James W. Austin

A Comparative Genetic Analysis of the Subterranean Termite Genus Reticulitermes (Isoptera: Rhinotermitidae)

Abstract DNA sequencing analysis of the mitochondrial DNA cytochrome oxidase II (COII) region was used to examine genetic variation in the termite genus Reticulitermes Holmgren. We examined 21 species and subspecies from three continents. Sequencing of a 677-bp region of a 780-bp amplicon from 41 individuals and from 17 sequences obtained from GenBank revealed 221 polymorphic sites within the genus. Tajima-Nei distances from species ranged from 0.9 to 12.7%, and parsimony and maximum likelihood analysis revealed several clades within the genus. Reticulitermes flavipes (Kollar) formed a distinct clade along with R. santonensis De Feytaud. European R. lucifugus (Rossi) formed a distinct clade with R. banyulensis (Béziers). Turkish R. lucifugus was distinct relative to European R. lucifugus, and along with R. clypeatus Lash from Israel formed a sister group with R. balkanensis Clément from Greece. This study provides support for the separation of Turkish R. lucifugus from European members of the species. This mitochondrial DNA marker was also able to identify several Reticulitermes specimens from Oklahoma, Texas, Missouri, and South Korea to R. flavipes, R. hageni Banks, R. virginicus (Banks), and R. speratus Shimizu.

Mitochondrial and Ribosomal Internal Transcribed Spacer 1 Diversity of Cimex lectularius (Hemiptera: Cimicidae)

Abstract Understanding genetic variation among populations of medically significant pest insects is important in studying insecticide resistance and insect dispersal. The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is widespread hematophagus insect pest around the world, including North America, and it has recently been identified as an emerging resurgent pest. To date, no studies have been conducted on genetic variation of this species. For this study, 136 adult bed bugs representing 22 sampled populations from nine U.S. states, Canada, and Australia were subjected to genetic analysis using polymerase chain reaction (PCR) to amplify and sequence a region of the mitochondrial DNA (mtDNA) 16S rRNA gene and a portion of the nuclear rRNA internal transcribed spacer (ITS) 1 region. For the 397-bp 16S marker, a 12 nucleotide sites in total were polymorphic, and 19 unique haplotypes were observed. Heterozygosity was observed within many of the sampled populations for the mtDNA marker. This suggests that bed bug populations did not undergo a genetic bottleneck as one would expect from insecticide control during the 1940s and 1950s, but instead, that populations may have been maintained on other hosts such as birds and bats. In contrast to the high amount of heterozygosity observed with the mitochondrial DNA marker, no genetic variation in the 589-bp nuclear rRNA marker was observed. This suggests increased gene flow of previously isolated bed bug populations in the United States, and given the absence of barriers to gene flow, the spread of insecticide resistance may be rapid.

Genetic Variation of Reticulitermes flavipes (Isoptera: Rhinotermitidae) in North America Applying the Mitochondrial rRNA 16S Gene

Abstract A molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA 16S gene was undertaken to determine the extent of genetic variation within Reticulitermes flavipes (Kollar) in North America. This study was done because differences in morphological variants (of R. flavipes) would presumably translate into genetic differences, and there are no prior studies that describe its genetic variation from the extent of its North American range. In total, 493 samples were analyzed from the United States, Canada, and Mexico. Nineteen nucleotide sites were variable in the 428-bp mitochondrial DNA (mtDNA) sequence, and 47 mtDNA haplotypes were observed. Nine haplotypes (19%) occurred only once, whereas the most common haplotype, F, occurred in 17% of the samples. Four haplotypes were found over a broad geographical range encompassing at least nine states each. The single haplotype found in Toronto, Canada, also occurs in Arkansas, whereas two of the three haplotypes found in Mexico are unique to that country. Based on this research, there seems to be numerous R. flavipes haplotypes that are widespread, perhaps due to human involvement, whereas other haplotypes may be more rare and could represent locally adapted populations.

