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SZ Alan Ski 2003
SZ Alan Ski 2003
SZ Alan Ski 2003
(Isoptera: Reticulitermatidae)
from South Central United States by PCR-RFLP
Author(s): Allen L. Szalanski, James W. Austin, and Carrie B. Owens
Source: Journal of Economic Entomology, 96(5):1514-1519. 2003.
Published By: Entomological Society of America
DOI: http://dx.doi.org/10.1603/0022-0493-96.5.1514
URL: http://www.bioone.org/doi/full/10.1603/0022-0493-96.5.1514
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HOUSEHOLD AND STRUCTURAL INSECTS
SPECIES OF THE GENERA Reticulitermes Holmgren are the economically important species (Su and Scheffrahn
major termite pests infesting wooden structures in the 1990). Similarly, in Europe and other parts of the
United States and in other countries around the world. world, Reticulitermes spp. are the most expensive and
Subterranean termite species, such as those found in damaging pest species. The cost of treatment against
the genera Reticulitermes, live in moist soil habitats, termites in Europe is expected to top one billion euros
feeding on wood buried beneath or in contact with the by 2005 (UNEP and FAO 2000). In the south central
soil. It has been estimated that more than $1.5 billion region of the United States, four species of Reticuli-
is spent annually for termite control in the United termes occur: R. flavipes, R. virginicus, R. tibialis, and
States, of which 80% is spent to control subterranean R. hageni (National Termite Survey 2002, Weesner
termites (Su 1993). More recent estimates by the Na- 1965).
tional Pest Management Association suggest this g- Correct identication is critical for pest insects,
ure to be closer to $2.5 billion (NPMA 2003). A break- such as termites, that may require very different con-
down by costs caused by termite species, reveal that trol methods depending on the target species. Iden-
the ve principal subterranean termite species in the tifying workers is nearly impossible and separating
United States are Reticulitermes flavipes (Kollar); Re- soldiers is especially difcult given that precise mea-
ticulitermes virginicus (Banks), the dark southern sub- surements are required and overlap may occur be-
terranean termite; Reticulitermes hesperus (Banks), tween species (Scheffrahn and Su 1994). Difculties
the western subterranean termite; Reticulitermes tib- arise in species determination at individual collection
ialis (Banks), the arid subterranean termite; and Cop- sites because the worker caste is the most abundant
totermes formansanus (Shiraki). Ninety percent of the caste. Finding an alate in a collection is seasonal and
termite control business involves these ve subterra- quite rare. Soldiers represent only 13% of Reticuli-
nean termite species (Forschler and Lewis 1997). Sub- termes colonies and are morphologically variable; use
terranean termites of the family Rhinotermitidae, ge- of this caste alone can result in equivocal species
nus Reticulitermes, cause the most structural damage determinations. Subtle clinal variations imposed by
in the United States, and are considered the most geographic boundaries can be misleading in correct
a
COII DNA sequences data for the entire gene.
species determination. Molecular genetic methods are mination of species from a given population of
able to differentiate species regardless of the caste subterranean termites. This is the rst application of a
encountered or locality obtained. molecular genetics technique for the identication of
Previous research on Reticulitermes genetics has Reticulitermes termite taxa from the south central
focused on genetic variation within and among pop- United States.
ulations, or phylogenetic relationships within Reticu-
litermes and Isoptera but not molecular diagnostics
Materials and Methods
(Austin et al. 2002; Miura et al. 1998; Jenkins et al. 1998,
2001; Clement et al. 2001). Research on genetic vari- Termites were eld collected continuously from
ation within Reticulitermes, using DNA sequencing of 2000 to present at various locations in Canada, The
a portion of the mtDNA COII gene, has revealed Bahamas, Europe, and the United States, and pre-
sufcient variation to distinguish many species from served in 100% ethanol (Table 1 and see Table 5).
