MOLECULAR GENETICS OF ECONOMICALLY IMPORTANT INSECTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0192515
• Project No.: ARK01954
• Project Start Date: Oct 1, 2007
• Project End Date: Sep 30, 2012

Project Director
Szalanski, A. L.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
ENTOMOLOGY

Non Technical Summary
A. Very little is known about the genetic structure of insects in the urban ecosystem. A. Genetic techniques can provide estimates of gene flow, dispersal, and molecular detection of insects in the urban ecosystem which can be useful for managing these pestiferous insects.

Animal Health Component

(N/A)

Research Effort Categories

Basic

50%

Applied

40%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
135	0613	1130	10%
136	0613	1080	10%
136	0613	1130	20%
721	5320	1080	20%
721	5320	1130	20%
722	5320	1130	20%

Knowledge Area
135 - Aquatic and Terrestrial Wildlife; 722 - Zoonotic Diseases and Parasites Affecting Humans; 136 - Conservation of Biological Diversity; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
0613 - Mixed conifer-broadleaf forests; 5320 - Houses (human residences), furniture, household equipment, non-textile furnishings;

Field Of Science
1080 - Genetics; 1130 - Entomology and acarology;

Keywords

urban ecosystem

molecular diagnostics

phylogeography

population genetics

termites

ants

bed bugs

flea

Goals / Objectives
Numerous insects are economically important in the urban environment including termites, wood destroying ants, and fleas. Recently, bed bugs have undergone a resurgence and are becoming a common urban insect pest across the United States. Very little is known about the phylogeography population genetic structure of these insects. Genetic techniques can provide estimates of gene flow, dispersal, and detection of locally adapted populations which can be useful for managing these pestiferous insects. Molecular genetics can also provide data useful for the development of molecular diagnostic keys to species, which can be used to identify species where morphological identification is difficult or insect samples are poorly preserved. Phylogeographic analysis can also be conducted using the DNA sequence data allowing detection of isolated populations and the description of new species. Genetic studies will be preformed to provide insight into the population structure of insects of urban importance. This can include estimates of gene flow, dispersal, and detection of locally adapted populations For example, if distinct populations are present, genetic analysis could assist in determining which population, local or immigrant, is primarily responsible for structural damage, or vectoring human pathogens. Genetic analysis which will be preformed will aid in determining if populations have different host preferences and if this can be used to assign individuals to specific populations. This information would likely have a direct bearing on the nature and implementation of management or control programs. Development of a database of pest insect genotypes will be preformed allowing this information to be applied in the future for the development of a reliable molecular diagnostic assay. The following insects will be studied under this objective. Subterranean Termites: Species of the genera Reticulitermes (Isoptera: Rhinotermitidae) are the major termites pests infesting wooden structures in the U.S. and in other countries around the world. Phylogenetic and population genetic analysis can provide insight into the distribution of these species in the United States, and gene flow among populations. Wood Destroying Ants: Wood destroying ants in the urban ecosysem include carpenter ants, Camponontus spp., as well as acrobat ants, Crematogaster spp., and Aphaenogaster ants. Despite their economic importance, few studies have been conducted on the phylogeography and population genetic structure of these species. Bed Bugs: Bed bugs, Cimex lectularius, are making a resurgence in the United States. Very little is known about the population genetic structure of this species and its blood hosts. Genetic analysis can provide insight into the dispersal of this species as well determining if the bed bugs are primarily feeding on human or other hosts. Fleas: Cat flea, Ctenocephalides felis (Bouche), and dog flea, Ctenocephalides canis (Curtis), are also important blood feeding insects in the urban environment. Little is known about the population genetic structure of these species.

