DNA Analyses Used to Understand the Effect of Acetamiprid And Acetamiprid plus Bifenthrin on Invasive and Non-Invasive Subterranean Termites Tracie. M. Jenkins 1, Bruce W. Ryser 2, Jim B. Ballard 3, Michael Fortunato 4, Kathryn A. Harman 1 and Kim Watson 3. 1) Dept. of Entomology, The University of Georgia, Griffin, GA 30223; 2) FMC Corporation, 9703 Cypress Pond Ave, Tampa, FL, (3) FMC Corporation, 1735 Marker St, Philadelphia, PA (4) Dept. of Chemistry, Pennsylvania State University, University Park, PA ABSTRACT We report on the preliminary results of the largest longitudinal efficacy study undertaken in the United States to date. The purpose of the study, which includes multiple sites in each of five states, is to evaluate the effectiveness of the liquid non-repellent insecticide F-5025 (acetamiprid) and F-4688 (acetamiprid + bifenthrin) against indigenous and exotic subterranean termites using mitochondrial sequence (haplotype) data. Baseline haplotypes for 82 Louisiana sites, including “pre-Katrina” collections in New Orleans, is presented as is an overview of protocols designed for FMC that preserve specimen DNA as well as provide easy voucher access for future studies. Pre-treatment DNA baseline data not only provided insights into post-treatment site-specific DNA results but also provided inference of insecticide transmission among collection sites. MATERIALS AND METHODS Samples were collected from termite infested structures in Louisiana with active mud tubes and trees in New Orleans that tested positive for triple-mark-release-recapture studies (Ryser, personal communication) (Table 1 and Figure 1). All samples were processed in the field according to established protocols that included putting all termites in 100% EtOH and then shipped to T. M. Jenkins’ UGA lab on the Griffin Campus where samples were emptied of old EtOH, cleaned of any debre, and transferred to new vials with 100% EtOH. Each collection was logged into a computer managed program designed to record collection-specific historical data, and track long-term storage for easy voucher retrieval. DNA extraction was accomplished on head and thorax from three to five individuals from each of 82 sites using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek, and quantified by gel electrophoresis with a Low Mass Ladder (Invitrogen, These field sites compose the baseline sites. Post-treatment collection haplotypes were compared to pre-treatment haplotypes and to other closely connected sites to determine if there was inference for insecticide transmission. Sequencing was accomplished on mitochondrial polymerase chain reaction (PCR) amplified products in both directions. The COII gene was amplified and sequenced using oligonucleotides TL2J3037 and TKN3785 (Leu and Bechenbach 1992; Simon et al. 1994). Sequencing was done at the University of Georgia sequencing facility in Athens, GA. The maternity of termites that appeared post-treatment (Table 1) with the same COII haplotype were confirmed by sequencing PCR amplified COI (Jenkins et al, 2001; Simon et al. 1994) and 16SrRNA gene fragments (Kambhampati and Smith 1995, Szalanski et al, 2004). Statistical analysis had four parts: 1) Sequencher was used for initial sequence alignment and analysis, to make contigs and identify consensus sequences; 2) CLUSTAL W, v.1.83, (Thompson et al., 1994) was used to align sequences prior to phylogeny analysis ( 3) maximum-likelihood phylogeny analysis was accomplished using PHYLIP 3.6 (Felsenstein, 1993) and, 4) haplotype, genera and species verification trees were generated in TREEVIEW (Page, 1966). RESULTS AND DISCUSSIOIN A haplotype differential between Reticulitermes species and Coptotermes formosanus was observed, as expected since C. formosanus is an exotic introduction. Thus the founder effect and haplotype pruning would operate on all C. formosanus populations (Avise 1994). We are exploring the question, therefore, of Coptotermes formosanus haplotype variation as it reflects efficacy. A haplotype differential between Reticulitermes species and Coptotermes formosanus was observed, as expected since C. formosanus is an exotic introduction. Thus the founder effect and haplotype pruning would operate on all C. formosanus populations (Avise 1994). We are exploring the question, therefore, of Coptotermes formosanus haplotype variation as it reflects efficacy. Transmission. Previous to this study B. Ryser discovered dead termites at untreated sites. He questioned if insecticide transmission could account for his observation. We discovered a single haplotype, LA-R4, at six locations within the Jackson Barracks complex (Table 1). These locations were identified (Table 1) by numbers and dates as 46 (5/17/04) the Museum, 47 (5/17/04) the Theater, 67 (6/11/04) the Officer’s Club in Building 53, 67#2 (3/8/05) the Officer’s Club in building 53, 68 (6/29/04) Building 39 and 84 (7/30/04) the Enlisted Men’s Club. F-5025 and F-4688 could have been transmitted through trophallaxis, which may account for the fact that termites with LA-R4 haplotype or any haplotype have not been collected at these sites for over a year. Numbers 157 through 160 also had a single haplotype, LA-C2, corresponding to affected trees in New Orleans City Park near Tad Gormley Stadium (Figure 1 a, b, c, d) and numbers 161, 162, 174 and 175 had haplotype GA1 (Figures 1 and 2), first characterized in Atlanta, GA (Jenkins et al 2002) as exotics possibly from New Orleans. LA-C2 and GA1 maternal lineages have been verified from 16SrRNA and COI gene fragment sequencing. These observations could also be indicative of transmittance through a single maternal population. We will continue to monitor the City Park sites as the water left behind by Katrina recedes. Other sites that show single haplotypes over a