North America, Europe, and Asia (Austin et al. 2002). Morphological identication of 32 Reticulitermes was
Although developments in molecular biology have performed applying the taxonomic keys of Krishna
improved our ability to differentiate species, identi- and Weesner (1969), Scheffrahn and Su (1994), and
cation by DNA sequencing can be both costly and Hostettler et al. (1995) (Table 1). Voucher specimens,
time consuming. Polymerase chain reaction-restric- preserved in 100% ethanol, are maintained at the Ar-
tion fragment length polymorphism (PCR-RFLP) has thropod Museum, Department of Entomology, Uni-
become established as a reliable molecular technique versity of Arkansas, Fayetteville, AR.
for identifying economically important insects includ- Alcohol-preserved specimens were allowed to dry
ing Rhagoletis (Diptera: Tephritidae) species (Salazar on lter paper, and DNA was extracted from individ-
et al. 2002), boll weevil, Anthonomus grandis Boheman ual worker, soldier, or alate heads using the Puregene
(Coleoptera: Curculionidae) populations (Roehrdanz DNA isolation kit D-5000A (Gentra, Minneapolis,
2001), and Muscidifurax wasps (Hymenoptera: Ptero- MN). Extracted DNA was resuspended in 50 l of
malidae) (Taylor and Szalanski 1999). We chose PCR- Tris:EDTA and stored at 20C. PCR was conducted
RFLP for this study because the technique is simple using two different mtDNA primer sets. The rst
and inexpensive, very reliable and repeatable, and primer set, tercoII-idf (5-TCTTCTTCCACGAY-
provides discrete character states for quantitative CAYACAYTAATAA-3) and primer tercoII-idr (5-
comparisons of two or more species. TTTATGGGTAGTACYATTCGYTT-3) were used to
In this study, a PCR-RFLP technique was developed amplify a 379-bp portion of the 5 end of the COII
to study restriction site variation of two regions of the gene. The other primer set, 16S-r (5-CGCCTGTT-
mtDNA genome. Digestion of PCR products with spe- TATCAAAAACAT-3) (Simon et al. 1994), and 16S-f
cic restriction enzymes yielded diagnostic patterns (5-TTACGCTGTTATCCCTAA-3) (Kambhampati
for the identication and differentiation of Reticuli- and Smith 1995) amplify an 428-bp region of the 16S
termes termites, regardless of caste. This is of great rRNA gene. PCR reactions were conducted using 1 l
importance because of the frequent lack of morpho- of the extracted DNA per Szalanski et al. (2000), with
logically distinct caste members collected, namely sol- a prole consisting of 35 cycles of 94C for 45 s, 46C
diers or alates, which are essential for accurate deter- for 45 s, and 72c for 45 s. Amplied DNA from indi-
1516 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 96, no. 5
Table 2. Reticulitermes genetic divergence (%), COII above, Table 3. Restriction fragment length polymorphisms from the
16S below diagonal COII gene
Table 5. Identification of unclassified Reticulitermes samples species, correct identication of the species involved
can assist professional pest managers with understand-
N Pattern Pattern ing of why their respective control efforts might have
Identication Collection site
Colonies COIIa 16Sa
failed. Numerous tactics used by professional pest
R. flavipes Fayetteville, AR 3 AA AA managers employ the biology and ecological demands
Chauvin, LA 1 AA AA
Houma, LA 1 AA AA
of various termite species (e.g., baiting regimes for
Beaver Co., OK 1 AA AA colony suppression or elimination), which might be
Greer Co., OK 2 AA AA jeopardized because of subtle differences that can
McCurtain Co., OK 2 AA AA occur between the various species and subspecies of
Norman, OK 1 AA AA
Oklahoma City, OK 2 AA AA
Reticulitermes.
