly established on dioecious or monoecious hydrilla in the U.S. ic plant species. Plant texture is cited as the primary reason (Grodowitz et al. 1997). The poor performance of H. balciu- for this preference (Sutton and Vandiver 1998). Apparently, nasi may be due to genetic differences between Australian and the soft tips of young tender hydrilla plants are more palat- U.S. hydrilla because survival and development of the leaf able to grass carp than other submersed plant species includ- miner was low on the U.S. strains of hydrilla when compared ing Brazilian egeria, hygrophila, and Eurasian watermilfoil. to the Australian biotype (Goodson 1997). The Australian bio- Likewise, growth and survival of the Asian leaf-mining fly H. type of hydrilla is genetically distinct from the Asian biotypes pakistanae and the Australian stem mining weevil B. hydrillae that are the source of the U.S. hydrilla (Madeira 1997). Bio- were enhanced on hydrilla plants with soft apical leaves or type mismatching could account for the inability of H. balciu- stems (Wheeler and Center 1996, 1997). nasi to become more widely established on the U.S. hydrilla. The role that plant nutrient content plays in the biologi- Competitive displacement by the better adapted H. pakistanae cal control of aquatic weeds has been examined extensively is another contributing factor (M. J. Grodowitz, pers. observ.). with insect natural enemies (Wheeler and Center 1996, New- Recently, Moody and Les (2002) suggested that newly dis- man et al. 1998, Grodowitz et al. 2004). Applying fertilizer of- covered hybrids of Eurasian and northern watermilfoil may ten increases the level of control by increasing host plant be more resistant to herbivory by the milfoil weevil. However, quality (Newman et al. 1998), but there may be a point of di- Roley and Newman (2006) recently found that weevil devel- minishing return. For instance, a study by Grodowitz et al. opment and size at eclosion were identical on all three taxa, (2004) suggests that increasing nutrient loads, especially ni- whereas juvenile survival was intermediate on the exotic Eur- trogen, may enhance the performance of two species of Hy- asian watermilfoil and the native northern watermilfoil hy- drellia flies that attack hydrilla. However, in an earlier tank brid. Different weevil populations also perform better on study that examined the effects of a single generation of the different plants (Tamayo and Grue 2004), suggesting an in- Asian leaf-mining fly H. pakistanae on hydrilla, Wheeler and teraction between weevil and plant gentoypes. The discovery Center (2001) showed that high densities of the leaf miner of hybrids and the identification of genetically distinct popu- reduced hydrilla biomass in plants subjected to a low fertiliz- lations suggest that more consideration of both agent and er treatment but not in plants grown under high fertilizer plant genotype is warranted. conditions. Under higher nutrient conditions, the plants outgrew the leaf miner damage. Carbohydrate Reserves In a recent greenhouse study, Shearer et al. (2007) showed that the nutritional quality of a target weed also may Knowledge of the phenology of an aquatic weed’s carbo- influence the performance of fungal pathogens used as weed hydrate allocation pattern may improve the effectiveness of biological control agents. For example, hydrilla shoots ob- biological control (Madsen and Owens 1998, Fox et al. tained from plants grown in high-nutrient sediment and ex- 2002). Presumably, an aquatic weed would be most vulnera- posed to the fungal pathogen M. terrestris were impacted ble to attack by a biological control agent when its total non- more by the fungus than shoots from low-fertility sediments. structural carbohydrate (TNC) reserves are at their lowest. In other words, the plant’s ability to regrow and recover from Genotypes the herbivore’s damage is dependent upon the stored TNCs (Madsen 1991). The TNC levels for the submersed aquatic Previous research has demonstrated that matching the cor- weed hydrilla are at their lowest in June or July (Madsen and rect biotype of a natural enemy with the variety or strain of the Owens 1998). However, the phenology of the hydrilla leaf- weed on which it evolved usually increases the likelihood the mining fly H. pakistanae in north central Florida indicates organism will establish and be an effective biological control that high larval populations do not occur in mid-summer agent in the weed’s introduced range (Harley and Forno when hydrilla would be most susceptible to herbivore dam- 1992). Retrospective studies of the origins of hydrilla and the age (Cuda et al. 1997, Wheeler and Center 2001). The mis- performance of the two Hydrellia spp. released for control of match between the phenology of hydrilla’s TNC reserves and this highly variable submersed aquatic weed in the U.S. sup- larval populations of H. pakistanae could explain the fly’s ap- port the “biotype matching concept.” Using random amplified parent ineffectiveness in controlling hydrilla. However, her- polymorphic DNA (RAPD) analyses, Madeira et al. (1997, bivory by the weevil E. lecontei reduces the TNC stored in the 2004) were able to determine that U.S. accessions of dioecious roots of Eurasian watermilfoil and probably lowers the and monoecious hydrilla are genetically similar to hydrilla plant’s overwintering survival and competitive ability (New- plants from southern India and Korea, respectively. Hydrellia man et al. 1996, Newman and Biesboer 2000). pakistanae is native to the same geographical region (Deonier 1993), and this natural enemy successfully established on the Biocontrol Agent Density U.S. dioecious hydrilla (Center et al. 1997b). From laboratory studies, the U.S. monoecious biotype of hydrilla also appears Successful biological control of a target weed is a function to be a suitable host plant for H. pakistanae (Dray and Center of the natural enemy’s capacity to reproduce on individual 1996, Goodson 1997). These findings suggest H. pakistanae is plants, and to increase in abundance to critically damage a capable of expressing its full reproductive potential on multi- plant population (Gassmann 1996, Julien 1997). However, ple biotypes of hydrilla that occur within its native range high population densities of an herbivore will not necessarily

22 J. Aquat. Plant Manage. 46: 2008. guarantee success. Effective biological control may only occur Recently, the proteobacterium Wolbachia was isolated from when the weed is being stressed concurrently by local climatic H. pakistanae and the native parasitic wasp T. columbiana that conditions (Vogt et al. 1992, Cilliers and Hill 1996, Bellows attacks it in the U.S. (Jeyaprakash and Hoy 2000). Wolbachia and Headrick 1999), competing plants (Sheppard 1996, New- are vertically transmitted reproductive parasites that can in- man et al. 1998, Van et al. 1998) or other natural enemies. duce cytoplasmic incompatibility, parthenogenesis, feminiza- Another aspect of biological control agent density is self- tion, or male killing in their hosts, and can be detrimental to evident. When biological control agents establish and in- the host’s reproductive success (O’Neill et al. 1997). The ori- crease in abundance in one geographical region, they are gin of the Wolbachia and its effects on reproduction in H. pa- likely to attain this same level of abundance after they are in- kistanae and T. columbiana are unknown. Further studies are troduced into ecoclimatically similar geographical regions needed to determine the implications of the Wolbachia infec- (Maywald and Sutherst 1997, Byrne et al. 2004). High popu- tion on the population dynamics of the introduced hydrilla lation densities of a weed biocontrol agent often observed biological control agent and its native parasitoid. soon after its release are usually attributed to an unlimited Newman et al. (2001) concluded that parasitoids (not food supply and the absence of coevolved parasites and pred- found) and pathogens were not important limiting factors ators in the new environment (Gassmann 1996, Keane and for the milfoil weevil. However, microsporidians and gre- Crawley 2002). This ecological concept is known as the ene- garines were found at low levels, and the fungus Beauvaria my release hypothesis (ERH) and is fundamental to classical bassiana (Balsamo) Vuillemin infected overwintering adults weed biological control (Williams 1954). The ERH is largely in the laboratory. Clearly, more work on the effects of parasi- responsible for the numerous examples of successful biologi- toids and pathogens on biocontrol agents is warranted. cal control of giant salvinia, water hyacinth and water lettuce Although difficult to document, predation by birds or fish in various tropical and subtropical regions worldwide (Julien is probably another important factor limiting the effective- and Griffiths 1998). ness of some insect biological control agents of aquatic weeds. For example, the adventive Asian hydrilla moth PREDATION, PARASITISM, AND DISEASES (Parapoynx diminutalis [Snellen]) severely damages cultivated hydrilla (Buckingham and Bennett 1996), but its effective- Although predators, parasitoids and diseases have been ness as a biological control agent in the field is limited due to identified as important factors contributing to the failure of predation, presumably by fish (Buckingham 1994, Center et biological weed control (Stiling 1993, Cullen 1995), the ef- al. 2002). Coots (Fulica americana [Gmelin]) and moorhens fect of native parasitoids on weed biocontrol agents may be (Gallinula chloropus L.) initially prevented or interfered with more important than previously thought (Hill and Hulley the establishment of H. pakistanae at several release sites by 1995, McFadyen and Jacob 2004). Great care is taken to en- selectively feeding on infested hydrilla placed at the sites sure candidate weed biological control agents are released (Center et al. 1997b). In Minnesota, predation by sunfish without their co-evolved natural enemies, but there is no way (Lepomis spp.) was identified as an important source of mor- to prevent local natural enemies from exploiting this new re- tality for the weevil E. lecontei, a natural enemy of Eurasian source. Herbivores introduced into new geographical re- watermilfoil (Sutter and Newman 1997, Ward and Newman gions as weed biocontrol agents often become prey items for 2006). Lakes with high densities of sunfish likely will not sup- resident natural enemies, usually generalist parasitoids or port adequate densities of control agents (Ward and New- predators (Cornell and Hawkins 1993). man 2006). Newman (2004) suggested that predation by fish Parasitism by the semi-aquatic wasp Trichopria columbiana may be one reason for the comparatively lower success rate (Ashmead), a pupal endoparasitoid of native Hydrellia spp., of biological control of submersed plants compared to the may be reducing the effectiveness of the Asian hydrilla leaf dramatic success on floating and emergent plants, where the miner H. pakistanae (Coon 2000, Wheeler and Center 2001, biocontrol agents would be immune to fish predation. Doyle et al. 2002), and preventing widespread establishment of the Australian leaf miner H. balciunasi in the U.S. (Gro- TECHNICAL FACTORS dowitz et al. 1997). This parasitic wasp has been recovered from established populations of H. pakistanae in Alabama Establishing approved natural enemies on the target weed (Grodowitz et al. 1997), Florida (Cuda et al. 1997, Wheeler is a critical step in classical weed biological control programs. and Center 2001), and Texas (Doyle et al. 2002). Laboratory Clearly, natural enemies must establish in the new environ- studies have confirmed that T. columbiana will effectively par- ment for the project to succeed. Establishment of newly re- asitize H. balciunasi (Coon 2000). Furthermore, predation by leased biocontrol agents depends not only on the damselflies was suspected of reducing populations of H. pa- aforementioned environmental factors that are beyond the kistanae at release sites in Florida (Center et al. 1997b). An investigator’s control, but also on technical aspects of the autoradiographic study was conducted by Cuda et al. (1997) project that can be influenced by the researcher, such as se- to examine field predation on H. pakistanae. Labelling larvae lection of release sites, release strategies, and the timing and of H. pakistanae with the radioactive isotope 35S confirmed size of releases (Buckingham 1994, Julien 1997, Center and that aquatic naiads (immature stages, or nymphs) of the in- Pratt 2004, Coombs 2004). sect Order Odonata (dragonflies and damselflies) are vora- Climate matching should be given a high priority when cious predators of larval H. pakistanae, whereas mosquitofish planning releases (Buckingham 1994, Julien 1997). Natural Gambusia sp. apparently do not feed on the larvae (Cuda et enemies preadapted to the climatic conditions in the release al. 1997). area will have a better chance of surviving in the new envi-

J. Aquat. Plant Manage. 46: 2008. 23 ronment. Computer modelling programs such as CLIMEX, Other studies have shown that fungal pathogens can en- DYMEX (Maywald et al. 2000), and GARP (Stockwell and Pe- hance the effectiveness of herbicides applied at lower than ters 1999) are valuable tools for selecting release sites. These the recommended rates. For instance, the microbial patho- modelling packages use available meteorological and/or life gen M. terrestris enhanced the performance of two herbicides table data to predict an organism’s global geographic distri- commonly used to control hydrilla. In both laboratory tests bution and population dynamics relative to climate. For ex- and small-scale field trials, integrating low doses of fluridone ample, the CLIMEX model was used to predict locations in or endothall with the pathogen increased the susceptibility Asia, Australia, Africa, and Europe where the South Ameri- of hydrilla to otherwise sublethal doses of these herbicides can alligatorweed flea beetle is most likely to establish and (Netherland and Shearer 1996, Nelson et al. 1998, Shearer successfully control alligatorweed (Julien et al. 1995). and Nelson 2002). Similar results were obtained when the fungus was combined with 2, 4-D for the control of Eurasian Release Strategies watermilfoil (Nelson and Shearer 2005). Shabana et al. (1998, 2003) explored the effects of com- In the southeastern United States, the successful establish- bined attacks by fungal pathogens and an insect biocontrol ment of the Asian hydrilla leaf-mining fly H. pakistanae was agent on hydrilla. They found that isolates of Botrytis sp., thought to be directly related to the type of release (caged vs. Cephalosporium sp., Fusarium culmorum, and an unidentified open) and the stage of the insect released (Center et al. fungus collected from hydrilla shoots or from soil and water 1997b, Center and Pratt 2004). For example, in the early surrounding hydrilla in ponds and lakes in Florida, were ca- stages of the project, open releases of small numbers of eggs pable of killing hydrilla in a test-tube bioassay. Fusarium cul- failed to establish persistent populations of H. pakistanae. morum, the most effective isolate, was examined further in an However, establishment of the insect eventually succeeded aquarium test. The interaction of F. culmorum and H. paki- when the release protocol was modified to include the use of stanae resulted in a higher level of damage on hydrilla shoots cages for releasing large numbers of late instar larvae. Appar- than either organism alone (Shabana et al. 2003). Maximum ently, the eggs and first instar larvae of H. pakistanae suffer shoot kill was achieved at 20 to 30 C compared to 15 or 35 C. higher intrinsic mortality rates in comparison to later instars Thus, it may be possible to integrate fungal and insect natu- (Center et al. 1997b). ral enemies to control hydrilla. In terrestrial environments, the ability of a weed to recover Formulation Issues from the effects of herbivory diminishes as competition from neighboring plants increases (Van Driesche and Bellows The fungal pathogen M. terrestris is a promising inundative 1996, Bellows and Headrick 1999). The interaction between biological control agent for hydrilla (Shearer 1996, 1998). plant competition and a weed’s natural enemies often under- This native pathogen, which is undergoing development as a lies successful weed biological control (Sheppard 1996). Rec- bioherbicide (Shearer 1998, Shearer and Jackson 2003), ef- ognition of the importance of plant competition to biological fectively reduced the biomass of hydrilla in laboratory, glass- control success is creating a paradigm shift in the design of house and small-scale field trials. It was recently discovered terrestrial biological weed control programs (McEvoy and that the dilution and contact-time problems normally associ- Coombs 1999). By placing greater emphasis on manipulating ated with applying a bioherbicide in an aquatic system could bottom-up effects such as interspecific plant competition, be overcome by formulating the pathogen in an EPA ap- minimizing disturbance, and introducing fewer but more ef- proved biocarrier that adheres to the plant (Shearer 1998). fective natural enemies, the potential for nontarget damage However, when production was scaled-up to meet the re- can be minimized (McEvoy and Coombs 1999). This conser- quirements for field-testing the new formulation, unantici- vative approach has the added benefit of ensuring that intro- pated changes in the formulation of the carrier itself ductions of safe natural enemies will continue in the future. rendered the fungus ineffective (Shearer 1999b). Current In the aquatic environment, there is evidence that inter- work is focusing on resolving the formulation problems, in- specific competition from native plants may be equally im- cluding developing new formulations that will not only stick portant to the successful biological control of rooted to hydrilla but can be applied with conventional herbicide submersed weeds. For example, the results of outdoor tank equipment (J. F. Shearer, pers. comm.). studies conducted in Florida indicate that selective herbivory by the ephydrid fly H. pakistanae and the stem-mining weevil Integration of Control Tactics Bagous hydrillae O’Brien, two introduced natural enemies of hydrilla, shifts the competitive balance in favor of eelgrass In general, the prospects for integrating biological con- (Vallisneria americana [Michx.]), a commonly occurring na- trol agents with herbicides are excellent. Several studies have tive species frequently associated with hydrilla (Van et al. demonstrated the successful integration of herbicides with 1998). More recent mescosm experiments in Mississippi and insects for controlling floating aquatic plants (e.g., Center et Texas using typical densities of H. pakistanae show a reduc- al. 1999). Maintaining untreated refuge areas is important tion in both biomass and tuber production when herbivory is for sustaining sufficient densities of insect biocontrol agents combined with competition from eelgrass (Doyle et al. (Haag and Habeck 1991, Julien and Storrs 1996). Plant 2007). In another study in Minnesota, competition from na- growth retardants also may increase the effectiveness of some tive plant species appears to be an important factor contrib- insect biocontrol agents (Van and Center 1994). uting to the sustained biological control of Eurasian watermilfoil by the weevil E. lecontei (Newman et al. 1998).