Project Methods
Sample Collection: Adult and immature insects will be obtained from collaborators, museum specimens, and from field collections. Field collected insects will be placed in ziplock bags or in vials containing 95% ethanol, and will be sent to the insect genetics laboratory. Samples will be preserved by freezing at -20C to -80C or by 95% ethanol. Morphological Analysis: Insect samples will be classified using existing keys, including those of Scheffrahn and Su (1994), Mackay (2007), Usinger (1966), and Warren and Rouse (1969). Voucher specimens will be maintained in the Arthropod Museum, , University of Arkansas. Longitude and latitude coordinates for each collection will be obtained using a Garmin GPS device. GPS data will then be visualized using Google Earth (earth.google.com). Measurements of specimens, will be made using a calibrated ocular micrometer to a maximum accuracy of +/-0.01 mm using a Wild MSA microscope. Photomicrographs will be obtained using a digitized three-dimensional imaging system (Auto-Montage, Syncroscopy Inc. Frederick, MD). Any holotype or morphotype samples will be deposited in the collection of the National Museum of Natural History, Washington, D.C. Paratype samples will be deposited in the Arthropod Museum, Department of Entomology, University of Arkansas, Fayetteville, Arkansas, and in the Insect Genetics Laboratory. DNA Extraction: Alcohol preserved specimens for DNA extraction will be allowed to dry on filter paper. Entire insects, or individual thoraces or heads (depending on the taxon studied) will be used as the source of DNA, and the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis, MN) will be used to extract DNA. Extracted DNA will be resuspended in 50 μl of Tris:EDTA and stored at -20C. Enough DNA will be available from each DNA extraction for approximately 25-50 PCR reactions. PCR: Polymerase chain reaction will be conducted using various primer sets which amplify specific portions of the nuclear and mitochondrial genomes. PCR products will be visualized by electrophoresis on 1% agarose gels per Taylor and Szalanski (1999). DNA Sequencing: Amplified DNA from individual insects will be purified, and concentrated using Microcon-PCR Filter Units (Millipore, Bedford, MA) per Szalanski et al. (2000). Samples will be sent to the University of Arkansas Medical School for direct sequencing in both directions using an ABI sequencer. Sequences will be edited and aligned using BioEdit software (Hall 1999). Phylogenetic and Population Genetic Analysis: Phylogenetic analysis will be conducted using PAUP* 4.0b10 (Swofford 2001) and BEAST v1.4.3 (Drummond and Rambaut 2006) for maximum parsimony, maximum likelihood and Bayesian analysis. Population genetic analysis will be conducted using DNAsp v3.51 (Rozas and Rozas 1999), Arlequin v. 2.0 (Schneider et al. 2000), and TCS (Clement et al. 2000) softward. Analysis will include haplotype diversity, analysis of molecular variation (AMOVA), pairwise FST analysis and genealogical analysis of haplotyes. Molecular diagnostics will be conducted using both PCR-RFLP (Taylor and Szalanski 1999), and species specific PCR (Szalanski et al. 2006).