Payne Co., OK 1 AA AA Molecular diagnostics will aid in the identication
Spiro, OK 1 AA AA of future introductions of Reticulitermes species to the
Stillwater, OK 2 AA AA United States or in ongoing genetic studies that de-
Wagoner Co., OK 1 AA AA
Addison, TX 1 AA AA
mand correct identication to ascertain what popu-
Arlington, TX 2 AA AA lation the species in question may have originated
Austin, TX 1 AA AA from. Molecular diagnostics is also more cost effective
Balch Springs, TX 1 AA AA than cuticular hydrocarbon examinations, which re-
Beaumont, TX 1 AA AA
Beeville, TX 1 AA AA
quire more costly chromatographic techniques and
Blanco, TX 1 AA AA materials, generally more termites per location, and
Bryan, TX 1 AA AA may have chemical polymorphisms that vary among
Carrollton, TX 2 AA AA castes. Additionally, based on DNA sequence data
College St., TX 1 AA AA
Combine, TX 2 AA AA
(Austin et al. 2002), the 379-bp marker used in this
Corpus Christi, TX 2 AA AA study could be used to identify Reticulitermes lucifigus
Dallas, TX 18 AA AA and Reticulitermes grassei from Europe and Asia. Eu-
DeSoto, TX 1 AA AA ropean introductions of R. flavipes from the United
El Paso, TX 2 AA AA
Ft. Worth, TX 3 AA AA
States have been documented to have occurred in
Hempstead, TX 1 AA AA Germany (Becker 1970) and Austria (Hrdy 1961).
Irving, TX 2 AA AA More recently, introductions of damaging exotic Re-
Manseld, TX 2 AA AA ticulitermes spp. are known to occur in South America,
Milano, TX 2 AA AA
Odessa, TX 1 AA AA
including: R. lucifugus in Uruguay (Aber and Fontes
Post, TX 1 AA AA 1993) and R. flavipes in Santiago and Valparaiso, Chile
Red Water, TX 1 AA AA (Clement et al. 2001) where they have established
Rowlette, TX 2 AA AA themselves as serious structural pest of wooden tim-
Stephenville, TX 2 AA AA
Taylor, TX 1 AA AA
bers. In Europe, discoveries of R. grassei in southwest-
The Woodlands, TX 2 AA AA ern England have prompted new studies to assist in
Suffolk, VA 2 AA AA understanding the risks associated with their occur-
R. virginicus Fayetteville, AR 8 CB BB rence and their associated threat to structural timbers
McGehee, AR 2 CB BB
Columbia, MO 1 CB BB
in the United Kingdom. The anthropogenic introduc-
Oklahoma City, OK 1 CB BB tions of hardy Reticulitermes species (existing beyond
Bryan, TX 1 CB BB what would be considered their normal habitation
Roanoke, VA 2 CB BB range) are likely to continue. Clearly, problems with
R. hageni Conway, AR 1 BB AB
Fayetteville, AR 2 BB AB
the classication of synonymous species are another
R. tibialis Ft. Worth, TX 1 AB AC area in which having the alternative viewpoint of this
El Paso, TX 1 AB AC technique is not only valid, but necessary. This has
a
been demonstrated when looking at the synonymy of
Restriction enzyme pattern in the order HinfI, Taq I for COII and the R. lucifugus complex in Europe and the Middle
Dra I, TspR I for 16S (see Tables 2 and 3).
East (Austin et al. 2002), the R. hesperus complex in
California and the southwest United States (Haverty
nique using unclassied eld-collected Reticulitermes, and Nelson 1997, Haverty et al. 1999), and the various
the method is more reliable than predictions based on complexes of R. tibialis in the southwest United States
putative mutation rates. As a diagnostic tool, this pro- (Myles 2000). Quick and accurate identication of
cess could eliminate much of the ambiguity involved these species will be a daunting task for inexperienced
in morphological identication of species within the professional pest managers operating in areas in which
genus Reticulitermes or with other taxa within the previously undescribed species are now occurring.
order Isoptera. The PCR-RFLP molecular diagnostic Molecular diagnostics is just one way that we might
method presented herein can be used with workers, gain insight as to the origins of newly introduced
soldiers, or alate specimens. The ability to identify species so that intervention may be directed in the
specimens to species regardless of caste is a great most economically effective manner (whether iden-
advantage over traditional morphological methods in tifying the source of introduction, discriminating spe-
which keys are designed primarily for soldiers and cies for the application of corrective treatment mea-
alates. Although the control tactic used for most Re- sures, or for cataloging species with the intent to
ticulitermes infestations is unlikely to vary between correctly classify species that will likely be discovered
1518 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 96, no. 5
later). It also supports the possibility of classifying Haverty M. L., and L. Nelson. 1997. Cuticular hydrocarbons
samples currently in museums that have not yet of Reticulitermes from California indicate undescribed
been resolved. For example, within the insect col- species. Comp. Biochem. Physiol. 118B: 9598.