24 J. Aquat. Plant Manage. 46: 2008. MANAGEMENT PRIORITIES tirely on the submersed weed should be identified early on in a project and dropped from further consideration. The submersed aquatic plants hydrilla, Eurasian watermil- Resources also are needed to screen plant pathogens col- foil and Brazilian egeria are widely recognized as three of the lected during the 1990s in Asia and Europe from both hydril- worst invasive aquatic weeds in the U.S., with millions of dol- la and Eurasian watermilfoil (Shearer 1997, Harvey and lars spent annually to control large infestations of these Varley 1996, Harvey and Evans 1997, Balciunas et al. 2002). plants in all types of waterbodies. Since 2000, several hydrilla These disease organisms, which are currently stored in a biotypes in Florida have developed resistance to the herbi- high security quarantine laboratory in Ft. Dietrick, Maryland, cide fluridone (Michel et al. 2004). As a result, interest in the may have potential to be used in inoculative releases or for submersed aquatic weed problem has increased dramatically development of new bioherbicides if they are found to be because of the herbicide resistance issue (Hoyer et al. 2005, sufficiently viable and host specific. Screening of the patho- Netherland et al. 2005). gen F. graminearum, recently discovered impacting Brazilian The discovery of fluridone resistance is cause for concern egeria in South America, should also be investigated and de- for several reasons. First, the resistance problem will make it veloped as soon as possible (Nachtigal and Pitelli 2000). Fi- difficult for aquatic plant managers in Florida to control hyd- nally, additional overseas surveys are needed to identify new, rilla in a cost-effective and selective manner. This can lead to currently undiscovered pathogens for all invasive submersed the eventual spread and establishment of resistant biotypes aquatic plants. throughout hydrilla’s introduced range. Secondly, fluridone is the only aquatic herbicide approved by the U.S. EPA for Taxonomic Expertise managing large infestations of the aforementioned sub- mersed aquatic weed species. However, the overuse of fluri- The loss of taxonomic expertise for natural enemies as done eventually could lead to similar resistance problems in well as their host plants must be addressed. Systematists high- Eurasian watermilfoil and Brazilian egeria. Finally, no other ly trained in traditional and molecular methods are needed registered herbicides are available with comparable environ- to better understand the genetic diversity of the weed targets mental, cost, and application characteristics to replace fluri- and their natural enemies. The need for continued advance- done (Hoyer et al. 2005). Therefore, biological control is a ment in taxonomic understanding is underscored by the suc- viable alternative because it is one of the few tactics currently cess of Cyrtobagous salviniae (originally misidentified) in available that is not only economical but can provide the controlling Salvina molesta (also originally misidentified; kind of selective control needed, without damaging nontar- Buckingham 1994, Forno and Julien 2000), the recent dis- get species or the environment. Furthermore, the public is covery of hybrid watermilfoils (Moody and Les 2002), and becoming increasingly more receptive to the development of the fact that many potential agents discovered in previous ex- effective nonchemical alternatives such as biological control ploratory surveys remain unidentified. Advances in taxonom- for managing aquatic weeds. ic expertise will facilitate the process of “biotype matching” by increasing the likelihood of selecting biological control Identification and Screening of New Classical agents highly adapted to a particular weed biotype, or possi- Biocontrol Agents bly a new hybrid.

Adequate long-term funding and agency commitment are Post-release Monitoring and Assessment needed to continue overseas surveys and screening of new natural enemies of hydrilla (e.g., Overholt and Cuda 2005), Effective monitoring is needed to evaluate new biological Eurasian watermilfoil, Brazilian egeria, and other widespread control projects to determine which agents are effective and invasive submersed plants such as parrotfeather (Cilliers what factors limit or enhance their success (Blossey and 1999) and curlyleaf pondweed (Potamogeton crispus L.). Pro- Skinner 2000, Forno and Julien 2000, Blossey 2004). Fund- grams targeting species of regional importance like water ing often has been limited to screening, introduction, estab- chestnut (Trapa natans L.; Pemberton 1999, 2002, Ding et al. lishment, and spread of agents, with little attention given to 2006a, b) and Indian swampweed (Hygrophila polysperma; Cu- quantifying their effectiveness or potential unanticipated ef- da and Sutton 2000) also will benefit by increased financial fects (McClay 1995, McEvoy and Coombs 1999, Kaslichuk et as well as agency support. Until recently, surveys for new bio- al. 2004). Monitoring programs are often underfunded or logical control agents of the aforementioned aquatic weeds inadequate in scope and do not identify where and why con- have been lacking, largely due to inadequate funding for for- trol is or is not successful (Blossey 2004). For example, de- eign exploration and screening. Overseas surveys should bates over the degree of success of biological control on adopt the scoring system recently proposed by Forno and water hyacinth and hydrilla in North America are largely due Julien (2000) for selecting and prioritizing new arthropods to poor monitoring after release. Development of objective as candidates for biological control of submersed aquatic evaluations of success also is needed (Forno and Julien 2000, weeds. This system can readily identify potentially effective Delfosse 2004). Toward this goal, monitoring should include natural enemies in the native range because greater impor- historical or untreated reference sites for comparisons, be tance is placed on objective criteria, such as the type of dam- long-term (>2 years), and evaluate the direct and indirect age caused by both adults and immatures, the duration of effects on target and nontarget plants as well as agent perfor- the attack, and number of generations annually. Candidate mance (Syrett et al. 2000, Blossey 2004). Monitoring also arthropods incapable of completing their development en- should take place at a regional scale to determine generality

J. Aquat. Plant Manage. 46: 2008. 25 of success and to further identify climatic and environmental or working with local correctional facilities and training in- factors that impact success. In addition to providing informa- mates to recognize/handle the biological control agents (Os- tion on the success or failure of specific projects or agents, borne 2005). Additional research on mass rearing of successful evaluation programs should identify the causes of different agents may result in effective inundative control or success or failure and development of better selection, re- better establishment of new populations. lease and management strategies (Syrett et al. 2000, Blossey 2004). Integration of Tactics Standardized procedures/techniques for post-release monitoring of biological control agents are essential to con- To gain greater acceptance by stakeholders and the gener- firm establishment and assess the effects on the target weed al public, more emphasis should be placed on integrating and the associated plant community (Forno and Julien 2000, biological control with other tactics (e.g., herbicides, revege- Blossey 2004). However, the growth habit of submersed tation). Research should be targeted at directly assessing in- aquatic weeds presents a challenge because it does not readi- tegrative approaches rather than ad hoc evaluation. For ly lend itself to sampling procedures normally used by terres- instance, addressing the fluridone resistance problem in hy- trial researchers to quantify biological control agent impacts. drilla will require additional resources for new research on Biological control researchers need to develop collaborative removal techniques for grass carp (Netherland et al. 2005), projects with aquatic plant ecologists and watershed manag- and combining lower stocking rates with a revegetation pro- ers who have the appropriate expertise to address this prob- gram may minimize adverse effects to native plant species. lem. Development of precise but efficient methods to assess This approach was proposed for Eurasian watermilfoil con- agent densities and their impacts on target plants and aquat- trol at Houghton Lake, Michigan (Getsinger et al. 2002), but ic plant community response, for both degree of control and was not adequately implemented. for nontarget impacts, will increase the likelihood of accu- More research is needed on revegetation as well as pro- rate assessment. Finally, to maintain the high standards of moting a positive native plant response in combination with the discipline, practitioners of classical biological control of compatible invasive plant control methods (e.g., biological aquatic weeds should adhere to the guidelines in the Inter- control) (Van et al. 1998, Doyle et al. 2007). Failure to incor- national Code of Best Practices for Classical Biological Con- porate this aspect into a management program may result in trol of Weeds (Balciunas 2000, Balciunas and Coombs 2004). reduced habitat, poor weed control, and/or replacement of one invasive with another (Newman et al. 1998, McEvoy and Further Assessment of Non-classical Biological Control Coombs 1999). Finally, funding should be made available so that industry can mass produce and market effective bioher- Continued work on the use of native and naturalized bicide products (e.g., M. terrestris). These commercially pro- agents is needed. For example, the native milfoil weevil E. le- duced bioherbicides could be used alone or in combination contei successfully controls Eurasian watermilfoil in some with lower concentrations of fluridone for controlling sus- lakes, but is limited by fish predation in others (Newman ceptible hydrilla. The combination could preserve the selec- 2004, Ward and Newman 2006). The extent and degree of tivity and cost effectiveness characteristics of this herbicide. this limitation, along with other limiting factors, is unknown even though the weevil is being stocked in more than 80 Foundational Research lakes (Maple 2006). Rigorous evaluation of these projects should result in more effective selection of lakes and more Greater emphasis should be placed on basic research in efficient use of resources. Testing of established hydrilla bio- support of biological control. For instance, studies on plant/ logical control agents, specifically the tip-mining midge Crio- biological control agent physiology, the influence of larval cotpus lebetis Sublette (Cuda et al. 2002) and the two Hydrellia and adult nutrition on reproduction, mechanisms of host lo- flies (Center et al. 1997b, Grodowitz et al. 1997) should be cation (Marko et al. 2005), and the effects of deleterious mi- completed as soon as possible to assess their developmental croorganisms (e.g., Wolbachia) on survival and reproduction and reproductive performance on the fluridone-resistant hy- are needed to gain insight into the factors impacting the ef- drilla biotypes. fectiveness of biological control agents. Foundational re- Trained personnel and funding are needed to conduct search on waterhyacinth, hydrilla and Eurasian watermilfoil statewide surveys to confirm the presence/absence of estab- has advanced our understanding of factors regulating suc- lished biological control agents of hydrilla, Eurasian water- cess in different systems and our ability to integrate biologi- milfoil, and other aquatic weeds outside their currently cal control with other management practices. Adoption and known range. Such surveys are essential because if a biologi- testing of new ecological niche models such as the Genetic cal agent is not present in a particular state, then additional Algorithm for Rule Set Production (GARP) can help to pre- host range testing of at-risk native plant species may be re- dict where biological control agents and their target weeds quired before the organism can be imported into that state. are likely to establish (Stockwell and Peters 1999). The GARP Mass rearing and release of large numbers of high quality model also could be used for early detection and rapid re- introduced and native biological control agents can lead to sponse to new weed problems before they reach the U.S. In- better control of the weed target in a shorter period of time, creasing the adoption of GARP would facilitate more rapid but can be an expensive enterprise (Grodowitz et al. 2004). biological control response by fostering collaboration with However, costs of rearing natural enemies can be reduced overseas researchers much earlier in those countries where dramatically by using outdoor ponds (Grodowitz et al. 2004) the plant is considered native.

