SYSTEMATICS Survey and Identification of Termites (Isoptera: Rhinotermitidae) in Indiana CHANGLU WANG,1,2,3 XUGUO ZHOU,3,4 SHUJUAN LI,5 MARGARET SCHWINGHAMMER,6 7 MICHAEL E. SCHARF, GRZEGORZ BUCZKOWSKI, AND GARY W. BENNETT Center for Urban and Industrial Pest Management, Department of Entomology, Purdue University, West Lafayette, IN 47907 Ann. Entomol. Soc. Am. 102(6): 1029Ð1036 (2009) ABSTRACT In total, 289 termite samples were collected from 45 counties in Indiana during 2002Ð 2004. Approximately 89% of the collection sites were associated with artiÞcial structures, and almost half of the samples were from inside homes. The rest of the samples were from forested areas. Termite samples were identiÞed based on their morphological characteristics, molecular characteristics, or both. Five species from the genus Reticulitermes were identiÞed, and the relative abundance (per- centage of the total collections) of these Þve species was Reticulitermes flavipes (Kollar) (90.0), Reticulitermes virginicus (Banks) (7.6), Reticulitermes arenincola (Goellner) (1.0), Reticulitermes tibialis (Banks) (1.0), and Reticulitermes hageni Banks (0.3). Based on the distribution map, R. flavipes was the dominant and the most widely distributed species in Indiana (44 counties); followed by R. virginicus (13 counties). The three other Reticulitermes species, R. arenincola, R. tibialis, and R. hageni, were encountered in only Þve counties. R. arenincola is considered a rare species and its distribution has been limited to sand dunes near Lake Michigan. However, in this study, two of the three R. arenincola samples were collected outside of its type location. R. tibialis was found in three counties, whereas R. hageni was only found in Evansville, IN. To complement the morphological identiÞcations, a 389-bp region of the mitochondrial DNA (mtDNA) 16S rRNA gene was ampliÞed and sequenced from all Þve Reticulitermes species. Based on species-speciÞc polymorphisms exhibited in mtDNA sequences, a polymerase chain reaction-restriction fragment length polymorphismÐbased diagnostic tool was developed to identify samples lacking of diagnostic morphological characters. KEY WORDS termite, survey, 16S rRNA, polymerase chain reaction-restriction fragment length polymorphism, Reticulitermes Termites are the most important and most efÞcient building material in the United States because it has lignocellulose (e.g., dead wood) decomposers (Sugi- been readily available in most parts of the country. moto et al. 2000). They play a vital role in recycling With sufÞcient moisture, these wood materials be- wood and plant materials, modifying soil condition, come the ideal food sources for subterranean termites. improving soil composition and fertility, and providing Drywood termites and subterranean termites are the food for other animals (Lee and Wood 1971). How- two major termite groups that have the greatest eco- ever, because of their large colony size, nesting be- nomic impacts in North America, with subterranean havior, and feeding preferences, termites can cause termites causing Ͼ80% of the damage, and drywood considerable damage to artiÞcial structures and com- termites causing the other 20% (Su and Scheffrahn modities. Su (2002) estimated that the overall damage 1990). Subterranean termites from the genera Reticu- and cost to control termites exceeds $20 billion an- litermes and Coptotermes arguably are the most eco- nually worldwide. Wood has long been the primary nomically important species. Furthermore, Reticuli- termes is the most widespread termite genus in North 1 Current address: Department of Entomology, Rutgers University, America, with seven described species, including Re- New Brunswick, NJ 08901. ticulitermes flavipes (Kollar), Reticulitermes virginicus 2 Corresponding author, e-mail: [email protected]. (Banks), Reticulitermes tibialis (Banks), Reticuli- 3 These authors contributed equally. 4 Current address: Department of Entomology, University of Ken- termes arenincola Goellner, Reticulitermes hesperus tucky, Lexington, KY 40546. Banks, Reticulitermes hageni Banks, and Reticulitermes 5 Current address: University of Arizona, Maricopa Agricultural malletei (nomen nudum) (Austin et al. 2007). Center, Maricopa, AZ 85238. Termite species vary in their basic biology and ecol- 6 Current address: 2121 Jamieson Ave., #910, Alexandria, VA 22314. 