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Tripodi, A.D., and A.L. Szalanski. 2012. The distributions and genetic diversity of the Xylocopa of the eastern United States. Annual meeting of the Southeastern Branch - Entomological Society of America, Little Rock, AR. Szalanski, A.L., and A.D. Tripodi. 2012. Genetic variation of the large carpenter bees, Xylocopa virginica and X. micans. Annual meeting of the North Central Branch - Entomological Society of America, Lincoln, NE. Szalanski, A.L., and A.D. Tripodi. 2012. Molecular diagnostics of Nosema from feral honey bees. Annual Meeting of the Entomological Society of America, Knoxville, TN. Tripodi, A.D., and A.L. Szalanski. 2012. Population genetics of bumble bees in central United States. Annual Meeting of the Entomological Society of America, Knoxville, TN. Trammel, C., A.D. Tripodi, and A.L. Szalanski. 2012. Genetic variation of the striped bark scorpion, Centruroides vittatus. Annual Meeting of the Entomological Society of America, Knoxville, TN. Kim, S-H.S., A.D. Tripodi, D.T. Johnson, and A.L. Szalanski. 2012. Spotted wing Drosophila: new pest in Arkansas, management, monitoring, and alternative identification. Arkansas Crop Protection Conference, Fayetteville, AR. Tripodi, A.D. and Szalanski, A.L. 2012. Genetic variation of the bumble bee, Bombus auricomus. Annual meeting of the Central States Entomological Society, Jonesboro, AR. Tripodi, A., R. Magnus, and A.L. Szalanski. 2010. Genetic diversity of bumble bees in south central United States. International Conference on Pollinator Biology, Health and Policy. State College, PA. Szalanski, A.L., J. Whitaker, and P. Cappy. 2010. Molecular diagnostics of Nosema ceranae and N. apis from honey bees in New York. North American Beekeeping Conference, Orlando, FL. Szalanski, A.L., and A.D. Tripodi. 2011. Assessing the utility of a molecular diagnostic marker for identification of Africanized honey bees in the United States. Annual meeting of the Entomological Society of America, Reno, NV. Szalanski, A.L., R. Magnus, and J.L. Bernhardt. 2010. Molecular association of tabanid lifestages using a mitochondrial DNA marker. Annual meeting of the Entomological Society of America, San Diego, CA. Szalanski, A.L. 2007. Phylogeography and population genetics of bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae), from North America. Annual meeting of the Entomological Society of America, San Diego, CA PARTICIPANTS: Allen Szalanski, Professor of Entomology, Univ. of Arkansas, Fayetteville Amber Tripodi, PhD Graduate Research Assistant, Univ. of Arkansas, Fayetteville, AR, Roxane Magnus, MS Graduate Research Assistant, Univ. of Arkansas, Fayetteville, AR. TARGET AUDIENCES: general public, master gardeners, pollinator conservation groups, botanical gardens, small organic farmers, pest control, beekeepers, regulatory agencies PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Molecular identification of Africanized honey bees, Apis mellifera scutellata (AHB) in the United States often involves the use of polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) on a portion of the mitochondrial DNA (mtDNA) cytochrome b gene. One problem with this technique is that the restriction enzyme site is only found in European honey bee subspecies (EHB) and not AHB. To enhance this technique, we developed a multiplex PCR-RFLP assay that can discriminate Africanized bees from European honey bees. Based on DNA sequence variation, a PCR primer specific for AHB was designed. Multiplex PCR-RFLP provides a more robust identification of maternally Africanized bees than PCR-RFLP alone. Genetic diversity of queen breeder honey bees relative to feral honey bees in the United States was conducted. This study revealed that are lower levels of mitochondrial DNA diversity of queen breeder populations in the United States relative to feral populations. Locally adapted feral populations may be a source of genetic diversity for queen breeders. Research was conducted on population genetic diversity of Africanized honey bees in the United States. This was the first study to use primarily DNA sequence data, and revealed high levels of genetic diversity (12 haplotypes) in the United States. This data may be used to detect the source of Africanized honey bee introductions in the United States. The bed bug, Cimex lectularius, is a widespread blood feeding pest of humans around the world, including North America, and has recently undergone a resurgence. A molecular diagnostic technique applying multiplex PCR was developed in order to distinguish bed bug eggs, leg fragments and degraded samples from other arthropods which frequently occur in human dwellings. This technique should provide a reliable, quick and economical technique for identifying C. lectularius, when morphological identification is not possible. The genetic diversity and population structuring of 3 bumble bee species from AR, NE and TN were analyzed using one mitochondrial and one nuclear marker. This regional assessment of B. pensylvanicus agrees with a recent national one; B. pensylvanicus seems to be declining at both spatial scales. On the other hand, B. bimaculatus appears stable nationally, but populations of B. bimaculatus throughout Arkansas, Nebraska and Tennessee are fragmented from one another, and the species may be declining in Tennessee. These results highlight the importance of scale in assessing the conservation status of these important native pollinators. Two termite species, Incisitermes minor and I. snyderi are common drywood termites in southwestern and the southern United States, respectively. A molecular genetics study involving DNA sequencing of a portion of the mtDNA 16S gene was undertaken to determine the extent of genetic variation in I. minor and I. snyderi. There appears to be enough genetic variation within these 2 species for a molecular phylogeographic study, which may provide insight into dispersal and introductions of these species.