26 J. Aquat. Plant Manage. 46: 2008. Outreach Balciunas, J. K., M. J. Grodowitz, A. F. Cofrancesco and J. F. Shearer. 2002. Hydrilla, pp. 91-114. In: R. G. Van Driesche, B. Blossey, M. Hoddle, S. Public education about the safety of biological control Lyon and R. Reardon (eds.). Biological control of invasive plants in the needs improvement (e.g., Scoles et al. 2005). At the same eastern United States. USDA Forest Service Publication FHTET-2002-04, Morgantown, WV. time, scientists need to continue developing innovative state- Balciunas, J. K. and E. M Coombs. 2004. International code of best practices of-the-art tools for biological control technology transfer for classical biological control of weeds, pp. 130-136. In: E. M. Coombs, (Grodowitz et al. 1996). For example, two computer-based J. K. Clark, G. L. Piper and A. F. Cofrancesco, Jr. (eds.). Biological con- information/expert systems have been developed and re- trol of invasive plants in the United States. Oregon State University Press, Corvallis. cently updated that contain information on biological con- Barreto, R. W. and H. C. Evans. 1996. Fungal pathogens of some Brazilian trol and other methods available for aquatic plant aquatic weeds and their potential use in biocontrol, pp. 121-126. In: V. C. management (Whitaker et al. 2004). These two training Moran and J. H. Hoffman (eds.). Proc. IX Int. Symp. Biol. Contr. Weeds. tools, including the Noxious and Nuisance Plant Manage- University of Cape Town, South Africa. ment Information System (PMIS) and the Aquatic Plant In- Barreto, R., R. Charudattan, A. Pomella and R. Hanada. 2000. Biological control of neotropical aquatic weeds with fungi. Crop Prot. 19:697-703. formation System (APIS), are easy to use, readily available, Bellows, T. S. and D. H. Headrick. 1999. Arthropods and vertebrates in bio- and will enable the general public to gain a greater apprecia- logical control of plants, pp. 505-516. In: T. S. Bellows, T. W. Fisher, L. E. tion for and acceptance of biological control technology. Caltagirone, D. L. Dahlsten, C. Huffaker, G. Gordh (eds.). Handbook of Other examples of successful outreach programs that pro- biological control. Academic Press, San Diego, CA. Bennett, C. A. and G. R. Buckingham. 1999. Biological control of hydrilla and vide training in biological control are the annual short cours- Eurasian watermilfoil- insect quarantine research, pp. 363-369. In: D. T. es held in California and Florida and teaching modules for Jones and B. W. Gamble (eds.). Proc. 1998 Joint Symp. Florida Exotic Pest primary and secondary schools developed by the University of Plant Council and Florida Native Plant Soc.: Florida’s garden of good and Florida’s, Institute of Food and Agricultural Sciences, Center evil. South Florida Water Management District, West Palm Beach, FL. for Aquatic and Invasive Plants (CAIPS 2005). Established in Bennett, C. A. and G. R. Buckingham. 2000. The herbivorous insect fauna of a submersed weed, Hydrilla verticillata (Alismatales: Hydrocharitaceae), 1978, CAIPS is a multi-disciplinary research, teaching and ex- pp. 307-313. In: N. R. Spencer (ed.). Proc. X Int. Symp. Biol. Contr. tension unit devoted to the study and management of fresh- Weeds. Montana State University, Bozeman. water aquatic and invasive plants. The Aquatic, Wetland and Blossey, B. and L. Skinner. 2000. Design and importance of post release Invasive Plant Information Retrieval System (APIRS) is the in- monitoring, pp. 693-706. In: N. R. Spencer (ed.). Proc. X Int Symp. Biol. Contr. Weeds, 4-10 July 1999. Montana State University, Bozeman. formation office for the Center. APIRS maintains the world’s Blossey, B. 2004. Monitoring in weed biological control programs, pp. 95- largest on-line aquatic and wetland plant research database, 105. In: E. M. Coombs, J. K. Clark, G. L. Piper and A. F. Cofrancesco, Jr. and produces a variety of educational materials relating to (eds.). Biological control of invasive plants in the United States. Oregon aquatic ecosystems. One of these is a companion website for State University Press, Corvallis. the general public, which was developed in collaboration with Buckingham, G. R. and E. A. Okrah. 1993. Biological and host range studies with two species of Hydrellia (Diptera: Ephyridae) that feed on hydrilla. the Florida Department of Environmental Protection, Bureau U.S. Army Engineer Waterways Experiment Station, Tech. Rept. A-93-7, of Invasive Plant Management. This website not only address- Vicksburg, MS. 58 pp. es biological control but also other aspects of aquatic plant Buckingham, G. R. 1994. Biological control of aquatic weeds, pp. 413-480. management. Finally, professional organizations such as the In: D. Rosen, F. D. Bennett and J. L. Capinera (eds.). Pest management in the subtropics: biological control—a Florida perspective. Intercept, Aquatic Plant Management Society (APMS 2006) and the Andover, U.K. Weed Science Society of America (WSSA 2005) should con- Buckingham, G. R. and C. A. Bennett. 1996. Laboratory biology of an immi- tinue producing educational materials to extend and develop grant Asian moth, Parapoynx diminutalis (Lepidoptera: Pyralidae), on public interest in biological control as the basis for integrated Hydrilla verticillata (Hydrocharitaceae). Fla. Entomol. 79:353-363. management of invasive aquatic plants. Buckingham, G. R. 1998. Surveys for insects that feed on Eurasian watermil- foil, Myriophyllum spicatum, and hydrilla, Hydrilla verticillata, in the People’s Republic of China, Japan, and Korea. U.S. Army Corps of Engineers LITERATURE CITED Waterways Experiment Station, Tech. Rept A-98-5, Vicksburg, MS. 36 pp. Byrne, M. J., J. Coetzee, A. J. McConnachie, W. Parasram and M. P. Hill. Allen, F. and T. D. Center. 1996. Reproduction and development of the bio- 2004. 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J. Aquat. Plant Manage. 46: 32-41 Improvements in the Use of Aquatic Herbicides and Establishment of Future Research Directions

KURT D. GETSINGER1, M. D. NETHERLAND2, C. E. GRUE3 AND T. J. KOSCHNICK4

ABSTRACT Emerging exotic weeds of regional concern such as egeria (Egeria densa Planch.), curlyleaf pondweed (Potamogeton crisp- Peer-reviewed literature over the past 20 years identifies us L.), and hygrophila (Hygrophila polysperma (Roxb.) T. significant changes and improvements in chemical control Anders), as well as native plants such as variable watermilfoil strategies used to manage nuisance submersed vegetation. (Myriophyllum heterophyllum Michx), and cabomba (Cabomba The invasive exotic plants hydrilla (Hydrilla verticillata L.f. caroliniana Gray) are invasive outside their home ranges. In Royle) and Eurasian watermilfoil (Myriophyllum spicatum L.) addition, there is always the threat of new plant introductions continue to spread and remain the plant species of greatest such as African elodea (Lagarosiphon major (Ridley) Moss) or concern for aquatic resource managers at the national scale. narrow-leaf anacharis (Egeria najas Planchon). The registra- tion of the bleaching herbicide fluridone in the mid 1980s for whole-lake and large-scale management stimulated numerous 1Research Biologist, Environmental Laboratory, U.S. Army Engineer lines of research involving reduction of use rates, plant selec- Research and Development Center, 3909 Halls Ferry Rd., Vicksburg, MS tivity, residue monitoring, and impacts on fisheries. In addi- 39180; [email protected]. tion to numerous advances, the specificity of fluridone for a 2Research Biologist, Environmental Laboratory, U.S. Army Engineer Research and Development Center, University of Florida Center for Aquatic single plant enzyme led to the first documented case of herbi- and Invasive Plants, Gainseville, FL 32653. cide resistance in aquatic plant management. The resistance 3U.S. Geological Survey, Associate Professor and Leader, Washington of hydrilla to fluridone has stimulated a renewed interest by Cooperative Fish and Wildlife Research Unit, School of Aquatic and Fisher- industry and others in the registration of alternative modes ies Sciences, University of Washington, Seattle, WA 98195. 4Aquatics Research Manager, SePRO Corp., Carmel, IN 46032. Received of action for aquatic use. These newer chemistries tend to be for publication June 1, 2007 and in revised form Oct 8, 2007. enzyme-specific compounds with favorable non-target toxicity

32 J. Aquat. Plant Manage. 46: 2008. BioControl DOI 10.1007/s10526-011-9418-y

Role of molecular genetics in identifying ‘fine tuned’ natural enemies of the invasive Brazilian peppertree, Schinus terebinthifolius: a review

J. P. Cuda • L. R. Christ • V. Manrique • W. A. Overholt • G. S. Wheeler • D. A. Williams

Received: 28 April 2011 / Accepted: 13 October 2011 Ó International Organization for Biological Control (IOBC) 2011

Abstract Brazilian peppertree, Schinus terebinthifo- host plant genotypes. The Brazilian peppertree model lius Raddi (Sapindales: Anacardiaceae), is a highly reviewed here could provide a useful framework for successful invasive species in the continental United studying biological control agents on other invasive States, Hawaiian archipelago, several Caribbean weed species that have exhibited intraspecific Islands, Australia, Bermuda, and a number of other hybridization. countries worldwide. It also is one of only a few invasive intraspecific hybrids that has been well Keywords Intraspecific hybridization Á Host-plant characterized genetically. The natural enemy complex genotypes Á Biological control Á Local adaptation Á of Brazilian peppertree includes two thrips and two Pseudophilothrips ichini Á Pseudophilothrips psyllids that appear to be highly adapted to specific gandolfoi Á Calophya terebinthifolii Á Calophya haplotypes or their hybrids. Successful biological latiforceps Á Thysanoptera: Phlaeothripidae Á control of Brazilian peppertree will require careful Hemiptera: Calophyidae Á Sapindales: Anacardiaceae matching of the appropriate natural enemies with their

Introduction Handling Editor: Ted Douglas Non-native plants often become invasive when intro- J. P. Cuda (&) Á L. R. Christ Entomology and Nematology Department, University duced outside their native ranges (Pimentel 2002). of Florida, Bldg. 970, Natural Area Drive, There are a number of hypotheses that have been PO Box 110620, Gainesville, FL 32611-0620, USA proposed to explain invasion success including pre- e-mail: jcuda@ufl.edu adaptation (Baker 1965; Parker et al. 2003; Richards V. Manrique Á W. A. Overholt et al. 2006), escape from natural enemies (Williams Biological Control Research and Containment 1954; Keane and Crawley 2002), propagule pressure Laboratory, University of Florida, 2199 South Rock Road, (Williamson 1996), empty niches (Elton 1958; Mac- Ft. Pierce, FL 34945, USA Arthur 1970), invasional meltdown (Simberloff and G. S. Wheeler Von Holle 1999), evolution of increased competitive USDA-ARS Invasive Plant Research Laboratory, ability (Blossey and No¨tzold 1995), novel weapons 3225 College Ave., Fort Lauderdale, FL 33314, USA (Callaway and Ridenour 2004), and diversity-invisi- bility (Elton 1958). Recently, there is an increasing D. A. Williams Department of Biology, Texas Christian University, emphasis on post-introduction evolution as an impor- 2800 S. University Dr., Fort Worth, TX 76129, USA tant determinant of invasion success (Sakai et al. 2001; 123 J. P. Cuda et al.

Lee 2002; Cox 2004; Prentis et al. 2008; Suarez and with marketing time (Pemberton and Liu 2009), and/ Tsutsui 2008). There is accumulating evidence that or may be a function of propagule pressure and invasive plants can undergo rapid adaptive evolution evolutionary change after introduction (Sakai et al. in their new range including the evolution of latitu- 2001). dinal or altitudinal clines (Maron et al. 2004, 2007; Historical records and genetic evidence indicate Keller et al. 2009), increased phenotypic plasticity that two genetic lineages of Brazilian peppertree were (Lavergne and Molofsky 2007), or other attributes that established in Florida, USA, one in Miami on the east improve colonization or competitiveness with native coast and a second near Punta Gorda on the west coast species (eg. Jain and Martins 1979; Prati and Bossdorf (Nerhling 1944; Morton 1978; Workman 1979). Since 2004; Kliber and Eckert 2005; Dlugosch and Parker arriving, the distributions of these two genotypes have 2008a, 2008b; Seifert et al. 2009, Xu et al. 2010). greatly expanded, and they have extensively hybrid- Hybridization between species or genetically ized (Williams et al. 2005, 2007). distinct populations of the same species can be an Brazilian peppertree was initially targeted for important factor leading to evolutionary change and biological control in Hawaii (Krauss 1963; Yoshioka successful invasions (Arnold 1997; Ellstrand and and Markin 1991), and later in Florida, USA (Bennett Schierenbeck 2000; Rieseberg et al. 2007; Schieren- et al. 1990; Cuda et al. 2006). Recent surveys for beck and Ellstrand 2009). For instance, hybridization natural enemies in Brazil resulted in the discovery of may produce novel genotypes that have a selective several insects, specifically thrips and psyllids, that are advantage in the introduced range. This could arise highly adapted to the two Brazilian peppertree geno- through producing traits that are intermediate between types and/or to their hybrids established in Florida the two parents, recombining parental traits, or by (Manrique et al. 2008; Christ 2010). More impor- producing transgressive (extreme) traits, some of tantly, natural enemies belonging to the thrips genus which might be favorable in the new environment Pseudophilothrips and the psyllid genus Calophya (Ellstrand and Schierenbeck 2000; Rieseberg et al. turned out to be a complex of cryptic species (Cuda 2007). Hybridization also may be particularly advan- et al. 2009; Mound et al. 2010; Burckhardt et al. 2011). tageous in exotic plant populations because there will In this review, the ecological significance of local be an initial lack of local adaptation. The benefits of adaptation of these natural enemies is discussed in the hybridization such as heterosis or the production of context of the different Brazilian peppertree popula- novel genotypes may then outweigh the cost of losing tions and their hybrids. gene combinations for local adaptation in the former native range (Verhoeven et al. 2011). There are now a number of examples of inter- and intra-specific Genetic structure of Brazilian peppertree hybridization that have led to invasiveness (Schieren- beck and Ellstrand 2009; Gaskin et al. 2009; Travis Both chloroplast sequence and nuclear microsatellite et al. 2010; Mukherjee et al., pers. comm.). data indicate there were two separate introductions Brazilian peppertree (Schinus terebinthifolius Rad- of Brazilian peppertree into Florida, one on the west di, Anacardiaceae) was introduced into Florida from coast (the A chloroplast haplotype) and one on the east South America in the 19th century as an ornamental coast (the B chloroplast haplotype) (Williams et al. plant (Morton 1978). Based on herbarium records and 2005, 2007). GIS mapping indicated that individuals available literature, it began to escape cultivation in with the highest ancestry were found close to the the 1950s, and is currently one of Florida’s most introduction sites and became progressively more invasive weeds (Schmitz et al. 1991; Cuda et al. 2004, admixed with increasing distance away from the 2006). The initial discovery of a naturalized popula- introduction sites (Williams et al. 2007). Although we tion of Brazilian peppertree in the Florida Keys do not know when the two lineages began hybridizing, 50–60 years after it was introduced confirms the long most Brazilian peppertrees in Florida are now hybrids lag period often exhibited by woody weeds before although there are still individuals in the Miami and they become invasive (Kowarik 1995). Lag periods Punta Gorda areas that retain a high proportion of for exponential growth and naturalization of horticul- ancestral DNA (ancestry coefficient, q [ 0.90) from tural plants like Brazilian peppertree can be correlated the original introductions (Williams et al. 2007). 123 Role of molecular genetics in identifying ‘fine tuned’ natural enemies

Brazilian peppertree has strong phylogeographic Miami haplotype B is northeastern Brazil (Williams, structure in its native range with most localities having unpublished). only one or several closely related chloroplast haplo- types differing by one to two mutational differences (Williams et al. 2005). A parsimony network revealed Genetic structure of natural enemies that the introduced haplotypes A and B were different from other haplotypes in Brazil (Fig. 1). Furthermore, Pseudophilothrips spp. haplotypes A and B are from allopatric populations, indicating that the Florida introductions are from An extensive investigation of the genetic structure two distinct regions in South America. The origin of of P. ichini and a recently described cryptic species the Punta Gorda haplotype A is southeastern Brazil Pseudophilothrips gandolfoi Mound, Wheeler and (Williams et al. 2005), whereas the origin of the Williams (Mound et al. 2010) was conducted using the mitochondrial cytochrome oxidase I gene (COI). Seven haplotypes were found in P. ichini from Bahia to Santa Catarina, Brazil, but only a single haplotype was found for the recently described P. gandolfoi (Fig. 2), which appears to be confined to more inland populations of Brazilian peppertree in the state of Parana´ (Mound et al. 2010). P. gandolfoi, previously referred to as Pseudophilothrips sp. near ichini (Manrique et al. 2008), is almost always associated with Brazilian peppertree populations characterized by haplotypes C and D and has very low survival on populations characterized by haplotype A from Brazil (Manrique et al. 2008).