7 Current address: Entomology and Nematology Department, Uni- ogy, including colony size, nesting, feeding, swarming, versity of Florida, Gainesville, FL 32611. and reproductive behavior. Proper identiÞcation of 0013-8746/09/1029Ð1036$04.00/0 ᭧ 2009 Entomological Society of America 1030 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6 termite species and knowledge of their distribution frahn et al. 1988), South Carolina (Hathorne et al. are the Þrst steps in developing environmentally 2000), Georgia (Scheffrahn et al. 2001), Mississippi compatible/sustainable integrated pest management (Wang and Powell 2001), Louisiana (Messenger et al. (IPM) strategies. Of the 2,600 known termite species, 2002), Ohio (Jones and Nuss 2002), Arkansas (Austin only a fraction (Ϸ40 species) have been identiÞed in et al. 2004), Oklahoma (Brown et al. 2004), Nebraska the United States (Kambhampati and Eggleton 2000). (Husen et al. 2006), and Missouri (Pinzon and House- IdentiÞcation of termite species is a challenging task man 2009). There is no termite survey data available due to the ambiguity in their morphological charac- for Indiana. To Þll this gap we have 1) investigated the ters, the difÞculty of collecting morphologically dis- diversity, distribution, and abundance of the termite tinct castes (e.g., soldiers and alates), geographical species in Indiana; 2) developed a termite identiÞca- variations, and the overall lack of termite systematic tion key with quantiÞable morphological characters; studies. This is especially true for the genus Reticuli- and 3) developed a PCR-RFLPÐbased molecular di- termes that notoriously lacks discrete morphological agnostic tool to complement morphological identiÞ- characters and rightfully deserves a thorough system- cations. atic revision (Nutting 1990, Scheffrahn and Su 1994). Developmental end points, such as soldier and alate Materials and Methods castes, have been used extensively in the morpholog- ical identiÞcation of Reticulitermes species. In con- Termite Survey. Survey letters and collection kits trast, the worker caste is indistinguishable morpho- were sent to pest management companies in Indiana logically. with help from the Indiana Pest Management Asso- Molecular techniques can circumvent the limita- ciation in 2002 and 2003. Each collection kit included tions of morphological assessments, and have been general instructions, 10 collection vials containing adopted for the study of termite classiÞcation/iden- 100% ethanol and two postage-paid return envelopes. tiÞcation, phylogenetic analyses (Kambhampati et al. Each vial was individually sealed in a plastic zip-lock 1996; Miura et al. 1998; Cle´ment et al. 2001; Austin et bag. Each bag was provided with a label for Þlling in al. 2004; Szalanski et al. 2003, 2004) and population the collection information, including the collection genetics (Jenkins et al. 1998, 2001; Marini and Man- date, county, mailing address, habitat type, and the tovani 2002; Austin et al. 2002, Ye et al. 2004). Mito- collectorÕs name. In total, 62 collection kits were sent chondrial genes are known to evolve more rapidly and out in April 2002 and 82 kits were sent in February their DNA to be more abundant relative to nuclear 2003. In total, 262 samples (vials) were returned. In genes; therefore, they are the most widely used addition, the authors collected 27 samples. Among marker in molecular identiÞcations (Simon et al. these 27 samples, 11 were from dead wood materials 1994). Polymerase chain reaction (PCR)-based re- at Dune Acres, Porter County, and 16 were from striction fragment length polymorphism (RFLP) infested structures in Tippecanoe County or sur- techniques readily separate closely related taxons by rounding counties. Dune Acres is located at the center their taxon-speciÞc mitochondrial DNA (mtDNA) of the R. arenincola distribution area described by markers and have been used extensively in molecular Goellner (1931). Each collection (vial) returned was systematic and phylogenetic analyses, especially after assigned a number for future reference. Subsequently, the introduction of universal primers (Simon et al. 1994, half of the termites in each vial were transferred to a Loxdale and Lushai 1998). rRNA (e.g., 12S and 16S), vial containing 75% ethanol for morphological iden- tRNA, and protein coding genes (e.g., cytochrome oxi- tiÞcation, and the other half were transferred to a vial dase [COI] I and II) are the primary mtDNA markers containing 100% ethanol for molecular identiÞcation. used in molecular identiÞcation of Reticulitermes species Morphological Identification. Collections contain- (Austin et al. 2002, Szalanski et al. 2003, Ye et al. 2004). ing soldiers, alates, or both were identiÞed to species Among them, 16S rRNA has been proven to be an ac- by their morphological characters under an SZ 60 curate, reliable, repeatable, and inexpensive mtDNA dissecting microscope (Olympus, Vienna, Austria). marker for the termite identiÞcation, especially for Re- Species determination was based on previous descrip- ticulitermes species (Szalanski et al. 2003; Austin et al. tions by Goellner (1931), Weesner (1965), Nutting 2005a,b; Tripodi