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Tripodi, A.D., and A.L. Szalanski. 2011. Population structuring of bumble bees from the Central United States. Annual meeting of the Southeastern Branch - Entomological Society of America, San Juan, Puerto Rico. Szalanski, A.L., and A.D. Tripodi. 2011. Assessing the utility of a molecular diagnostic marker for identification of Africanized honey bees in the United States. Annual meeting of the Entomological Society of America, Reno, NV. Austin, J.W., A.L. Szalanski, and R.H. Scheffrahn. 2011. Molecular biogeography of Incisitermes minor (Hagen) and Incisitermes snyderi (Light): two distinct urban termite pests. Annual meeting of the Entomological Society of America, Reno, NV. Tripodi, A.D., and A.L. Szalanski. 2011. Population structuring and genetic diversity of bumble bees from the central United States. Annual meeting of the Entomological Society of America, Reno, NV. PARTICIPANTS: Allen Szalanski, Professor of Entomology, Univ. of Arkansas, Fayetteville Amber Tripodi, PhD Graduate Research Assistant, Univ. of Arkansas, Fayetteville TARGET AUDIENCES: general public, master gardeners, pollinator conservation groups, botanical gardens, small organic farmers, pest control, beekeepers, regulatory agencies. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Native populations of bumble bee species freely provide indispensable pollination services, and their capacity for buzz pollination makes them particularly useful agricultural pollinators. The genetic diversity and population structuring of three bumble bee species from Arkansas, Nebraska and Tennessee were analyzed using one mitochondrial and one nuclear marker. Bombus pensylvanicus showed lower genetic diversity (Hd = 0.19) than B. impatiens or B. bimaculatus (Hd = 0.78 and 0.79, respectively) and exhibited lower heterozygosity than expected (F = 0.29, p = 0.03). High levels of population structure were seen in B. bimaculatus (FST = 0.33, p = 0.01), and populations in Tennessee had lower genetic diversity (Hd = 0.57), than those in Nebraska (Hd = 0.73, not significant) or Arkansas (Hd = 0.79, p = 0.03). This regional assessment of B. pensylvanicus agrees with a recent national one; B. pensylvanicus seems to be declining at both spatial scales. On the other hand, B. bimaculatus appears stable nationally, but populations of B. bimaculatus throughout Arkansas, Nebraska and Tennessee are fragmented from one another, and the species may be declining in Tennessee. These results highlight the importance of scale in assessing the conservation status of these important native pollinators. The western drywood termite, Incisitermes minor (Hagen) and the light southern drywood termite I. snyderi (Light) are common drywood termites in southwestern and the southern United States, respectively. Despite the economic importance of these 2 species, no information exists on the mitochondrial genetic diversity of I. minor and I. snyderi. A molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA 16S rRNA gene was undertaken to determine the extent of genetic variation in I. minor and I. snyderi. There appears to be enough genetic variation within these 2 species for a molecular phylogeographic study, which may provide insight into dispersal and introductions of these species. The bed bug, Cimex lectularius L. is a widespread blood feeding pest of humans around the world, including North America, and has recently undergone a resurgence. A molecular diagnostic technique applying multiplex PCR was developed in order to distinguish bed bug eggs, leg fragments and degraded samples from other arthropods which frequently occur in human dwellings. A 410-428 bp region of the mitochondrial DNA 16S rRNA gene was used. To design C. lectularius specific PCR primers, DNA sequences of various bed samples from the United States, Canada and Australia, along with sequences of other Cimicidae and arthropods which often occur in dwellings, were considered. Based on DNA sequence variation, one reverse PCR primer specific for C. lectularius was identified. Multiplex PCR using three primers will yield a 417, and 140 bp amplicon for C. lectularius and a single 410 to 428 bp amplicon for other taxa. This assay was successful in identifying C. lectularius eggs, leg fragments and degraded samples. This technique should provide a reliable, quick and economical technique for identifying C. lectularius, when morphological identification is not possible.