Calophya spp.

To date, the genetic structure of only a small sample of C. terebinthifolii from Santa Catarina (n = 20) was investigated with the COI using methods similar to those reported in Mound et al. (2010). During a recent survey trip in March 2010, psyllids collected on Brazilian peppertree in northeastern Brazil (Salvador, Bahia) were identified as a new species, Calophya latiforceps Burckhardt, using both morphological and molecular characters (Burckhardt et al. 2011). After sequencing this new species (n = 4 individ- uals) at the COI gene and calculating the Kimura 2-parameter (K2P) genetic distance both between and within the two psyllid species Kimura (1980), the results indicated that all individuals tested from southern Brazil have identical mitochondrial COI haplotypes and that the four psyllids sequenced from Salvador only differ at 0.2–0.7% of their sequence. Fig. 1 CpDNA haplotype network of Schinus terebinthifolius However, C. latiforceps from Salvador, Bahia, was illustrating relationships between the different haplotypes. found to be genetically different from C. terebinthifolii Each connecting line indicates one nucleotide difference and unlabeled nodes are inferred intermediates. Figure redrawn from collected in southern Brazil with a 13.4% sequence Williams et al. (2005) divergence. The morphological and genetic evidence 123 J. P. Cuda et al.

Fig. 2 Relationships of Pseudophilothrips species inferred using the neighbor- joining method and K2P pairwise distances of mitochondrial COI sequences. Bootstrap values are shown next to the branches. The scale bar indicates the number of base substitutions per site. Figure from Mound et al. (2010); reprinted with permission

confirmed the two psyllids are distinct species peppertree in a laboratory study conducted in Brazil (Burckhardt et al. 2011). (Christ 2010). Calophya terebinthifolii performed significantly better on plants with haplotype A, which occur in Florida, than the other populations character- Performance of natural enemies on Brazilian ized by haplotypes O, D, K, and M (G-test, G = 7.63; peppertree genotypes P \ 0.01) (Christ 2010). There was over a 75% success rate when this psyllid was raised on its natal plants Pseudophilothrips spp. with haplotype A, a 20% success rate on plants with haplotype O, and 0% success on plants characterized Performance (survival, development time, and adult by haplotypes D, K, and M (Fig. 3). Because haplo- longevity) of P. ichini and P. gandolfoi has been types M and O are only one base-pair different from investigated in the laboratory (Manrique et al. 2008). haplotype K (D.A. Williams, unpublished data), these P. ichini was originally collected on Brazilian pep- data suggest that populations of C. terebinthifolii also pertrees in the city of Ouro Preto, Minas Gerais, Brazil are highly adapted to specific Brazilian peppertree in November 2007. Peppertrees in this region are haplotypes. The performance of the newly described characterized by haplotype A. In contrast, P. gandolfoi C. latiforceps from Salvador has not yet been tested on was collected on Brazilian peppertrees in the vicinity the different Brazilian peppertree haplotypes. of Curitiba, Parana´, Brazil, in January 2007. Pepper- trees here carry either haplotype C or D. Ouro Preto is located 830 km northeast of Curitiba. Conclusions The two Pseudophilothrips spp. differed in their ability to accept the Florida populations as their Most of Florida’s Brazilian peppertrees are the result host plants. For instance, P. gandolfoi exhibited low of intraspecific hybridization between two introduc- survival (0–4%) and short adult longevity (\ten days) tions (haplotypes A and B) from distinct source when reared on the original Florida populations regions in Brazil (Williams et al. 2005, 2007; characterized by haplotypes A and B, or their hybrids Mukherjee et al., pers. comm.). Common garden between the original invasive populations, whereas studies recently conducted in Florida suggest that higher survival (*50%) and longevity (*30 days) these hybrid individuals have higher growth rates were observed for P. ichini on these same haplotypes than the parental types, which may have facilitated (Manrique et al. 2008). the invasion (Geiger et al. 2011). Our research on the genetics and performance of two thrips and two Calophya spp. psyllid natural enemies of Brazilian peppertree has shown that these insects exhibit what Harley and Performance (% rearing success) of C. terebinthifolii Forno (1992) referred to as ‘fine tuned’ adaptation was investigated on five native haplotypes of Brazilian to specific populations and genotypes of their host 123 Role of molecular genetics in identifying ‘fine tuned’ natural enemies

80

70

60

50

40

30

20 Rearing Success (%)

10

0 AOKDM cpDNA BP Haplotype

Fig. 3 Performance of Calophya terebinthifolii on five different haplotypes of Brazilian peppertree. Data from Christ (2010). Sample sizes for haplotypes were A (n = 12), O (n = 5), K (n = 2), D (n = 2), and M (n = 1) plants. On-going studies have not yet revealed a References negative effect of hybridization per se on the perfor- mance of biological control agents. However, our Arnold ML (1997) Natural hybridization and evolution. Oxford studies indicate that it may be necessary to match University Press, Oxford Ayres DR, Ryan FJ, Grotkopp E, Bailey J, Gaskin J (2009) biocontrol agents with the specific Brazilian pepper- Tumbleweed (Salsola, section Kali) species and speciation tree geographic populations and/or their hybrids that in California. Biol Invasions 11:1175–1187 occur in the introduced range. Baker HG (1965) Characteristics and modes of origins of weeds. Successful invasive species often are introduced In: Baker HG, Stebbins GL (eds) The genetics of colo- nizing species. Academic Press, New York, pp 147–172 multiple times from distinct source regions in the Bennett FD, Crestana L, Habeck DH, Berti-Filho E (1990) native range, so intraspecific hybridization may be Brazilian peppertree—prospects for biological control. quite common (Dlugosch and Parker 2008a, 2008b; In: Delfosse ES (ed) Proceedings of the VII International Schierenbeck and Ellstrand 2009). Several studies also Symposium on biological control of weeds, pp 6–11 Blossey B, No¨tzold R (1995) Evolution of increased competitive have shown that some pathogens and herbivores can ability in invasive nonindigenous plants–a hypothesis. be adapted to specific genotypes or populations of J Ecol 83:887–889 their host plants (Hasan 1972; Karban 1989; Karban Burckhardt D, Cuda JP, Manrique V, Diaz R, Overholt WA, and Strauss 1994; Gaskin and Schaal 2002; Goolsby Williams DA, Christ LR, Vitorino MD (2011) Calophya latiforceps, a new species of jumping plant lice (Hemip- et al. 2006; Ayres et al. 2009; Gaskin and Kazmer tera: Calophyidae) associated with Schinus terebinthifo- 2009). Our studies on the Brazilian peppertree inva- lius (Anacardiaceae) in Brazil. Florida Entomol 94: sion in Florida and fine scale adaptation of some of 489–499 its natural enemies provide further evidence of this Callaway RM, Ridenour WM (2004) Novel weapons: invasive success and the evolution of increased competitive ability. phenomenon. Future biological control programs Front Ecol Environ 2:436–443 could benefit from population genetic studies on both Christ LR (2010) Biology, population growth, and feeding the invasive species and potential biological control preferences of Calophya terebinthifolii (Hempitera: Psy- agents, especially when the specific origin(s) of the llidae), a candidate for biological control of Brazilian Peppertree, Schinus terebinthifolius (Anacardiaceae). MS weed are in doubt or there is evidence of differential Thesis, University of Florida, USA attack by natural enemies. Cox GW (2004) Alien species and evolution: the evolutionary ecology of exotic plants, animals, microbes and interacting Acknowledgments We thank two anonymous reviewers for native species. Island Press, Washington DC, USA their comments on an earlier draft of this manuscript. These Cuda JP, Habeck DH, Hight SD, Medal JC, Pedrosa-Macedo JH projects were supported by grants from the Florida Department (2004) Brazilian peppertree, Schinus terebinthifolius: of Environmental Protection, South Florida Water Management Sumac family-Anacardiaceae. In: Coombs E, Clark J, Piper District, and Florida Exotic Pest Plant Council. G, Cofrancesco A (eds) Biological control of invasive 123 J. P. Cuda et al.

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Rieseberg LH, Kim SC, Randell RA, Whitney KD, Gross BL, Yoshioka ER, Markin GP (1991) Efforts of biological control Lexer C, Clay K (2007) Hybridization and the colonization of Christmas Berry Schinus terebinthifolius in Hawaii. In: of novel habitats by annual sunflowers. Genetica 129: Center T, Doren RF, Hofstetter RL, Myers RL, Whiteaker 149–165 LD (eds) Proceedings, Symposium of Exotic Pest Plants, Sakai AK, Allendorf FW, Holt JS, Lodge DM, Molofsky J, With 2–4 November 1988, Miami, Florida, pp 377–385 KA, Baughman S, Cabin RJ, Cohen JE, Ellstrand NC, McCauley DE, O’Neil P, Parker IM, Thompson JN, Weller Author Biographies SG (2001) The population biology of invasive species. Ann Rev Ecol Syst 32:305–332 Schierenbeck K, Ellstrand N (2009) Hybridization and the J. P. Cuda received his PhD in entomology from Texas A&M evolution of invasiveness in plants and other organisms. 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123 BIOLOGICAL CONTROLÑWEEDS Fundamental Host Range of Pseudophilothrips ichini s.l. (Thysanoptera: Phlaeothripidae): A Candidate Biological Control Agent of Schinus terebinthifolius (Sapindales: Anacardiaceae) in the United States

1,2 1 1 1 3 J. P. CUDA, J. C. MEDAL, J. L. GILLMORE, D. H. HABECK, AND J. H. PEDROSA-MACEDO

Environ. Entomol. 38(6): 1642Ð1652 (2009) ABSTRACT Schinus terebinthifolius Raddi (Sapindales: Anacardiaceae) is a non-native perennial woody plant that is one of the most invasive weeds in Florida, Hawaii, and more recently California and Texas. This plant was introduced into Florida from South America as a landscape ornamental in the late 19th century, eventually escaped cultivation, and now dominates entire ecosystems in south-central Florida. Recent DNA studies have conÞrmed two separate introductions of S. terebin- thifolius in Florida, and there is evidence of hybridization. A thrips, Pseudophilothrips ichini s.l. (Hood) (Thysanoptera: Phlaeothripidae), is commonly found attacking shoots and ßowers of S. terebinthifolius in Brazil. Immatures and occasionally adults form large aggregations on young terminal growth (stems and leaves) of the plant. Feeding damage by P. ichini s.l. frequently kills new shoots, which reduces vigor and restricts growth of S. terebinthifolius. Greenhouse and laboratory host range tests with 46 plant species in 18 families and 10 orders were conducted in Parana´, Brazil, and Florida. Results of no-choice, paired-choice, and multiple-choice tests indicated that P. ichini s.l. is capable of reproducing only on S. terebinthifolius and possibly Schinus molle L., an ornamental introduced into California from Peru that has escaped cultivation and is considered invasive. Our results showed that P. ichini s.l. posed minimal risk to mature S. molle plants or the Florida native Metopium toxiferum L. Krug and Urb. In May 2007, the federal interagency Technical Advisory Group for Biological Control Agents of Weeds (TAG) concluded P. ichini s.l. was sufÞciently host speciÞc to recommend its release from quarantine.

KEY WORDS Brazilian peppertree, Christmas berry, natural enemy, risk assessment

Schinus terebinthifolius Raddi (Sapindales: Anacardi- In Florida, S. terebinthifolius is listed as a noxious aceae), commonly known as Brazilian peppertree or weed (FLDACS 1999), a prohibited plant (FLDEP Christmasberry in the United States and aroeira in 1993), and a Category I invasive species by the Florida Brazil, is an invasive weed that is threatening biodi- Exotic Pest Plant Council (FLEPPC 2007). The plant versity in Florida (Austin 1978; Loope and Dunevitz is widely naturalized in central and south Florida, as 1981a, b), California (Randall 2000), Hawaii (Hight et well as further north along coastal regions where more al. 2003), Texas (Gonzalez and Christoffersen 2006), moderate air temperatures favor its growth (Anony- and the Bahama Islands (Hammerton 2003). This pe- mous 2007a, b; Wunderlin and Hansen 2008). Recent rennial shrub/tree is native to Argentina, Brazil, and estimates based on aerial surveys indicate that Ϸ3,000 Paraguay (Barkley 1944, 1957). Historical accounts km2 of all terrestrial ecosystems in central and south indicate that S. terebinthifolius was introduced into Florida have been invaded by this invasive weed Florida from Brazil as an ornamental around 1898Ð (Cuda et al. 2006). During FY 2005Ð2006, the state of 1900 (Morton 1978, Workman 1979, Mack 1991); once Florida spent more than $40 million controlling on the west coast in Charlotte Co. (Punta Gorda) and Ͼ2,000 ha of S. terebinthifolius on state lands (FLDEP, once on the east coast in Miami-Dade Co. (Miami) BIPM 2006). (Williams et al. 2005). Although the plant was com- The invasiveness of S. terebinthifolius is attributed to mon in cultivation, it was a rare component of the ßora its enormous reproductive potential. Large quantities in south Florida until the late 1950s, when the Þrst of fruits (or drupes) are produced per plant, and naturalized plants were discovered on Big Pine Key, wildlife disperse seeds in their droppings (Morton Monroe County (Austin and Smith 1998). 1978, Toops 1979). S. terebinthifolius outcompetes na- tive plants because of its tolerance to conditions of extreme moisture (Ewe and Sternberg 2002, 2003) and 1 Department of Entomology & Nematology, University of Florida, salinity (Mytinger and Williamson 1987), its capacity Bldg. 970, Natural Area Drive, Gainesville, FL 32611. 2 Corresponding author, e-mail: jcuda@uß.edu. to grow in shady environments (Ewel 1978), and 3 Federal University of Parana´, Curitiba, Parana´ 80035-010, Brazil. allelopathic effects on neighboring plants (Gogue et