Susceptibility of the Bed Bug Cimex lectularius L. (Heteroptera: Cimicidae) Collected in Poultry Production Facilities to Selected Insecticides 1

Abstract Cimex lectularius L. is a widespread hematophagus insect pest around the world and is currently experiencing a reemergence as a public health pest of concern. One possible source of bed bugs to the human environment is the movement of bed bugs from poultry facilities to human structures by poultry workers. No recent studies have been conducted on the susceptibility of this insect to a wide range of insecticides. In addition, populations of bed bugs from poultry facilities have not been screened against insecticides for over 15 yr. Adult bed bugs collected from three poultry facilities in northwest Arkansas were exposed for 24 or 48 h (25°C) to glass vials treated with various dilutions of 12 insecticides dissolved in acetone to determine the concentration–response relationship. The order of toxicity, from most to least based on the LC50s was: λ-cyhalothrin, bifenthrin, carbaryl, imidacloprid, fipronil, permethrin, diazinon, spinosyn, dichlorvos, chlorfenapyr, and DDT. Significant differences in LC50 and LC90 values for diazinon was observed among the three populations due to the previous history of repeated exposure to a mixture of tetrachlorvinphos and dichlorvos over a 10 yr period when compared to the LC50s of two populations that had been exposed to the tetrachlorvinphos and dichlorvos mixture during 2–3 flock cycles. Bed bugs in each of the three populations exhibited high levels of DDT resistance, LC50 > 100,000 ppm, which confirms that resistance to this insecticide continues in bed bug populations. This study documents baseline toxicological data for 12 insecticides in three populations of bed bugs and provides the first data on bed bug susceptibility to fipronil, spinosyn, and imidacloprid.

Genetic evidence for two introductions of the Formosan Subterranean Termite, Coptotermes formosanus (Isoptera: Rhinotermitidae), to the United States.

Abstract Exotic introductions of Formosan Subterranean Termite (FST) to the United States from Asia have had significant economic consequences. Multiple introductions through marine transport have been proposed, but identification of these routes has yet to reveal more than one lineage in the continental U.S. DNA sequencing of a 640-bp cytochrome oxidase II (COII) mitochondrial DNA (mtDNA) marker to 60 disjunct populations, revealed two independent lineages spanning the continental U.S., Hawaii, Japan, and China. Limited genetic variation was observed with this marker. Group I constitutes a largely Asian clade, while Group II is comprised of both Asian and southern U.S. populations. This is the first study which has documented 2 distinct lineages to continental United States and Hawaii.

MOLECULAR DIAGNOSTICS OF THE FORMOSAN SUBTERRANEAN TERMITE (ISOPTERA: RHINOTERMITIDAE)

Abstract Formosan subterranean termite, Coptotermes formosanus Shriaki, is a serious pest of structures in portions of United States. A 467-bp region of the mtDNA 16S rRNA gene was subjected to DNA sequencing from 12 Coptotermes species, including 64 populations of C. formosanus. Genetic diversity among species ranged from 1.8% to 7.0%, with C. formosanus at least 3.0% divergent to the other Coptotermes taxa. No genetic variation was detected among the C. formosanus populations for this marker making it ideal for diagnostics. Comparison of nucleotide sequence of mitochondrial rRNA 16S was used to design polymerase chain reaction (PCR) primers specific for C. formosanus. The diagnostic assay consists of two independent PCR runs of the 16S primer pair along with the C. formosanus primer set. PCR product from samples that are not C. formosanus can be subjected to DNA sequencing and compared with the database of termite 16S sequences on GenBank for identification. This technique provides a non-morphological method to identify field collected termites and may facilitate future quarantine programs for C. formosanus.

MITOCHONDRIAL DNA VARIATION AND DISTRIBUTION OF THE SUBTERRANEAN TERMITE GENUS RETICULITERMES (ISOPTERA: RHINOTERMITIDAE) IN ARKANSAS AND LOUISIANA

Abstract Limited information exists on genetic variation and distribution of Reticulitermes from the south central United States. Focusing on molecular sequence data from the mitochondrial DNA 16S gene, this study records the distribution and genetic variation of Reticulitermes species in Arkansas and updates the current distribution in a neighboring State, Louisiana. Termite samples were collected from the field, subjected to DNA analysis with Polymerase Chain-Reaction (PCR), and sequenced. Reticulitermes sp. sequence data were aligned, genetic distances recorded, and their respective haplotypes were evaluated for possible geographic structure. From 35 Arkansas counties, 59 R. flavipes, 13 R. hageni, and seven R. virginicus were identified. In Arkansas, 11 mitochondrial haplotypes were observed in R. flavipes, three in R. hageni and three in R. virginicus. Among the 12 Louisiana parishes sampled, 13 R. flavipes, three R. virginicus, and one R. tibialis were identified with six, three, and one haplotypes for each species, respectively. Genetic variation among the R. flavipes haplotypes from both States ranged from 0.2 to 0.9%. Reticulitermes flavipes haplotype diversity observed in Arkansas and Louisiana was lower than observed in Texas and Oklahoma.