Publications

• Austin, J.W., Szalanski, A.L., C. Solorzano, R. Magnus, and R.H. Scheffrahn. 2012. Mitochondrial DNA genetic diversity of the drywood termites Incisitermes minor and I. snyderi. Florida Entomologist 95:(1)(in press).
• Austin, J.W., A.L. Szalanski, T.G. Myles, P.A.V. Borges, L. Nunes, and R.H. Scheffrahn. 2012. First record of Reticulitermes flavipes (Isoptera: Rhinotermitidae) from Terceira Island (Azores, Portugal). Florida Entomologist 95:(1) (in press).
• Tripodi, A.D., and A.L. Szalanski. 2011. Further range expansion for Xylocopa micans Lepeletier (Hymenoptera: Apidae). Journal of the Kansas Entomological Society 84: 77-78.
• Szalanski, A.L., A. Tripodi, and J.W. Austin. 2011. Multiplex PCR diagnostics of the bed bug, Cimex lectularius L. Journal of Medical Entomology 48: 937-940.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The following research presentations were made to disseminate the results to communities of interest: Tripodi, A., R. Magnus, and A.L. Szalanski. 2010. Genetic diversity of bumble bees in south central United States. International Conference on Pollinator Biology, Health and Policy. State College, PA.; Szalanski, A.L., J. Whitaker, and P. Cappy. 2010. Molecular diagnostics of Nosema ceranae and N. apis from honey bees in New York. North American Beekeeping Conference, Orlando, FL.; Szalanski, A.L., R. Trout, J.W. Austin, R. Magnus, D. Charlwood. 2010. Temporal genetic variation, and bloodmeal identification of Anopheles funestes and An. gambiae mosquitoes from Mozambique. 4th Annual Arthropod Genomics Symposium, Kansas City, MO. Szalanski, A.L., R. Magnus, and J.L. Bernhardt. 2010. Molecular association of tabanid lifestages using a mitochondrial DNA marker. Annual meeting of the Entomological Society of America, San Diego, CA. ; Singh, N., D.T. Johnson, C.D. Steelman, and A.L. Szalanski. 2010. Baseline dose-response of lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae) to insect growth regulators. Annual meeting of the Entomological Society of America, San Diego, CA.; Szalanski, A.L. 2010. Molecular research on bed bugs. Annual meeting of the Entomological Society of America, San Diego, CA. (invited).; Tripodi, A.D., R. Magnus, and A.L. Szalanski. 2010. Genetic diversity of bumble bees from central United States. Annual meeting of the Entomological Society of America, San Diego, CA.; Trout, R.T., A.L. Szalanski, J.W. Austin, R. Magnus, D. Charlwood. 2010. Temporal genetic variation, and bloodmeal identification of Anopheles funestes and An. gambiae mosquitoes from Mozambique. Annual meeting of the Entomological Society of America, San Diego, CA.; Magnus, R., and A.L. Szalanski. 2010. Mitochondrial DNA variation of queen breeder and feral honey bee populations in the United States. Annual meeting of the Entomological Society of America, San Diego, CA. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Medical, Veterinary, and Urban Entomology. Published papers on: genetic variation of the louse fly, Lipoptena mazamae from Arkansas; identification of Borrelia burgdorferi and Borrelia lonestari in Arkansas ticks, canines, and white-tailed deer; genetic diversity of the mosquito, Aedes vexans, in New Orleans: Pre and post Katrina and Genetic diversity of ants (Hymenoptera: Formicidae) from the Ozark-St. Francis National Forest, Arkansas. Papers were submitted on molecular diagnostics of bed bugs, Cimex lectularius, in the United States as well as isolation of Rickettsia pathogens from Arkansas canines, white-tailed deer, and their associated ticks (Acari: Ixodidae). Honey bees. Completed and published research on genetic diversity of queen breeder honey bees in the United States. This study revealed that there is a lack of mitochondrial DNA diversity of queen breeder populations in the United States relative to feral populations. Locally adapted feral populations may be a source of genetic diversity for queen breeders. This research was a part of a MS student thesis. Published a study on population genetic diversity of Africanized honey bees in the United States. This was the first study to use primarily DNA sequence data, and revealed high levels of genetic diversity (12 haplotypes) in the United States. This data may be used to detect the source of Africanized honey bee introductions in the United States. We also conducted studies on the occurrence of the honey bee pathogens Nosema ceranae and N. apis from apiaries located in South Dakota and New York. We found the invasive pathogen N. ceranae to be the only Nosema found in South Dakota using PCR molecular diagnostics, and that it accounted for 97% of Nosema positive samples in New York. Also published a paper on molecular detection of Nosema ceranae from Turkish honey bees.