0046-225X/09/1642Ð1652$04.00/0 ᭧ 2009 Entomological Society of America December 2009 CUDA ET AL.: HOST RANGE OF BRAZILIAN PEPPERTREE THRIPS P. ichini S.L. 1643 al. 1974, Morgan and Overholt 2005, Donnelly et al. are ovoid in shape and golden in color (length, Ϸ0.45 2008). In Florida, the plant readily invades disturbed mm; width, Ϸ0.18 mm), and the chorion is sculptured sites (e.g., fallow farmlands) as well as natural com- with irregular hexagons. Neonates hatch in 7Ð8 d at munities such as pinelands, hardwood hammocks, and 24ЊC. After hatching, immature thrips undergo two mangrove forests (Cuda et al. 2006), and is a major instars that are the active feeding/damaging stages of invader of Everglades National Park (Ewel et al. 1982). this insect. First and second instars last 6 and 11Ð12 d, It also serves as a host for other invasive species, respectively. The wingless larvae exhibit polymor- including redbanded thrips, Selenothrips rubrocinctus phism; they are mostly red but occasionally orange or Giard. (Morton 1978), the root weevil Diaprepes ab- yellow in color. As soon as the larval feeding phase is breviatus L. (McCoy et al. 2003), and black spiny- completed on the host plant, the remainder of the life tailed iguana, Ctenosaura similis Gray (Jackson and cycle occurs on or in the soil to a depth of Ϸ0.5 cm. P. Jackson 2007). Recently, pollen from S. terebinthifolius ichini s.l. and other thrips belonging to the family was discovered clogging the drain tube in a solid Phlaeothripidae are unique in that they undergo three rocket booster of one of NASAÕs space shuttles (Jarzen nonfeeding pupal stages (the propupa, pupa I, and and Nelson 2008). pupa II) instead of two (Mound and Marullo 1996). Schinus terebinthifolius is naturalized in Ͼ20 coun- These nonfeeding pupal stages each require Ϸ8dto tries worldwide, including American Samoa, Australia, complete their development. Adults of P. ichini s.l. are Bermuda, Fiji, Island of Mauritius, Micronesia, New black, winged, and relatively small (2Ð3 mm in length). Caledonia, Reunion Island, South Africa, and Tahiti. In Males are generally smaller than females (Hood 1949). the Caribbean region, the plant occurs in the Bahamas, After transformation to the adult stage, females un- Commonwealth of Puerto Rico and U.S. Virgin Islands dergo a 5- to 15-d preoviposition period and can ovi- (Ewel et al. 1982, Habeck 1995, USDA NRCS 2008). It posit up to 220 eggs during their lifetime (45Ð78 d). was Þrst identiÞed as a suitable target for introductory Mating is not required to produce offspring; P. ichini or classical biological control in Florida in 1978 (Del- s.l. can reproduce parthenogenetically through arrhe- fosse 1979). Biological control is an appropriate man- notoky. In Brazil, adults overwinter on the underside agement tactic because the invasive characteristics of S. terebinthifolius leaßets. Under laboratory condi- exhibited by this weed are consistent with increased tions, life cycle from egg to egg was completed in 76 d resource availability and release from natural enemies at 18ЊCand38dat24ЊC. Females survived for Ϸ 8d in the invaded areas (Williams 1954, Blumenthal et al. at 23.1ЊC, and up to four generations per year have 2009). been observed in Brazil. Several natural enemies are being studied as can- Other plants associated with S. terebinthifolius were didates for biological control of S. terebinthifolius examined by Garcia (1977) for the presence of P. (Cuda et al. 2004, 2006; McKay et al. 2009). One of ichini s.l., including local species in the genera of these is a shoot and ßower attacking thrips Pseudophi- Baccharis L., Baccharidastrum Cabrera, Lantana L., lothrips ichini (Hood) s.l. (Thysanoptera: Phlaeothri- Solidago L., Vernonia Shreb., and Ocotea Aubl., but the pidae) (Johansen 1981, Mound and Marullo 1996). thrips was not found attacking these plants. Because Hood (1949), who described the species in the genus this insect was associated only with S. terebinthifolius Liothrips, listed “Ichinus terebinthifolius Raddi” as the in Þeld surveys, Garcia (1977) suggested that P. ichini host plant of the insect. Later, Silva et al. (1968) s.l. may be a good candidate for biological control of corrected the misspelling of the genus name and listed this invasive weed. S. terebinthifolius as the only host plant for P. ichini s.l. In this study, we report results of fundamental host According to Hood (1949), P. ichini s.l. is native to range studies on P. ichini s.l. The Þndings reported southeastern Brazil. Type locality for the taxon used in herein convinced the U.S. federal interagency Tech- the species description is Jacarepagua, which is lo- nical Advisory Group (TAG) for Biological Control cated in Rio de Janeiro state. Garcia (1977) studied the Agents of Weeds to recommend Þeld release of P. biology, ecology, and Þeld host range of P. ichini s.l. ichini s.l. in Florida in May 2007. south of Rio de Janeiro in Parana´ State (Curitiba municipality). Recently, the native range of P. ichini Materials and Methods s.l. was found to encompass the entire east coast of Brazil from Pernambuco state in the north to Rio Thrips Collections. Larvae and adults of P. ichini s.l. Grande do Sul in the south. New morphological and were Þeld collected on S. terebinthifolius in southeast- molecular data obtained from thrips samples collected ern Brazil from June 1994 to January 2007. Excised in this region indicate the existence of perhaps three shoot tips with adults and larvae were transferred to distinct taxa (Manrique et al. 2008, W. A. Overholt, 185-ml (50-dram) plastic snap cap vials with small personal communication)Ñhence the designation “P. holes punched in the lids and transported under per- ichini s.l.” mit (hand carried) to Florida. A small piece of Kim- The life cycle of P. ichini s.l. was described in Por- wipe (Kimberly-Clark, Neenah, WI) was sandwiched tuguese by Garcia (1977) in his MS thesis but never between the lid and the vial of insects to prevent published. In early spring (September in Brazil), fe- buildup of excess moisture inside the container. On males begin laying eggs singly or in small groups on the arrival into the Florida Biological Control Laboratory underside of the winged petioles and leaßets of S. (FBCL) containment facility, Gainesville, FL, adults terebinthifolius or on new tender shoot growth. Eggs of P. ichini s.l. were transferred individually with a 1644 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 6 small brush to S. terebinthifolius plants in a maximum ilies of the Order Sapindales that are native to the security room. Plants were originally collected in Caribbean archipelago (4). south Florida, propagated in 3.8-liter (1 gal) nursery Experimental Design. The “reverse order se- pots, and maintained in an outdoor nursery at the quence” method proposed by Wapshere (1989) was Entomology and Nematology Department, University used to delineate the fundamental host range of P. of Florida, Gainesville, FL. Potted plants were trans- ichini s.l. Larval tests were conducted initially under ported to the FBCL 24Ð48 h before the arrival of thrips no-choice conditions, which were followed by adult shipments from Brazil. A clear acrylic cylinder (14 cm oviposition tests under close conÞnement (no-choice) diameter by 45 cm height) with six screened ventila- and Þnally loose conÞnement (paired and multiple tion holes was used to conÞne the adult thrips on the choice). The value of this approach is that some plants potted plants for oviposition. Details on rearing pro- capable of supporting development in the more con- cedures were reported by Cuda et al. (2008). servative larval no-choice tests may be unacceptable Field-collected larvae of P. ichini s.l. were allowed to females in the no-choice, paired-choice, and mul- to complete their development on the excised shoot tiple-choice oviposition tests. Similarly, plants se- tips after the plant material was transferred to a clean lected for oviposition may be unsuitable for larval plastic vial. New shoot tip cuttings obtained from the development. nursery grown S. terebinthifolius plants were added to Host range tests were conducted initially at the the vial, as needed, so that larvae always had access to FBCL and at the Universidade Federal do Parana´, fresh host plant material. After the larvae completed Curitiba, Brazil. Only no-choice tests (larval feeding) their development in the vials, they were transferred were conducted in these earlier trials. No-choice, as adults to potted plants for colony production. paired-choice, and multiple-choice tests (oviposition Voucher specimens of P. ichini s.l. and associated nat- and larval development) were conducted exclusively ural enemies were deposited in the Florida Depart- at the FBCL. Finally, a second series of no-choice tests ment of Agriculture and Consumer Services State Col- (oviposition and larval development) was conducted lection of Arthropods, Gainesville, FL. The identity of at the FBCL because of the potential for migration of P. ichini s.l. was conÞrmed by specialists who prepared weed biological control agents from the Caribbean and examined slide-mounted specimens. into Florida (Pemberton 1995) and vice versa (Pratt et al. 2006, 2008). These supplemental tests included In total, 48 shipments of adults and larvae of P. ichini primarily nontarget species in the Anacardiaceae na- s.l. from Brazil were processed in the FBCL from June tive to the Caribbean, as well as other plants in the 1994 to January 2007. Thrips collections (total num- Order Sapindales closely related phylogenetically to ber) were made in the municipalities of Curitiba (37), the family Anacardiaceae that were not in the original Juruqui (4), and Juveve (1) in Parana state; Tangua in ´ ´ ´ test plant list. All host range tests conducted in Brazil Rio de Janeiro state (4); Tabao in Mato Grosso state ˜ and in Florida included an equal number of S. tere- (1); and Vicosa in Minas Gerais state (1). binthifolius plants as the positive control. Test Plant List. At the beginning of the project, the No-Choice Larval Survival Test (Brazil). This test test plant list (Table 1) was developed using the cen- was conducted from March 1995 to June 1996 in a trifugal phylogenetic method (Wapshere 1974). Later, laboratory greenhouse with either 30 or 50 neonates the list was modiÞed by incorporating families and placed on caged potted plants (n ϭ 3 replications with species from published molecular phylogenies of the one exception). Cages consisted of glass cylinders (35 Order Sapindales (Savolainen et al. 2000, Soltis et al. cm diameter by 70 cm height) with the top covered by 2000) and Anacardiaceae (Miller et al. 2001, Yi et al. a Þne mesh fabric and sealed with rubber bands. Lar- 2004). The list was compiled by starting with the target val thrips were transferred to a single test plant, and weed (S. terebinthifolius), and adding representative daily observations were made on their feeding activity species (total number) based on the following cate- and survival. After 14 d, most of the actively feeding gories: (A) North American species in the same genus Þrst and second instars began to pupate, the cylinders as the target weed (3); (B) North American species in were removed, and plants were examined to deter- other genera in the same family as the target weed mine larval mortality and feeding responses. Seven (13); (C) threatened and endangered species in the plant species in three families were tested in Brazil; six same or closely related families to the target weed (5); of these were retested in Florida (see Table 1). (D) North American species in other families in the No-Choice Larval Survival Test (Florida). Thrips same order that have some phylogenetic, morpholog- larvae collected in Brazil were exposed to 29 plant ical, or biochemical similarities to the target weed (3); species in 11 families at the FBCL from March 1995 to (E) North American species in other orders that have June 1996. Because all plants could not be tested si- some morphological or biochemical similarities to the multaneously, larvae were exposed to plants in a series target weed (2); (F) any plant on which the biological of nine separate laboratory trials. Thirty larvae (Þrst control agent or its close relatives (within the same and second instars) were caged on individual potted genus) have been previously found or recorded to plants 50Ð150 cm tall (n ϭ 3 or 4 replications). Cages feed and/or reproduce (2); (G) plants not closely consisted of Þne mesh fabric bags (15 cm height by 8.8 related to weed, which have agricultural signiÞcance cm diameter) opened at one end. Larvae were trans- and are grown in the same range as the weed in North ferred to the test plants with a camel hair brush, the America (14); and (H) representative species in fam- mesh bag was placed over the thrips, and the open end December 2009 CUDA ET AL.: HOST RANGE OF BRAZILIAN PEPPERTREE THRIPS P. ichini S.L. 1645

Table 1. Test plant list used to predict the field host specificity of the thrips P. ichini s.l.

No. of ScientiÞc name Common name Status Categorya U.S. distributionb tests Sapindales Anacardiaceae Schinus terebinthifolius Brazilian peppertree Introduced A Southern United States 30c Raddi Schinus molle L. California peppertree Introduced A Southern United States 4c Schinus polygamus Chilean peppertree Introduced A CA 3 (Cav.) Cabrera Cotinus obovatus Raf. American smoketree Native, special concern TN C Southern United States 3 Mangifera indica L. Mango Introduced B Southern United States, 7c Caribbean Rhus aromatica Aiton Fragrant sumac Native B Eastern, midwestern 3 United States Rhus copallinum L. Winged sumac Native B Eastern, midwestern 6 United States Rhus glabra L. Smooth sumac Native, special concern CT B Continental United States 3 Rhus michauxii Sarg. MichauxÕs sumac Native, endangered US C Southeastern United 3 States Rhus typhina L. Staghorn sumac Native B Eastern, midwestern 5 United States Toxicodendron Poison ivy Native B Eastern, midwestern 4 radicans L. United States Toxicodendron Poison oak Native B Southeastern, midwestern 3 pubescens Mill. United States Toxicodendron vernix Poison sumac Native, endangered KY C Eastern, midwestern 3 L. Kuntze United States Metopium toxiferum L. Poison wood Native B FL, PR 4 Krug and Urb Spondias dulcis Jocote Introduced B PR 3 Parkinson Spondias purpurea L. Purple mombin Introduced B FL, PR 3 Pistacia chinensis Chinese pistachio Introduced B Southern United States 3 Bunge Pistacia vera L. Cultivated pistachio Introduced B CA, AZ 3 Anacardium Cashew Introduced B Southern United States, 3c occidentale L. Caribbean Comocladia dodonaea Poison Ash Native H Caribbean, PR, VI 3 L. Urban Staphyleaceae Staphylea trifolia L. American bladdernut Native, endangered FL, C Eastern, midwestern 3 NH United States Sapindaceae Litchi chinensis Sonn. Lychee Introduced D FL, HI 3 Hypelate trifoliata Sw. Inkwood Native H Southeastern United 3 States, Caribbean Meliaceae Sweetenia mahogani L. Mahogany Native, endangered FL H FL, Caribbean 3 Jacq. Aceraceae Acer saccharinum L. Silver Maple Native D Eastern, midwestern 3 United States Burseraceae Bursera simaruba L. Gumbo limbo Native H FL, Caribbean 3 Sarg. Simaroubaceae Leitneria floridana Corkwood Native, threatened FL C Southeastern United 3 Chapman States Simarouba glauca DC Bitterwood Native D FL, Southeastern United 3 States Rutaceae Citrus sinensis L. Sweet orange Introduced F FL, TX, CA 3 Osbeck Aquifoliales Aquifoliaceae Ilex cassine L. Dahoon Holly Native E Southern United States 3 Ariales Apiaceae Daucus carota L. Carrot Introduced E Continental United States 2 Fabales Fabaceae Arachis hypogaea L. Peanut Introduced G Continental United States 3 Phaseolus vulgaris L. Pinto bean Introduced G Eastern United States 4 Vigna unguiculata L. Cowpea Introduced G Eastern, midwestern 5 Walp. United States

Continued on following page 1646 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 6

