P1251328617MxNYU

1
DNA Analyses Used to Understand the Effect of
Acetamiprid And Acetamiprid plus Bifenthrin on
Invasive and Non-Invasive Subterranean Termites
Tracie. M. Jenkins1, Bruce W. Ryser2, Jim B.
Ballard3, Michael Fortunato4, Kathryn A. Harman1
and Kim Watson3. 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.
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.
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, www.omegabiotek.com) and
quantified by gel electrophoresis with a Low Mass
Ladder (Invitrogen, www.invitrogen.com). 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 (http//align.genome.jp) 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).
REFERENCES Avise, J. C. 1994. Molecular Markers,
Natural History and Evolution. Chapman Hall,
NY Clément, J. L. (1986). Sociobiology 11
311-323. Forschler, B. T. and R. M. Jenkins.
(2000). Trends in Entomol. 2 71-80. Jenkins, T.
M., Basten, C. J., Dean, R., Mitchell, S. e.,
Kresovich, S., and Forschler, B. T. (1998).
Sociobiology 33 239-263. Jenkins, T. M, R.E.
Dean, R. Verkerk, and B.T. Forschler. (2001)
MPE 20 286- 293 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. 4 233-236. Page, R.
D., M. (1996). Comput. Appl. Biosci. 12
357-358. Simon, C, Frati, F., Beckenbach, A.,
Crespi, B., Liu, H. and Flook, F. (1994) Ann.
Entomol. Soc. Am. 87 651-7-1 Szalanski, A. L.,
Scheffrahn, R. H., Austin, J. w., Krecek, J. and
Su, N-Y. (2004). Ann. Entomol. Soc. Am .
97556-566) Thompson, J. D., Higgins, D. G., and
Gibson, T. J. (1994). Nucleic Acid Res. 22
4673-4680.
 
 
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. 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 Officers Club in Building 53, 672 (3/8/05)
the Officers Club in building 53, 68 (6/29/04)
Building 39 and 84 (7/30/04) the Enlisted Mens
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.
 
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.
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.