Publications

• Magnus, R., and A.L. Szalanski. 2010. Mitochondrial DNA diversity of honey bees, Apis mellifera L. (Hymenoptera: Apidae) from queen breeders in the United States. Journal of Apicultural Science (accepted).
• Whitaker, J., A.L. Szalanski, and M. Kence. 2010. Molecular detection of Nosema ceranae from Turkish honey bees. Apidologie 41: (in press).
• Szalanski, A.L., J.A. McKern, C. Solorzano, and J.W. Austin. 2010. Genetic diversity of ants (Hymenoptera: Formicidae) from the Ozark-St. Francis National Forest, Arkansas, USA. Sociobiology 56: 601-610.
• Solorzano, C., A.L. Szalanski, C.B. Owens, and C.D. Steelman. 2010. Genetic diversity of Aedes vexans in New Orleans: Pre and post Katrina. Biochemical Genetics 48: 711-726.
• Trout, R.T., C.D. Steelman, A.L. Szalanski, and P.C. Williamson. 2010. Identification of Rickettsia parkeri and Rickettsia amblyommii in Amblyomma maculatum from Arkansas, USA. Emerging Infectious Diseases 16: 830-832.
• Szalanski, A.L., and R.M. Magnus. 2010. Mitochondrial DNA characterization of Africanized honey bee (Apis mellifera L.) populations from the USA. Journal of Apicultural Research 49: 177-185.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The following research presentations were made to disseminate the results to communities of interest: Solorzano, C.D., A.L. Szalanski, R.H. Scheffrahn, J.W. Austin and R.E. Gold. 2009. Mitochondrial DNA genetic diversity of the drywood termites, Incisitermes minor and I. snyderi. Ann meeting of the ESA, Indianapolis, IN.; Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2009. Phylogenetics and population genetics of the louse fly, Lipoptena mazamae from Arkansas, USA. Ann meeting of the ESA, Indianapolis, IN.; Whitaker, J.T., and A.L. Szalanski. 2009. Distribution and molecular detection of Nosema ceranae in US honey bees. Annual meeting of the ESA, Indianapolis, IN.; Magnus, R., and A.L. Szalanski. 2009. Genetic variation in honey bees (Apis mellifera) from central and southern United States. Ann meeting of the ESA, Indianapolis, IN.; Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2009. Genetic and spatial identification of Borrelia and Rickettsia pathogens from Arkansas ticks and mammals. The 64th International Conference on Diseases in Nature Communicable to Man. Fort Worth, TX. (invited); Trout, C.D. Steelman, A.L. Szalanski, and P.H. Williamson. 2009. Genetic and spatial identification of Rickettsia in Arkansas, Livestock Insect Workers Conf, French Lick, IN. ; Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2009. Population genetics and phylogeography of Ixodes ticks from Arkansas. Livestock Insect Workers Conference, French Lick, IN.; Chapon, L., M.D. Ellis, and A.L. Szalanski. 2009. Nosema and tracheal mites in the north central region - 2008 survey. American Bee Research Conference, Gainesville, FL.; Magnus, R. and A.L. Szalanski. 2009. Genetic variation of honey bees from central United States. Third Annual Arthropod Genomics Symp Kansas City, MO.; Solorzano, C., and A.L. Szalanski. 2009. Genetic diversity of honey bees from Turkey. Third Annual Arthropod Genomics Symp, Kansas City, MO; Whitaker, J., and A.L. Szalanski. 2009. Molecular detection and occurrence of Nosema ceranae from Turkish honey bees. 3rd Annual Arthropod Genomics Symp, Kansas City, MO; Szalanski, A.L., and R.M. Magnus. 2009. Genetic variation of Africanized honey bees in the United States. Annual meeting of the North Central Branch - ESA. ; Ellis, M.D., L. Chapon, and A.L. Szalanski. 2009. Nosema and tracheal mite prevalence in honey bee colonies in the North Central Region of the U.S. Annual meeting of the North Central Branch - ESA; Trout, R.T., C.D. Steelman, A.L. Szalanski, and P.C. Williamson. 2009. Borrelia pathogens isolated from canines, deer, and their associated ticks. Annual meeting of the South Eastern Branch - ESA; Magnus, R.A., and A.L. Szalanski. 