Table 1. Continued

No. of ScientiÞc name Common name Status Categorya U.S. distributionb tests Malvales Malvaceae Gossypium hirsutum Upland cotton Native G Southern United States, 4 L. Caribbean Abelmoschus Okra Introduced G Southern United States 2 esculentus L. Moench Solanales Capsicum annuum L. Bell pepper Native & introduced G Continental United States 6c Solanum tuberosum L. Potato Introduced G Continental United States 4 Lycopersicon Tomato Introduced G Continental United States 7c esculentum Miller Convolvulaceae Ipomoea batatas L. Sweet potato Introduced G Continental United 7c Lam. States, Caribbean Brassicales Brassicaceae Brassica oleracea L. Broccoli/caulißower Introduced G Continental United States 8 Magnoliales Lauraceae Persea americana Mill. Avocado Introduced F FL, Caribbean 3 Poales Poaceae Zea mays L. Corn Introduced G Continental United States 4 Oryza sativa L. Rice Introduced G Continental United States 3 Saccharum officinarum Sugar cane Introduced G Southern United States 3 L. Violales Caricaceae Carica papaya L. Papaya Introduced G FL, Caribbean 4

a See test plant list in Materials and Methods section for explanation of categories. b Distribution codes: AZ, Arizona; CA, California; FL, Florida; HI, Hawaii; PR, Puerto Rico; TX, Texas; VI, Virgin Islands. c Species also tested in Brazil. was closed using a 7- to 8-cm-long twist tie. Plants cages, which consisted of a piece of molecular porous inoculated with thrips (treatments) were arranged in membrane tubing (Spectra/Por, Spectrum Laborato- a completely randomized design and were maintained ries, Rancho Dominguez, CA)(20 cm height by 3.4 cm in the FBCL greenhouse at a temperature of 26.0 Ϯ diameter) placed over a shoot tip and closed on the 2.0ЊC and 60Ð70% RH. After 7 and 14 d, which coin- ends with a 3.4-cm-diameter foam stopper. After 10 d, cided with the development of the actively feeding each plant shoot was thoroughly examined with a Þrst and second instars, cages were removed, and dissecting microscope for the presence of eggs. Plants plants were examined to determine larval mortality with eggs were monitored for larval development and and to record feeding responses. adult eclosion. No-Choice Oviposition Tests. In total, 20 adults (10 From March to April 2002, a Þnal oviposition test males and 10 females, n ϭ 4 replications) were ex- was conducted with Cotinus obovatus Raf. using the posed to four plant species in four different genera of aforementioned acrylic cylinder experimental proce- Anacardiaceae from December 2000 to January 2001. dure. C. obovatus was not included in the original test Plants used in these tests either supported partial or plant list but is considered an important nontarget complete development in the no-choice larval survival native species. Only one replication was performed tests or were considered critical test plants (e.g., with C. obovatus in this test because of the difÞculty threatened, endangered, or sympatric native species in obtaining plant specimens (but see below). not included in the larval survival tests). Thrips were No-Choice Oviposition Tests: Caribbean and Other sexed on the basis of size (Hood 1949) and set up on Native Plants. In these tests conducted from June 2003 individual plants enclosed in clear acrylic cylinders to November 2005, adults (10 males and 10 females) (15 cm diameter by 50Ð60 cm height). The top and the were exposed to an additional 17 plant species (n ϭ 3 six circular ventilation holes (6 cm diameter) of the replications except for S. terebinthifolius, n ϭ 18) in cylinder were covered with nylon 40/42 mesh screen eight families of Sapindales native to North America (Nitex Ltd., SoÞa, Bulgaria) to prevent insects from (including C. obovatus, n ϭ 2) and the Caribbean. escaping. These plants were recommended for testing by the A slightly different procedure was used for the TAG because they were not included in the original larger test plants, i.e., Mangifera indica L. (mango), test plant list. Thrips were placed on individual potted and Metopium toxiferum L. Krug and Urb. (poison- plants enclosed in the same clear acrylic cylinders wood). Adult thrips were transferred to dialysis tube described previously and allowed to feed and oviposit December 2009 CUDA ET AL.: HOST RANGE OF BRAZILIAN PEPPERTREE THRIPS P. ichini S.L. 1647 on the plants. Larval development and adult eclosion beginning of each replication (n ϭ 4), 70 adults thrips were recorded for each test plant species. (approximately equal number of males and females) Paired-Choice Oviposition Tests. Paired-choice ovi- were caged on a S. terebinthifolius plant inside one of position tests were conducted from December 2000 to the clear acrylic cylinders described previously. After June 2005 with three species because of their ecolog- a visibly large population of thrips larvae was estab- ical or ornamental value; the federal endangered Rhus lished on the target weed (148, 106, 137, and 108 larvae michauxii Sarg. (USFWS 1989), the California orna- in each of the four acrylic cylinder cages, respec- mental S. molle L., and the Florida native M. toxiferum. tively), each infested plant was placed inside a larger In each test, S. terebinthifolius was paired with one of aluminum screen cage along with a similar sized M. the three test plant species. toxiferum plant. Screen cages (61 by 61 by 61 cm) had For the test with R. michauxii, a single potted plant three sides completely covered with translucent plas- of each test species was placed in a wooden sleeve tic (5 mil) and partially covering all but a rectangular cage (55 by 50 by 45 cm) with a Plexiglas (Professional opening (55 by 15 cm) on the fourth side, which was Plastics, Inc., Fullerton, CA) top and screened back. covered with Þne mesh screen for ventilation. Tops of The screened back was covered with clear plastic the cages were covered with a clear acrylic sheet, and wrap to maintain humidity and the top was covered the ßoor of the cage was aluminum. Plants were ar- with shade cloth to prevent heat buildup inside the ranged randomly so that no foliage of either plant sleeve cage during exposure to the sun in the FBCL touched the sides of the cage or the other plant. As the glasshouse. In total, 35 adults of P. ichini s.l. (18 females condition of S. terebinthifolius plants deteriorated and 17 males) were added to the cage from 7 to 12 from larval feeding and new adults appeared in each December 2000. The thrips adults were placed in the cage, the paired M. toxiferum plant was checked for center of the cage midway between each test plant. the presence of P. ichini s.l. Plants were checked twice After 2 wk, both plants were removed from the sleeve weekly, and the number of eggs, larvae, and adults cage, dissected, and examined with a stereo micro- observed on the M. toxiferum plants was recorded. As scope for the presence of eggs and larvae. Only one larvae began to develop on the M. toxiferum plants, replication of this particular test was conducted be- they were collected and placed in the same plastic cause the remaining specimens of R. michauxii in the vials used for colony rearing (Cuda et al. 2008) except outdoor nursery had already senesced before a sufÞ- fresh shoot tips of M. toxiferum were used as a food cient number of thrips adults had emerged from the source. Each time larvae were collected from M. pupal stage. However, R. michauxii was challenged toxiferum plants, an equal number of larvae was ran- again in the multiple-choice oviposition tests (see domly collected from S. terebinthifolius plants to be below). reared as controls. Four replications of the paired- For the S. molle test conducted from December choice test were conducted under the following en- 2001 to February 2002, two potted plants of each vironmental conditions: temperature of 23.2Ð24.0ЊC, species (four per cage) were arranged opposite each 55Ð75% RH, and a 16 L:8 D photoperiod. other by placing the pots into a Plexiglas (Professional Multiple-Choice Oviposition Tests. Adults obtained Plastics) sheet with four equidistant circular holes from larvae of P. ichini s.l. collected in March 2001 supported by PVC legs. Plants were placed inside a from Brazil were used to conduct two separate mul- Þne mesh square screen cage supported by a PVC tiple-choice oviposition tests in the FBCL from March frame (60 by 60 by 60 cm). Rims of the nursery pots, to June 2001. The Þrst test was conducted from March which were the same diameter as the circular holes, to May and the second from May to June 2001. Ex- were positioned to be almost ßush with the Plexiglas perimental design consisted of the same cage setup (Professional Plastics) sheet. This experimental de- used in the aforementioned paired-choice oviposition sign allowed the thrips to have equal access to both tests. Test plants included in the multiple choice tests specimens of each plant species. Twenty adult P. ichini were those that produced positive results in the no- s.l. (10 males and 10 females) were added to the center choice larval feeding and oviposition tests or were of each screen cage (n ϭ 3). After 2 wk, each plant was considered critical nontarget species in Florida. Each removed from the sleeve cage and covered with an cage (n ϭ 3) was provided with the following test acrylic cylinder to monitor thrips development. The plants: S. terebinthifolius (control), the federal listed cages were maintained on a greenhouse bench in the endangered R. michauxii, the cultivated M. indica, and FBCL. The average temperature was 24.5 Ϯ 5.0ЊC and the native M. toxiferum. The plants were randomly the RH was Ϸ53%. assigned to different positions in each cage. Twenty Metopium toxiferum is a native species commonly adults (10 males and 10 females) were released into associated with S. terebinthifolius in south Florida the center of three cages. Adult feeding tests were not (Austin and Smith 1998, Wunderlin and Hansen 2008). conducted because of the difÞculty in distinguishing Because P. ichini s.l. accepted M. toxiferum as an ovi- larval from adult feeding damage. positional host and some larval development occurred Both of the R. michauxii plants in Reps I and III of in one of the no-choice trials, a Þnal series of tests was the Þrst test died soon after the experiment was ini- conducted from March to June 2005 to assess risk of tiated and were replaced with new specimens. The damage to the native M. toxiferum if thrips were to dead plants were examined under a microscope on 21 build up high populations on S. terebinthifolius and March 2001 for the presence of eggs or larvae of P. spill over onto this south Florida native shrub. At the ichini s.l. On 5 June 2001, the second test was termi- 1648 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 6 nated and the plants in all three cages were dissected and examined under a microscope for the presence of thrips eggs and larvae. Data Analysis. Numbers of eggs, larvae, and adults of P. ichini s.l. observed in each test are reported as means Ϯ SE. Where appropriate, means were ana- lyzed with analysis of variance (ANOVA; PROC GLM; SAS Institute 2002) or StudentÕs t-test (PROC TTEST; SAS Institute 2002) at P ϭ 0.05. Survival data (%) were normalized with the arcsine transformation before analysis (Snedecor and Cochran 1989).