lection at Texas A&M University, College Sta- Haverty, M. L., L. Nelson, and B. Forschler. 1999. New
tion, TX (http://entowww.tamu.edu/new/research/ cuticular hydrocarbon phenotypes of Reticulitermes from
the United States. Sociobiology 34: 121.
systematics/collection.html), there are presently 227
Heiman, M. 1997. Webcutter 2.0. (http://www.ccsi.com/
Reticulitermes samples, of which only 96 have been rstmarket/cutter/cut2.html).
classied to species (85% R. flavipes, 7% R. virginicus, Hostettler, N. C., D. W. Hall, and R. H. Scheffrahn. 1995.
7% R. hageni, and 1% R. tibialis). Fifty-eight percent, Intracolony morphometric variation and labral shape in
representing 131 vials, have not yet been classied to Florida Reticulitermes (Isoptera: Rhinotermitidae) sol-
species. diers: signicance for identication. Flor. Entom. 78: 119
The PCR-RFLP method presented herein was de- 129.
veloped to provide researchers and professional pest Hrdy, I. 1961. Contribution to the knowledge of European
managers with a rapid and simple molecular method species of the genus Reticulitermes. Act faun. Entomol.
for accurately differentiating four species of Reticuli- Mus. Nat. Prague 7: 97107.
termes encountered in the south central United States, Jenkins, T. M., C. J. Basten, R. Dean, S. E. Mitchell, S.
Kresovich, and B. T. Forschler. 1998. Matriarchal ge-
and could be used to identify Reticulitermes species
netic structure of Reticulitermes (Isoptera: Rhinotermiti-
that may be introduced to the United States. Field- dae) populations. Sociobiology 33: 239 263.
collected samples can be preserved in ethanol at the Jenkins, T. M., R. E. Dean, R. Verkerk, and B. T. Forschler.
collection site and stored for several years until iden- 2001. Phylogenetic analysis of two mitochondrial genes
tication is required. The PCR amplication is robust, and one nuclear intron region illuminate European sub-
and the use of a small amplicon, 450 bp, facilitates the terranean termite (Isoptera: Rhinotermitidae) gene ow,
use of specimens that have not been optimally pre- taxonomy, and introduction dynamics. Molec. Phylog.
served (Taylor et al. 1996). The restriction enzymes Evol. 20: 286 293.
used in this study are economical, costing less than Kambhampati, S., and P. T. Smith. 1995. PCR primers for
$12.50 per 1,000 U, and yield fragments that can be the amplication of four insect mitochondrial gene frag-
readily visualized under less-than-optimal PCR and ments. Ins. Molec. Biol. 4: 233236.
Krishna, K., and F. M. Weesner. 1969. Biology of termites,
electrophoretic conditions. The entire set of proce-
vol. 1. Academic, New York.
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be completed within a single working day. niques to identify freshwater eels: RFLP analysis of PCR-
amplied DNA fragments and allele-specic PCR from
mitochondrial DNA. Zool. Stud. 41: 421 430.
Miura, T., K. Maekawa, O. Kitade, T. Abe, and T. Matsumoto.
Acknowledgments
1998. Phylogenetic relationships among subfamilies in
We thank R. Gold, B. Foster, B. Kard, M. Haey, C. Casta- higher termites (Isoptera: Termitidae) based on mito-
lano, U. Noldt, R. Houseman, T. Myles, G. Henderson, D. chondrial COII gene sequences. Ann. Entomol. Soc. Am.
Miller, and R. Scheffrahn for providing samples. C. D. Steel- 91: 515523.
man provided helpful suggestions and critical reviews of the Myles, T. G. 2000. Evaluation of the occurrence of termite
manuscript. Research was supported in part by the University species and potential termite burrowing depths in the
of Arkansas, Arkansas Agricultural Experiment Station. area of the Nevada test site: present day and for the next
10, 000 years. Special Report, Neptune and Company,
Inc., Los Alamos, NM, Supplemental for review of the
NTS GCD PA by Sandia National Laboratory. Document
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