2009. Genetic structure of honey bee populations in south central United States. University of Arkansas Grad Student Res Symp, Fayetteville, AR; Magnus, R.A., and A.L. Szalanski. 2009. Genetic structure of honey bee populations in south central United States. 2009 University of Arkansas GSD Student Poster Competition, Fayetteville, AR; Solorzano, C., and A.L. Szalanski. 2009. Phylogeography of honey bee populations in Turkey. 2009 University of Arkansas GSD Student Poster Competition, Fayetteville, AR. PARTICIPANTS: A.L. Szalanski, C. Solorzano, J. McKern, B. Trout, R. Magnus, C.D. Steelman, University of Arkansas TARGET AUDIENCES: Molecular biologist, Social insect specialists PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
To determine the effect that hurricane Katrina had on the temporal genetic variation of Aedes vexans from New Orleans, we compared genetic diversity of mosquito specimens collected during 2005 (n= 99) with specimens from 2006 (n= 103), after the hurricane. A 423bp region of the NADH5 mtDNA gene was sequenced, resulting in 39 and 58 nucleotide polymorphic sites, and 38 and 62 haplotypes observed for 2005 and 2006, respectively. Average haplotype diversity (Hd) was high (Hd > 0.88) in 2005 and 2006 for both New Orleans and Jefferson parishes. Neither genetic differentiation nor isolation by distance was observed across parishes, and gene flow and/or population growth was considerable. It does not appear that Hurricane Katrina had any impact on genetic diversity and, despite the intense efforts to control mosquitoes in New Orleans. Research is continuing on a molecular phylogenetic revision of the drywood termite genus Incisitermes. This is the first molecular revision of this economically important genus and there is genetic evidence for six new species of Incisitermes in North, Central, and South America. Research is continuing on the genetic variation of honey bees in the central, south central and southern United States. DNA sequencing of the mtDNA COI-COII gene has not been conducted in the United States on honey bees prior to now and results from this study can be used for honey bee biodiversity. So far, approximately 469 samples from feral and managed colonies have been sequenced. Results show that for mtDNA sequence analysis from several queen breeders we have sampled has revealed six mitotypes. These mitotypes account for 88% of the overall 469 samples that we have sequenced to date, 90% from managed colonies, and 74% from feral colonies. From the DNA sequences we analyzed from 14 central, south central, and southern states, we observed 33 different mitotypes. Lineages observed include Eastern European (C 14 mitotypes); Western European (M 5 mitotypes); Near East and Middle East (O 4 mitotypes); and African (A 10 mitotypes). Mitotype data that has been obtained is being placed on a website: http://comp.uark.edu/~aszalan/Apis/Honey_bee_mitotype_map.html. Based on this difference in genetic variation, introduction of new genetic lineages to queen breeders may increase the genetic diversity of honey bees in the United States. Molecular diagnostics of the invasive honey bee pathogen, Nosema ceranae, was conducted on honey bees from AR and NY. N. ceranae is the prevalent form of Nosema in NY, N. ceranae occurs throughout NY and is more prevalent in commercial vs sideliner and hobbyist beekeepers. A preliminary molecular phylogenetic analysis of N. ceranae from NE, WI, SD, and NY revealed that it is similar to N. ceranae from Europe and Australia. Genetic analysis of Africanized honey bees from the state of UT was initiated in 2009 in corporation with the UT Department of Agriculture. From 120 samples collected from 7 counties in Utah, a total of 44 samples from 3 counties were AHB. DNA sequence analysis revealed 5 distinct haplotypes of AHB in UT.