Results Fig. 1. Results of replicated paired-choice oviposition No-Choice Larval Survival Tests. Results of the no- test with Schinus spp., December 2001 to January 2002. When choice larval survival tests indicated a narrow host given a choice, females of P. ichini s.l. did not exhibit a range for P. ichini s.l. In both Brazil and Florida tests, preference for S. molle (SCMO) over S. terebinthifolius larvae fed and survived for 2 wk only on their natural (SCTE) as an ovipositional host in a laboratory cage envi- host S. terebinthifolius and S. molle. In the Brazil test, ronment. Means followed by the same letter are not signif- icant (t ϭ 0.94, P ϭ 0.22). there was no difference in survival on S. terebinthifo- 4 lius (86%) or S. molle (81%). A similar pattern was observed in the Florida test; 40 and 56% of the larvae Acceptability of S. molle as a potential host plant for were still alive and feeding on S. terebinthifolius and S. P. ichini s.l. was clearly shown in this test (Fig. 1). molle, respectively, after 2 wk. The higher survival When given a choice between S. molle and S. terebin- observed on S. molle was not statistically different thifolius under caged conditions, females showed no from S. terebinthifolius (t ϭ 1.79, P ϭ 0.052). ϭ ϭ 10 preference for either species (t4 0.94, P 0.22), No-Choice Oviposition Tests. Under no-choice con- even though a higher proportion of the eggs (76%) was ditions, females oviposited on S. terebinthifolius and S. deposited on S. molle (21 Ϯ 15 eggs) compared with molle and also on the native M. toxiferum. Although it S. terebinthifolius (6 Ϯ 5 eggs). The suitability of S. appeared that signiÞcantly more eggs were deposited molle as a developmental host also was conÞrmed by on M. toxiferum (22 Ϯ 20) compared with S. terebin- the appearance of two F1 adults on one of the test thifolius (8 Ϯ 4) and S. molle (9 Ϯ 4), the results were plants Ϸ1 mo after exposure to P. ichini s.l. ϭ ϭ not statistically different (F3,12 0.81, P 0.051). Results of the paired-choice oviposition test with More importantly, none of the larvae that hatched the native M. toxiferum are shown in Table 2. High from the eggs on M. toxiferum developed to the adult larval populations allowed to build up on S. terebin- stage on this nontarget species, whereas the majority thifolius plants eventually caused a complete die back of neonates on S. terebinthifolius completed their de- of these plants. As expected, adult thrips moved over velopment. to adjacent M. toxiferum plants. However, the latter In the unreplicated test with C. obovatus, only Þve sustained only minor adult feeding damage on grow- eggs were deposited on this native species, but none ing tips and quickly recovered. Normal development of the eggs were viable. In contrast, almost 80 imma- and survival of P. ichini s.l. to the adult stage was tures of P. ichini s.l. (17 eggs and 61 larvae) were observed only on its natural host S. terebinthifolius observed on the single S. terebinthifolius test plant (Table 2). In two of the four replications (Reps I and when the experiment was terminated. II), thrips adults that spilled over onto M. toxiferum No-Choice Oviposition Tests: Caribbean and Other plants successfully oviposited but none of the 29 larvae Native Plants. In total, 148 Ϯ 46 larvae and 81 Ϯ 30 that emerged completed development on this nontar- adults of P. ichini s.l. were produced from eggs depos- Ͼ get species. In contrast, 60% of the F1 generation ited on S. terebinthifolius in this supplemental host range test. None of the Caribbean or other native plant species were attacked in this no-choice test. Even the Table 2. Survival of P. ichini s.l. exposed to S. terebinthifolius introduced S. polygamus (Cav.) Cabrera, which is in and M. toxiferum in a paired choice test the same genus but a different subgenus (Duvaua) S. terebinthifolius M. toxiferum from S. terebinthifolius (Barkley 1944, 1957), was not Rep. no. Larvae Adults Percent Larvae Adults Percent a a accepted as a host plant by P. ichini s.l. in this test. (F1) (F2) survival (F1) (F2) survival Paired-Choice Oviposition Tests. In this prelimi- 1 26 14 53.8 26 0 0 nary unreplicated test with the federal endangered R. 2 3 2 66.7 3 0 0 michauxii, no eggs or larvae of P. ichini s.l. were ob- Means Ϯ SEM 60.3 Ϯ 8.0 0 served on this listed species, whereas 11 eggs were a deposited on S. terebinthifolius. Viability of the eggs on Number of F1 larvae developing from the parental generation ϭ S. terebinthifolius was subsequently conÞrmed by the (n 70) exposed to both test plants. This value represents the total no. of F1 larvae produced on the M. toxiferum plants and setup as appearance of neonates on the plant when it was controls on S. terebinthifolius to match the no. of larvae developing on removed from the cage. the test plants. December 2009 CUDA ET AL.: HOST RANGE OF BRAZILIAN PEPPERTREE THRIPS P. ichini S.L. 1649 larvae on S. terebinthifolius completed development to plants taller than 3 m, it is unlikely that mature S. molle the adult stage on their natural host. trees planted for shade or ornamental purposes in Multiple-Choice Oviposition Tests. In both multi- California would be attacked. Fourth, even if P. ichini ple-choice oviposition tests, no eggs were deposited s.l. eventually migrates to California and becomes es- on the federally endangered R. michauxii or the trop- tablished on both Schinus spp., only tender new shoot ical fruit tree M. indica. Although oviposition occurred tips (and ßowers, if any are present) would be dam- on M. toxiferum in the aforementioned no-choice ovi- aged. The thrips does not attack fruits directly or cause position test, females selected M. toxiferum as an ovi- any observable damage to existing foliage. Because positional host in only one of the multiple-choice tests. fruit production would be inhibited through ßower In this test, the number of eggs deposited per plant on abortion, invasiveness of S. molle would be reduced by S. terebinthifolius (101 Ϯ 43) was 14-fold greater than P. ichini s.l. without affecting the shade value of mature on M. toxiferum (7 Ϯ 7), but the difference was not S. molle trees. Finally, even though S. molle is a popular ϭ ϭ signiÞcant (F3,8 4.04, P 0.051). ornamental shade tree in western United States, the California Invasive Plant Council includes this species on their invasive plant list along with S. terebinthifolius Discussion because both species have escaped cultivation in Cal- Results of this study conÞrmed the conclusion by ifornia (Cal-IPC 2006). Garcia (1977) that the thrips P. ichini s.l. is a potential According to Pemberton (2002), the potential risk biological control agent of S. terebinthifolius. P. ichini of biological control agents to native plants should be s.l. was found to be a narrow specialist capable of of greater concern than the risk to non-native orna- reproducing only on two of the three non-native Schi- mentals like S. molle because native species are irre- nus spp. that have escaped cultivation in the United placeable. The results of this study showed that P. States. Laboratory studies conducted in Brazil and ichini s.l. was unable to reproduce on the sympatric M. Florida showed that larvae fed and completed their toxiferum, which is native to Florida and the Carib- development only on S. terebinthifolius and S. molle, a bean (USDA, NRCS 2008), even under high thrips congener that has been widely planted as an orna- population densities when females would occasionally mental tree in southern California and Arizona (Nil- oviposit on this plant. None of the larvae produced son and Muller 1980a, b). from the few eggs laid on M. toxiferum survived. Al- This Þnding was not unexpected because the con- though the native M. toxiferum sustained some minor generic S. molle belongs to the same subgenus Eus- adult feeding damage from P. ichini s.l., our results also chinus as the target weed (Barkley 1944, 1957), and showed it may not be attacked by the thrips when acceptance of both Schinus spp. was observed in pre- females are given a choice between S. terebinthifolius vious studies (Cuda et al. 2008, Manrique et al. 2008). and other plant species. Therefore, P. ichini s.l. should Although laboratory studies showed that the funda- pose minimal risk to M. toxiferum if it was released for mental host range of P. ichini s.l. includes S. molle, the biological control of S. terebinthifolius in Florida. actual “measured” risk to this nontarget species may be Recently, we discovered that two distinct geno- much lower than the “potential” risk as suggested by types of S. terebinthifolius were introduced into Flor- Briese (2005) for several reasons. First, there is a wide ida early in the 20th century (Williams et al. 2005). geographic separation between S. molle in California After they established, extensive hybridization oc- and S. terebinthifolius in Florida, the proposed release curred over time that produced novel genotypes site. Second, in numerous Þeld surveys conducted unique to Florida (Williams et al. 2007). We also found from 1994 to 2002 in the states of Parana´, Rio Grande that populations of P. ichini s.l. from two geographic do Sul, and Santa Catarina, Brazil, where S. terebin- locations in Brazil differed in their ability to attack thifolius and S. molle populations are sympatric, P. Florida genotypes of S. terebinthifolius (Manrique et ichini s.l. was collected only from S. terebinthifolius and al. 2008). Our laboratory colony was established ini- never on S. molle. Further evidence for the preference tially with thrips collected near Curitiba, Parana´ state, of P. ichini s.l. for S. terebinthifolius was obtained while Brazil. However, contrary to what was reported in visiting a glasshouse containing the two Schinus spp. in Manrique et al. (2008), the colony was later supple- Curitiba, Brazil, in January 2003. One of us (J.C.M.) mented with thrips obtained from several source pop- discovered a thrips infestation on one of the S. tere- ulations during the course of this study (see Materials binthifolius plants. The glasshouse contained Ϸ60 S. and Methods). In retrospect, we observed higher sur- terebinthifolius plants and 7 S. molle plants for research vival and reproduction of the thrips on Florida S. purposes. On closer inspection, 11 of the S. terebin- terebinthifolius plants in the laboratory in 2003 (Cuda thifolius plants were infested with P. ichini s.l., whereas et al. 2008), which coincided with the addition of none of the S. molle plants were attacked. Third, es- thrips obtained from these other geographic locations. tablished S. molle trees (those Ͼ3 m tall) that are Thrips populations from Rio de Janeiro, Minas Gerais, valued as shade trees in California probably would be and Parana´ states appear to be distinct taxa. The pop- immune from attack because of the feeding habits of ulation from Minas Gerais in particular was found to the thrips. According to Garcia (1977), 90% of larval be better adapted to the Florida genotypes yet exhib- populations examined on S. terebinthifolius in Brazil ited the same preference for S. terebinthofolius and S. were found on plants ranging from only 20 cm to 2 m molle (Manrique et al. 2008). These Þndings empha- in height. Because no larval infestations were found on size the importance of matching the appropriate taxa 1650 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 6 of P. ichini s.l. with Florida genotypes of S. terebinthi- P. ichini s.l. in Florida should reduce the competitive folius to increase the likelihood of establishment and ability of S. terebinthifolius and contribute to the sus- successful biological control of this invasive weed. tainable management of this invasive weed in Florida DeLoach et al. (2008) had a similar experience with and perhaps California, Hiwaii, and Texas. the leaf beetle Diorhabda elongata (Brulle´) (Co- leoptera: Chrysomelidae) released for biological con- trol of saltcedars, Tamarix spp., in western United Acknowledgments States. Beetles from different source populations were We thank the following former graduate and undergrad- identiÞed postrelease as Þve genetically distinct taxa, uate students for their technical assistance: J. Abordo, R. i.e., D. elongata s.l. that have different climatic re- Granja, D. A. Harmuch, R. Loinaz, J. Marquez, O. Moeri, K. quirements but the same restricted host speciÞcity. Nichols, and M. Vitorino. We also thank H. Frank, B. Over- According to McFadyen and Jacob (2004), it is holt, and two anonymous reviewers for comments on an difÞcult to assess a priori the effect of novel natural earlier draft of this manuscript. The research reported herein enemies on a weed biological control agent like P. was supported by grants from the Florida Department of ichini s.l. before it is released. However, a survey of the Environmental Protection, Florida Exotic Pest Plant Council, and the South Florida Water Management District. arthropod fauna of S. terebinthifolius in Florida by Cassani (1986) showed that most arthropod species identiÞed were predacious (51.3%); the most fre- References Cited quently encountered predatory groups were ants and spiders. Generalist predators, especially when they are Anonymous. 2007a. Other news: Brazilian pepper expands abundant, could negatively affect populations of P. its range. Wildland Weeds 10: 29. Anonymous. 2007b. Panhandlers beware! Wildland Weeds ichini s.l. However, Garcia (1977) reported that large 11: 22. larval aggregations of up to 300 individuals on a single Austin, D. F. 1978. 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Whitten, K. J. Wurdack, and Yi, T., A. J. Miller, and J. Wen. 2004. Phylogenetic and M. W. Chase. 2000. Phylogeny of the eudicots: a nearly biogeographic diversiÞcation of Rhus (Anacardiaceae) in complete familial analysis based on rbcl gene sequences. the northern hemisphere. Mol. Phylogenet. Evol. 33: 861Ð Kew Bull. 55: 257Ð309. 879. Silva, A.G., A. G. d’Araujo, D. M. Goncalves, D. M. Galvao, A.J.L. Goncalves, J. Comes, M. N. Silva, and L. Simoni. 1968. Quarto Catalogo dos Insetos que Vivem nas Plantas Received 9 April 2009; accepted 3 August 2009. J. Aquat. Plant Manage. 54: 20–25 Compatibility of an insect, a fungus, and a herbicide for integrated pest management of dioecious hydrilla

JAMES P. CUDA, JUDY F. SHEARER, EMMA N. I. WEEKS, EUTYCHUS KARIUKI, JULIE BANISZEWSKI, AND MIHAI GIURCANU*

ABSTRACT INTRODUCTION

During the past 15 yr dioecious hydrilla (Hydrilla Hydrilla verticillata (L.f.) Royle, Hydrocharitaceae (hereaf- verticillata) in Florida developed resistance to fluridone ter hydrilla) is a federally listed noxious weed and one of the and endothall, two registered herbicides approved for worst invasive aquatic plants in the United States, with aquatic use. An integrated pest management approach millions of dollars spent annually to control large infesta- could mitigate the effects of herbicide resistance and tions in all types of water bodies. A dioecious female strain improve the sustainability of dioecious hydrilla manage- of hydrilla was introduced into Florida from Sri Lanka in the early 1950s through the aquarium trade (Schmitz et al. ment in Florida. In this study, we tested a reduced-risk 1991, Langeland 1996). This aggressive submersed plant was method for dioecious hydrilla control by integrating spread intentionally by growers of aquarium plants and selective insect herbivory with a disease organism or low unintentionally by boaters from one watershed to another concentrations of a new herbicide recently registered for (Balciunas et al. 2002). Florida currently spends approxi- aquatic use. Two rates of the fungal pathogen Mycoleptodiscus mately $15 million annually controlling dioecious hydrilla terrestris (Mt) and the acetolactate synthase-inhibiting in its public waters (Haller 2014). Nationally, established herbicide imazamox, and two densities of the hydrilla tip- populations of monoecious or dioecious hydrilla biotypes mining midge Cricotopus lebetis alone and in combination occur in 28 states as far north as Maine on the Atlantic were randomly applied to aquaria containing established coast, and Washington on the Pacific coast (Lietze and hydrilla plants and replicated three times. Hydrilla shoots in Weeks 2014). Balciunas and Chen (1993) predicted that each tank were harvested ~30 d after the treatments were hydrilla could colonize any water body in North America. applied. Hydrilla biomass produced in each treatment was Their prediction was validated when hydrilla infestations compared. Results showed that combining the hydrilla tip- were recently discovered in the Midwest, as far north as mining midge C. lebetis with either the Mt fungus or Wisconsin (EDDMaps 2014). herbicide imazamox significantly reduced hydrilla growth A major factor contributing to the negative impacts of and the effects in some treatments were synergistic. dioecious hydrilla is its pattern of growth. This submersed Furthermore, C. lebetis was compatible with the herbicide weed grows as a sparsely branched erect rooted plant until it imazamox; adult emergence of C. lebetis was similar in reaches the water surface, where it forms numerous side aquaria treated with imazamox compared with untreated branches. Dense surface mats that are produced comprise up to 20% of the plant’s biomass (Haller and Sutton 1975). controls. Incorporating biological control agents like Mt These mats not only displace native vegetation, which and the tip-mining midge C. lebetis into an integrated weed- affects native fish and zooplankton communities and alters management strategy could reduce overreliance on herbi- water temperature and chemistry, but also interfere with cides and provide a more sustainable solution to Florida’s navigation and flood control (Haller and Sutton 1975, Colle dioecious hydrilla problem. and Shireman 1980, Canfield et al. 1983, Schmitz and Key words: Cricotopus lebetis, herbicide-resistance manage- Osborne 1984). Furthermore, hydrilla is a major substrate ment, Hydrilla verticillata, imazamox, integrated weed man- for a new species of cyanobacterium that produces a agement, Mycoleptodiscus terrestris. neurotoxin that causes avian vacuolar myelinopathy in birds (Wilde et al. 2005). *First, third, fourth, and fifth authors: Professor, Assistant Research Management of dioecious hydrilla is difficult because of Scientist, Graduate Student, and Undergraduate Student, Entomology & its growth rate, which may exceed 30 cm per day (Glomski Nematology Department, University of Florida, Gainesville, FL 32611- 0620. Second author: Research Plant Pathologist, Environmental and Netherland 2012), and its ability to regenerate from Laboratory, U.S. Army Engineer Research & Development Center, fragments (Silveira et al. 2009). Because of the diversity of Waterways Experiment Station, Vicksburg, MS 39180-6199. Sixth author: water resource uses (e.g., fishing, hunting, recreation, flood Coordinator, IFAS Statistics, University of Florida, Gainesville, FL control, aquaculture, and crop irrigation), effective hydrilla 32611-0339. Current address of fifth author: Plant and Soil Sciences Department, University of Kentucky, Lexington, KY 40546-0312. control is difficult to achieve because of a limited number of Corresponding author’s E-mail: jcuda@ufl.edu. Received for publication environmentally sound options for integrated pest man- October 22, 2014 and in revised form October 2, 2015. agement (Hoyer et al. 2005). Current efforts for controlling