Synonymy of Two Arboreal Termites (Isoptera: Termitidae: Nasutitermitinae): Nasutitermes corniger from the Neotropics and N. polygynus from New Guinea

Abstract Morphological examination of soldiers and imagos assigned to Nasutitermes polygynus from New Guinea were determined to be conspecific with the neotropical species, N. corniger. A portion of the mtDNA 16S rRNA gene was sequenced from nine N. corniger samples and found to be congruent with that reported for N. polygynus. Complementary biological, behavioral, chemical, and reproductive ecology data further support this synonymy. Nasutitermes corniger was likely introduced to New Guinea as a result of accidental human transport.

FIRST RECORD OF RETICULITERMES FLAVIPES AND RETICULITERMES HAGENI IN OREGON (ISOPTERA: RHINOTERMITIDAE)

The majority of pestiferous subterranean termites in North America belong to the endemic genus Reticulitermes (Isoptera: Rhinotermitidae). Reticulitermes flavipes (Kollar), the eastern subterranean termite, is the most economically important (Su 1993) and widespread termite (Austin et al. 2005a) in the United States. Existing taxonomic studies provide information on only 1 Reticulitermes spp. in Oregon, R. hesperus (Banks), the western subterranean termite, which is the most common termite pest species found from southern British Columbia to central California (Snyder 1954; Weesner 1965). Distribution studies on Pacific Northwest species of Reticulitermes have been addressed by Castle (1928) and Light & Pickens (1934). We report herein findings of two unreported Reticulitermes spp. (R. flavipes and R. hageni) from Oregon. Soldiers, if available, and worker termites were collected from a total of 79 different colonies from 34 locations in Oregon located in the following counties: Clackamas, Coos, Jackson, Klamath, Lane, Linn, Marion, Multnomah, Polk, Umatilla, Washington, and Yamhill by our own collection efforts, by Pest Management Professionals (PMPs), and through the 2002 national termite survey. A 428-bp region of the mt-DNA 16s rRNA gene was amplified by PCR from 79 samples consisting of 1 worker from each colony and subjected to DNA sequencing per Szalanski et al. (2003). Two R. flavipes soldier specimens collected from a colony at a collection site in Keizer, OR were identified morphologically, applying keys of Scheffrahn & Su (1994) and by evaluating soldier labra (Hostettler et al. 1995), and confirmed genetically via sequence data from a worker specimen (Szalanski et al. 2003). A worker specimen from a collection site in Salem, OR was identified genetically from sequence data as R. hageni. Soldiers were not collected from this colony and morphological identification was not performed. Both Keizer and Salem are located within 50 km from Portland, OR. Reticulitermes flavipes is the most widely distributed Reticulitermes, and is found in the entire eastern region of North America as far as Ontario, Canada, and south to Florida (Snyder 1954; Weesner 1965). The known western distribution of the species extends through the central plains to the Rocky Mountains and down to Monterrey, Mexico (Banks & Snyder 1920; Snyder 1954; Weesner 1965; Messenger 2003). Austin et al. (2005a) reported the first occurrence ofR. flavipes in California and Nevada, extending its distribution westward. The presence of R. flavipes in western states has subsequently been independently verified (Su et al. 2006; Tripodi et al. 2006). The R. flavipes 16S rRNA haplotype was FF (GenBank Accession D2001958), which is predominately found in the eastern United States (Austin et al. 2005a). Because this is outside of the previou ly known distributions of both R. flavipes and R. hageni (Banks & Snyder 1920; Snyder 1954; Weesner 1965; Messenger 2003; Austin 2005a), eastern introductions to Oregon from anthropogenic sources are implicated. Because R. flavipes is a primary pest of structures in the United States (Austin et al. 2005a) and around the world (Scheffrahn et al. 1999; Austin et al. 2005b; Su et al. 2006), assessment of this pest should be carefully evaluated to determine whether its establishment in western Ore-

Genetic Evidence for a New Subterranean Termite Species (Isoptera: Rhinotermitidae) from Western United States and Canada

Abstract Genetic evidence for a new subterranean termite species herein named Reticulitermes okanaganensis is provided based on DNA sequence analysis. Partial sequences of the mitochondrial DNA rRNA 16S gene were obtained from 27 samples of R. okanaganensis from British Columbia, Idaho, Oregon, Nevada and California. Five nucleotide sites were variable among the four observed haplotypes. One haplotype occurred only once, while the most common haplotype, O3, occurred in 37% of the samples. Molecular phylogenetic analysis of R. okanaganensis relative to five other North American Reticulitermes species has clarified its distinct position within the genus.