larger range (radius of 5 miles) include Reticulitermes haplotypes in Baton Rouge, LA-R20 (15 sites), LA-R22 (8 sites), LA-R15 (10 sites). All of these haplotypes are being verified for maternal lineage by sequencing the COI gene fragment. Pre- and post-treatment haplotypes. Site 119 treated with F-4688 had a pre-treatment haplotype of LA-C3 (Coptotermes) and a post-treatment haplotype of LA-R7 (Reticulitermes). Sites 51, 61 and 70 also showed different haplotypes pre- and post-treatment with F-5025 and F-4688 respectively, but the haplotypes both represented indigenous Reticulitermes. The time between pre- and post-treatment for sites 51, 61, 71, and 119 in months was 23, 8, 12, and 8.2 respectively. Thus, the original termites could have been cleared before new termites moved into the void. This demonstrates the importance of a longitudinal study in order to keep data analysis from being assumption driven (Forschler and Jenkins 2000). We continue to monitor these sites monthly. Sites 157, 159 and 160 all had the same pre-and post-treatment haplotype (LA-C2) (Table 1). Since it was less than a month (0.9 months) between treatment and sampling, and the object of treatment was an oak tree, this could indicate that instead of monthly checks we should check these sites every two months in order to give treatments time to work. Multiple haplotypes were observed for five Reticulitermes flavipes. Two of these sites were at the Jackson Barracks complex, 51 (2/7/03) and 67 (3/8/05) and three were at Baton Rouge sites, 105, 132 and 136. These observations could be indicative of open, meta-colonies (Clement 1986; Jenkins et al. 1999; Forschler and Jenkins 2000) that future DNA studies will help to delineate. This result shows why multiple termites per site were sampled. REFERENCES Avise, J. C Molecular Markers, Natural History and Evolution. Chapman & Hall, NY Clément, J. L. (1986). Sociobiology 11: Forschler, B. T. and R. M. Jenkins. (2000). Trends in Entomol. 2: Jenkins, T. M., Basten, C. J., Dean, R., Mitchell, S. e., Kresovich, S., and Forschler, B. T. (1998). Sociobiology 33: Jenkins, T. M, R.E. Dean, R. Verkerk, and B.T. Forschler. (2001) MPE: 20: Felsenstein, J. PHYLIP (Phylogeny Inference Package) Manual (University of Washington, Seattle 1993). Distributed by the Author Felsenstein, J. PHYLIP (Phylogeny Inference Package) Manual (University of Washington, Seattle 1993). Distributed by the Author Kambhampati, S., and P. T. Smith. (1995). Insect Mol. Biol. 4: Page, R. D., M. (1996). Comput. Appl. Biosci. 12: Simon, C, Frati, F., Beckenbach, A., Crespi, B., Liu, H. and Flook, F. (1994) Ann. Entomol. Soc. Am. 87: Szalanski, A. L., Scheffrahn, R. H., Austin, J. w., Krecek, J. and Su, N-Y. (2004). Ann. Entomol. Soc. Am. 97: ) Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). Nucleic Acid Res. 22: ACKNOWLEDGEMENTS We are grateful to Drs. Gary Pederson and Ming Li Wang, USDA/ARS Plant Genetics Resource Unit, for their continued support of this work by providing lab space and consultation on molecular techniques. We also appreciate John D. Youmans for editing and printing this poster. We are grateful to Drs. Gary Pederson and Ming Li Wang, USDA/ARS Plant Genetics Resource Unit, for their continued support of this work by providing lab space and consultation on molecular techniques. We also appreciate John D. Youmans for editing and printing this poster. Figure 1. Aerial Views of Tad Gromley and Roosevelt Mall sites before and after Hurricane Katrina (August 29, 2005): a) Tad Gromley Stadium before Katrina with trees 1, 2 and Roosevelt Mall tree 6 identified, which correspond to numbers 156, 157 and 174 respectively in Table 1; b) Tad Gromley Stadium and Roosevelt Mall Trees 6 and 7 after Katrina corresponding to numbers 174 and 175 respectively in Table 1; c) Tad Gromley Trees 1 (156) and 2 (157) and Roosevelt Mall trees 6 (174) and 7 (175) before Katrina; d) Tad Gromley Stadium trees 1 and 2 and Roosevelt Mall trees 6 and 7 after Katrina. INTRODUCTION Although we report preliminary results for a single state, Louisiana, the overall purpose of this study was to use cytochrome oxidase II (COII) mitochondrial DNA (mtDNA) sequence haplotypes to evaluate the long term efficacy (Jenkins et al. 1999) of F-5025 (acetamiprid) and F-4688 (acetamiprid + bifenthrin) for treating termite infestations. The study was designed for a minimum period of two years. The objectives were to: 1) develop a termite collection protocol designed to preserve specimen DNA, 2) delineate through graphs and photos all collection sites for future reference, 3) design a voucher system for all samples collected that facilitates easy access and DNA processing, 4) edit all DNA sequence data and use phylogeny analysis to confirm genera, species and define haplotypes (Jenkins et al 2001, 2002), 5) establish pre-treatment DNA baselines for collection sites in order to evaluate post-treatment collections and transmission among termites; and, 6) confirm the maternity of a cytochrome oxidase II (COII) haplotype that appears pre- and post-treatment by sequencing 700 bp of a cytochrome oxidase I (COI) and 450 bp of a 16SrRNA. gene fragment. Table 1. Louisiana haplotype data: Pre-and post-treatment sites identified in red when post-treatment haplotype was different and in blue when the pre- and post-treatment haplotypes were the same. Only the baseline (pre-treatment) haplotype is recorded unless post-treatment termites were observed. The percentages in parentheses under treatment refer to the proportion of bifenthrin in F a b c d Figure 2. a) Aerial view of Roosevelt Mall, trees 6 and 7, numbers 174 and 175 respectively, after Hurricane Katrina. Each had haplotype GA1 first observed in Atlanta, GA; b) close-up view of tree 6 (175) one week after Hurricane Katrina.