20 J. Aquat. Plant Manage. 54: 2016 dioecious hydrilla in Florida rely primarily on herbicides 1990). A virulent strain of Mt has been studied extensively as (FWC 2011) and nonselective biological control using grass an inundative biological control agent (Shearer 1996, 1998). carp Ctenopharyngodon idella Val. (Cassani 1996, Sutton and During the past 10 yr, Mt has been under development as a Vandiver 1998, Dibble and Kovalenko 2009). Although mycoherbicide by the National Center for Agricultural various chemical, mechanical, and biological methods have Utilization Research (NCAUR), U.S. Department of Agri- been investigated for managing hydrilla infestations to culture–Agricultural Research Service (USDA-ARS) Labo- control the explosive growth of the weed (Gettys et al. 2014, ratory, Peoria, IL (Shearer and Jackson 2006), and the Weeks and Lietze 2014), none was as effective as the SePRO Corporation, Carmel, IN (Heilman 2012). herbicide fluridone. Until recently, the herbicides fluridone Imazamox is a systemic herbicide registered in 2008 for and endothall formed the basis of most publicly funded aquatic use (Netherland 2014). This herbicide targets the hydrilla control programs in Florida and elsewhere (Mac- plant-specific enzyme acetolactate synthase (ALS), which Donald 2012, Netherland 2014). plays a critical role in the production of amino acids In 2000, aquatic plant researchers discovered that required for protein synthesis (Netherland 2014). Treating dioecious hydrilla in Florida was developing resistance to hydrilla with imazamox reduces the plant’s biomass and fluridone in some water bodies (MacDonald et al. 2001). This suppresses growth for up to 7 mo (Netherland 2014). More finding confirmed field observations of declining hydrilla important, there are no restrictions for drinking water and control by public and private aquatic plant managers after minimal restrictions for irrigation (Netherland 2014). large-scaleandrepeateduseoffluridoneforhydrilla In this study, we assessed the efficacy of integrating control in the Kissimmee Chain of Lakes in Osceola County, herbivory by the naturalized meristem-mining midge C. Florida through the 1990s. This is the first case of a plant lebetis with either the native fungal pathogen Mt or the ALS- developing resistance to a carotenoid biosynthesis inhibitor, inhibiting herbicide imazamox for controlling hydrilla. or bleaching-type herbicide (Michel et al. 2004, Dayan and Netherland 2005). Fluridone resistance in Florida was not MATERIALS AND METHODS anticipated because of the naıve¨ assumption that dioecious hydrilla could not develop resistance. Nevertheless, at least The hydrilla tip-mining midge C. lebetis was collected at six clones have been identified with a two- to sevenfold Lake Rowell, Bradford County, FL (29855016.9600 N; increase in resistance to fluridone (Puri et al. 2006), and the 82809032.8500 W) and reared according to procedures de- level of resistance appears to be stable over time, even in the scribed by Cuda et al. (2002). The fungal inoculum of Mt1 absence of fluridone selection pressure (Puri et al. 2007). (USDA ARS Culture Collection [NRRL] #30559) was The discovery of fluridone resistance in Florida dioecious prepared using protocols described by Shearer and Jackson hydrilla led to several local and national workshops/ (2006) and shipped to the Engineer Research and Develop- meetings with concerned researchers, aquatic plant manag- ment Center (ERDC), Vicksburg, MS. Appropriate 100-ml ers, and other stakeholders to establish priorities for future dilutions of imazamox2 were mixed with distilled water in research directions (Hoyer et al. 2005, Netherland et al. 125 ml-screw-cap bottles3 at the University of Florida, 2005, Cuda et al. 2008, Systma 2008). One of the priority Gainesville, FL. Eggs/neonates of C. lebetis were transferred areas from these workshops was improving integration of to plastic screw-cap scintillation vials (20 ml) containing chemical control technology with other aquatic plant water from the aquaria at ERDC before the experiments. management practices, e.g., biological control. Bottles containing the insects and the imazamox dilutions Cricotopus lebetis Sublette (Diptera: Chironomidae) is a were delivered to the ERDC via ground transportation 24 h herbivorous midge whose larvae mine apical meristems of before initiating the aquarium tests. hydrilla, using living plant material as a food source (Epler et al. 2000, Cuda et al. 2002). Feeding damage by larvae of C. Compatibility of the hydrilla tip-mining midge C. lebetis lebetis stunts the growth of hydrilla and changes the plant’s with the fungus Mt architecture (Cuda et al. 2011). Cricotopus lebetis is widely distributed in peninsular Florida, albeit at relatively low Experiments were conducted in 55-L aquaria located in a densities (Stratman et al. 2013b). Recent studies have shown controlled-environment growth chamber at the ERDC, that C. lebetis is not a hydrilla specialist in laboratory tests Vicksburg, MS. Growth chamber conditions were main- (Stratman et al. 2013a), but it has been collected only from tained for optimal hydrilla growth: 25 C 6 1 C and a 14 : 10- hydrilla in field samples. The insect’s short generation time, h light : dark photoperiod. Aquaria (0.9 m tall 3 0.09 m2) high reproductive rate, and ease of mass rearing (Cuda et al. were filled with a water-based culture solution (Smart and 2002, Baniszewski et al. 2015) make it an ideal candidate for Barko 1985). Plastic cups (946 ml) containing fertilized an augmentation program (Cuda et al. 2008). Using both topsoil were drenched with reverse-osmosis water and four niche and physiological modeling approaches, Stratman et 15-cm apical cuttings from dioecious hydrilla were planted al. (2014) predicted that C. lebetis would complete up to 11 in each cup and placed in the aquaria (four cups per generations per year in Florida, and that much of the aquarium). Aquaria were gently aerated to provide circula- southeastern United States was climatically suitable for tion. Plants were allowed to grow in the aquaria for establishment of the midge. approximately 28 d, by which time they had formed a The indigenous fungus Mycoleptodiscus terrestris (Gerd.) canopy. Dry inoculum of the fungus Mt was applied by Ostazeski (incertae sedis: Magnaporthaceae) (hereafter Mt), scattering it evenly onto the water surface and allowing it to isolated in Texas in the 1980s, is pathogenic to hydrilla (Joye naturally dissipate over the hydrilla. As the rehydrated

J. Aquat. Plant Manage. 54: 2016 21 Figure 1. Biomass of hydrilla 28 d after application of different combinations of the plant pathogenic fungus Mycoleptodiscus terrestris (Mt) and the hydrilla tip-mining midge Cricotopus lebetis in 55-L aquaria compared with untreated controls. Bars with different letters are statistically different (ANOVA, Fisher’s LSD test, a , 0.05). granules fell through the water column they became lodged Data analysis on leaves and in leaf axils. Neonates of C. lebetis were applied the same day. Data are reported as means 6 standard error. Hydrilla Treatments included effective rates of 0.02 and 0.06 g L1 biomass and emergence of adult midges in the imazamox of Mt alone (Shearer and Nelson 2009), 40 and 80 neonates tests were subjected to ANOVA (SAS Version 9.2, 2011). of C. lebetis alone (Cuda et al. 2011), all combinations of Mt When significant treatment effects were detected, means and C. lebetis, and untreated controls. Aquaria were covered were separated using Fisher’s LSD test at the 0.05 with screens and each treatment was replicated three times. significance level. Synergistic effects between midge and At 28 d after treatment, hydrilla shoot biomass was Mt treatments and between midge and imazamox treat- harvested, dried for 4 d at 60 C, and dry weight (gm) ments were analyzed using a nonlinear mixed-model recorded. procedure (Blouin et al. 2004). Significant interactions were indicated when Colby estimates were positive (synergistic effect) or negative (antagonistic effect), and means were Compatibility of the hydrilla tip-mining midge C. lebetis separated by NLMIXED t tests at the 0.05 significance level. with the herbicide imazamox Using the same experimental setup, a second experiment RESULTS AND DISCUSSION was conducted to determine if the hydrilla tip-mining midge C. lebetis was compatible with the herbicide imaza- Compatibility of the hydrilla tip-mining midge C. lebetis mox, which causes hydrilla to branch at low concentrations with the fungus Mt (M. D. Netherland, pers. comm.). Treatments included low and high rates of imazamox (10 and 50 lgL1) alone Results from combining two rates of Mt with two (Shearer and Nelson 2009), 40 and 80 neonates of C. lebetis densities of the midge C. lebetis are presented in Figure 1. alone, all combinations of imazamox and C. lebetis, and Hydrilla biomass produced in the Mt low or high aquaria untreated controls. After imazamox was added to the tanks, (9.65 6 2.40 g and 8.04 6 1.36 g, respectively) did not differ midge neonates were added 2 wk later to allow the herbicide statistically from the controls (11.03 61.47 g). Similarly, to induce branching and ensure that the minimum plant biomass in the midge low- and high-treatment aquaria exposure/half-life times were met (Netherland 2014). Each (9.91 6 1.06 g and 10.51 6 3.40 g, respectively) was not treatment was replicated three times. Hydrilla biomass was statistically different from the controls. The nonsignificant harvested 28 d after aquaria were inoculated with midge increase in biomass observed in the midge high-treatment larvae. Plant material was dried for 4 d at 60 C, and dry aquaria was not unexpected as feeding damage by the weight recorded. The number of adult midges that emerged developing larvae often stimulates the formation of new was monitored in this experiment to determine if exposure shoot tips (Buckingham 1994). However, aquaria containing of developing larvae to imazamox negatively affected their the Mt high and midge low treatment produced significantly development and survival. less biomass (4.50 6 1.43 g) compared with the controls (df ¼

22 J. Aquat. Plant Manage. 54: 2016 Figure 2. Biomass of hydrilla 28 d after application of different combinations of the acetolactate synthase herbicide imazamox and the hydrilla tip-mining midge Cricotopus lebetis in 55-L aquaria compared with untreated controls. Bars with different letters are statistically different (ANOVA, Fisher’s LSD test, a , 0.05).

8, F ¼ 5.73, P , 0.001). This treatment combination lower hydrilla biomass compared with the controls (df ¼ 8, F significantly reduced hydrilla biomass by almost 60% ¼ 6.11, P , 0.0001). Furthermore, the imazamox high-midge compared with the untreated controls. In addition, a high treatment combination reduced hydrilla biomass by synergistic effect on hydrilla biomass reduction from this 81% and the effect was synergistic (df ¼ 27, C ¼ 6.4046, t ¼ fungus–insect combination was indicated (df ¼ 27, C ¼ 3.93, P , 0.001). 9.7009, t ¼ 3.70, P , 0.001). However, for the high Mt and In previous laboratory tests and small-scale field trials, high midge combination, the biomass produced (11.95 6 integrating low doses of fluridone, endothall, or imazamox 2.26 g) was statistically the same as the controls, suggesting with Mt increased the susceptibility of hydrilla to low doses an antagonistic effect (df ¼ 27, C ¼1.6755, t ¼0.7, P . of these herbicides (Netherland and Shearer 1996, Shearer 0.05). and Nelson 2002, 2009). For example, combining Mt fungus with fluridone significantly reduced hydrilla biomass by Compatibility of the hydrilla tip-mining midge C. lebetis 92% compared with the untreated control and by over 80% with the herbicide imazamox when compared with individual treatments (Netherland and Shearer 1996). In this study, we showed that integrating 1) Results of combining two midge densities with two high rates of Mt with low densities of the tip-mining midge imazamox rates are shown in Figure 2. As in the previous C. lebetis and 2) low or high rates of imazamox with high experiment, hydrilla biomass produced in the midge low- densities of C. lebetis significantly reduced hydrilla growth. In and high-treatment aquaria (14.65 6 2.93 g and 16.49 6 the first experiment, tissue damage from insect herbivory 2.30 g, respectively) did not differ statistically from the probably increased the Mt infection process by creating new controls (17.68 6 2.12 g). However, a significant antagonistic entry wounds in hydrilla for the fungus to infect, resulting interaction was observed in the imazamox low-midge low in pathogenesis. Shabana et al. (2003) observed that hydrilla aquaria (df ¼ 27, C ¼10.2592, t ¼ 4.71, P , 0.001). Hydrilla damage was significantly greater when the fungus Fusarium biomass produced in this treatment combination (14.66 6 culmorum (W. G. Smith) Sacc. (Hypocreales: Nectriaceae) was 2.70 g) was not statistically different from controls. Subtle integrated with the hydrilla leaf-mining fly Hydrellia pak- midge feeding damage and low herbicide rate probably istanae Deonier (Diptera: Ephydridae). stimulated the production of new shoots that neutralized In the second experiment, it is unclear how the the treatment effects. However, the imazamox low- and interaction between insect and herbicide affected hydrilla high-treatment aquaria as well as the remaining imazamox– growth. As expected, midge larvae were not adversely midge treatment combination aquaria yielded significantly affected by exposure to imazamox. The observed increase

J. Aquat. Plant Manage. 54: 2016 23 in adult emergence in some of the aquaria may be the result LITERATURE CITED of additional shoot tips created by branching of hydrilla Balciunas JK, Burrows DW, Purcell MF. 1996. Comparison of the after imazamox treatment (M. D. Netherland, pers. comm.). physiological and realized host-ranges of a biological control agent Imazamox-induced branching of hydrilla could provide from Australia for the control of the aquatic weed, Hydrilla verticillata. additional feeding/development sites for the midge larvae to Biol. Control 7:148–158. exploit, which would account for greater plant damage and Balciunas JK, Chen PP. 1993. Distribution of hydrilla in northern China: Implications on future spread in North America. J. Aquat. Plant an increase in the production of adult midges. Further Manage. 31:105–109. research will investigate whether efficacy of imazamox will Balciunas JK, Grodowitz MJ, Cofrancesco AF, Shearer JF. 2002. Hydrilla, pp. be enhanced by applying the herbicide simultaneously or 91–114. In: R. Van Dreische, S. Lyon, B. Blossey, M. Hoddle, and R. after midge establishment has occurred. Reardon (eds.). Biological control of invasive plants in the eastern United States. FHTET-2002-04, USDA Forest Service, Morgantown, WV. On the basis solely of laboratory host range tests, C. lebetis Baniszewski J, Weeks ENI, Cuda JP. 2015. Impact of refrigeration on eggs of may be unsuitable for redistribution outside the state of the hydrilla tip miner Cricotopus lebetis: Larval hatch rate and subsequent Florida, although it currently is established on hydrilla in development. J. Aquat. Plant Manage. 53:209–215. Louisiana (Epler et al. 2000). The native Canadian water- Berger S, MacDonald G. 2011. Suspected endothall tolerant hydrilla in Florida. Proc South. Weed Sci. Soc. 64:331. [Abstract] weed, Elodea canadensis Michx., and the introduced Brazilian Blouin DC, Webster EP, Zhang W. 2004. Analysis of synergistic and elodea, Egeria densa Planchon, were good laboratory (phys- antagonistic effects of herbicides using nonlinear mixed-model methol- iological) hosts for C. lebetis (Stratman et al. 2013a), yet the ogy. Weed Technol. 18:464–472. insect only has been field collected from hydrilla in Buckingham GR. 1994. Biological control of aquatic weeds, pp. 413–480. In: D. Rosen, F. D. Bennett, and J. L. Capinera (eds.). Pest management in the Louisiana and Florida (Epler et al. 2000, Stratman et al. subtropics: Biological control—A Florida perspective. Intercept, Ando- 2013b). It is noteworthy that the Australian hydrilla stem- ver, UK. boring weevil Bagous hydrillae O’Brien (Coleoptera: Curcu- Canfield DE, Jr., Langeland KA, Maceina MJ. 1983. Trophic state lionidae) that was released in Florida in 1991 and recently classification of lakes with aquatic macrophytes. Can. J. Fish. Aquat. Sci. 40:1713–1718. discovered in Louisiana (Center et al. 2013) exhibits a Cassani JR (ed.). 1996. Managing aquatic vegetation with grass carp: A guide similar field (or realized) host specificity. Both Elodea for water resource managers. American Fisheries Society, Introduced canadensis and Egeria densa were suitable laboratory hosts Fish Section, Bethesda, MD. 196 pp. for B. hydrillae but were not attacked in the field in Australia Center TD, Parys K, Grodowitz M, Wheeler GS, Dray FA, O’Brien CW, Johnson S, Cofrancesco A. 2013. Evidence of establishment of Bagous (Buckingham 1994, Balciunas et al. 1996). The B. hydrillae hydrillae (Coleoptera: Curculionidae), a biological control agent of case study clearly illustrates how laboratory tests often Hydrilla verticillata (Hydrocharitales: Hydrocharitaceae) in North Amer- overestimate field host range. ica? Fla. Entomol. 96:180–186. The recent discovery in Florida of endothall resistance in Colle DE, Shireman JV. 1980. 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