Publications

• Magnus, R.M. and A.L. Szalanski. 2010. Genetic evidence of honey bees belonging to the Middle East lineage in the United States. Sociobiology 55:(1) (in press).
• Trout, R.T., C.D. Steelman, A.L. Szalanski, and K. Loftin. 2010. Establishment of Amblyomma maculatum (Gulf Coast Tick) in Arkansas, U.S.A. Florida Entomologist (in press).
• Trout, R., C.D. Steelman, and A.L. Szalanski. 2010. Phylogenetics and population genetics of the louse fly, Lipoptena mazamae from Arkansas, USA. Medical and Veterinary Entomology (in press).
• Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2010. Population genetics and phylogeography of Amblyomma americanum L. (Acari: Ixodidae) collected from Arkansas. Journal of Medical Entomology (in press).
• Bae, C.H., A.L. Szalanski, and R.T. Robbins. 2009. Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP nuclear rRNA ITS regions. Journal of Nematology (in press).
• McKern, J.A., A.L. Szalanski, J.W. Austin, and R.E. Gold. 2009. Genetic diversity of field populations of the cat flea, Ctenocephalides felis and the human flea, Pulex irritans, in the United States. Journal of Agricultural and Urban Entomology (in press).
• Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2009. Population genetics and phylogeography of Ixodes scapularis collected from Arkansas canines and deer. Southwestern Entomologist 34: 273-287.
• Bae, C.H., A.L. Szalanski, and R.T. Robbins. 2009. Phylogenetic analysis of the Hoplolaiminae inferred from combined D2 and D3 expansion segments of 28S rDNA. Journal of Nematology 41: 28-34.
• Bae, C.H., R.T. Robbins, and A.L. Szalanski. 2009. Molecular identification of Hoplolaimus species based on multiplex PCR and PCR-RFLP analysis. Nematology 11: 471-480.
• Solorzano, C.D., A.L. Szalanski, M. Kence, J.A. McKern, J.W. Austin and A. Kence. 2009. Phylogeography and population genetics of honey bees (Apis mellifera L.) from Turkey based on COI-COII sequence data. Sociobiology 53: 237-246.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The following research presentations were made to disseminate the results to communities of interest; Solorzano, C., A.L. Szalanski, M. Kence, A. Kence, and J.W. Austin. Phylogeography and population genetics of honey bees (Apis mellifera L.) from Turkey based on COI-COII sequence data. Annual meeting of the Entomological Society of America, Reno, NV. Trout, R.T., C. D. Steelman, A.L. Szalanski, K. Kvamme, and P. Williamson. Genetic identification and environmental modeling of Borrelia pathogens isolated from Arkansas ticks and mammals. Annual meeting of the Entomological Society of America, Reno, NV. Magnus, R., A.L. Szalanski, and C. Solorzano. Genetic diversity of honey bees, Apis mellifera, from south central United States. Annual meeting of the Entomological Society of America, Reno, NV. McKern, J., A.L. Szalanski, and J.W. Austin. Genetic variation of the cat flea. Annual meeting of the Entomological Society of America, Reno, NV. Trout, R., A.L. Szalanski, C.D. Steelman. Population genetics and phylogeography of Arkansas-collected ticks. Annual meeting of the Entomological Society of America, Reno, NV. (invited). Szalanski, A.L. Genetic diversity of Reticulitermes termites from western United States. National Conference on Urban Entomology, Tulsa, OK. Solorzano, C.D., and A.L. Szalanski. 2008. Genetic variation of Incisitermes drywood termites. Annual meeting of the Arkansas Entomological Society, Fayetteville, AR. Magnus, R.A., and A.L. Szalanski. 2008. Genetic variation of honey bees from south central United States. Annual meeting of the Arkansas Entomological Society, Fayetteville, AR. Szalanski, A.L. and R.A. Magnus. 2008. Genetic diversity of Africanized honey bees from Oklahoma and Arkansas. Annual meeting of the Arkansas Entomological Society, Fayetteville, AR. Solorzano, C.D., and A.L. Szalanski. 2008. Detection of Dirofilaria immitis in Aedes vexans populations. Annual meeting of the SEB-ESA, Jacksonville, FL. Mckern, J.A., and A.L. Szalanski. 2008. Survey of Nosema in honey bees from south central United States. Annual meeting of the SEB-ESA, Jacksonville, FL. Trout, R.T., C.D. Steelman, and A.L. Szalanski. 2008. Population genetics and phylogeography of Amblyomma ticks. Annual meeting of the SEB-ESA, Jacksonville, FL. PARTICIPANTS: A.L. Szalanski, C. Solorzano, J. McKern, B. Trout, R. Magnus, C.D. Steelman, University of Arkansas TARGET AUDIENCES: Molecular biologist, Social insect specialists PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
High levels of mtDNA COI and COII genetic variation was observed from honey bees collected from Turkey. These populations may represent a source of genetic diversity for other locations. Molecular genetic analysis of Africanized honey bees from New Mexico, Oklahoma and Arkansas revealed that there are two distinct COI-COII lineages in the United States. This has also been observed in Mexico and South America. Molecular diagnostics to detect Africanized honey bees using multiplex PCR of the cyt b maker was still effective for both lineages. Populations of bed bugs in the United States have high levels of mitochondrial DNA variation within and among populations but a lack of nuclear (ITS1) genetic variation. This provides support that populations in the United States have not undergone a genetic bottleneck but have been maintained on alternative host species.

Publications

• Szalanski, A.L., J.W. Austin, J.A. McKern, C.D. Steelman and D. Miller. 2006. Time course analysis of bed bug, Cimex lectularius L., (Hemiptera: Cimicidae) blood meals using PCR. Journal of Agricultural and Urban Entomology 23: 237-241.
• Szalanski, A.L., J.W. Austin, J.A. McKern, C.D. Steelman, and R.E. Gold. 2008. Mitochondrial and ribosomal internal transcribed spacer (ITS1) diversity of the bed bug Cimex lectularius L. (Heteroptera: Cimicidae). Journal of Medical Entomology 45: 229-236.
• Szalanski, A.L., J.W. Austin, and J.A. McKern. 2008. Genetic diversity of Reticulitermes termites from Lake Wedington, Arkansas. Sociobiology 52: 95-106.

Progress 01/01/07 to 12/31/07

Outputs
Larvae of many species of Sesiidae, the clearwing moths, are important pests in commercial nurseries, urban landscapes, timber stands, vineyards and orchards. They cause economic loss by larval boring in stems and roots of herbaceous and woody plants. PCR was used to amplify a 606 bp region of the mtDNA COI and COII genes. PCR-RFLP analysis was conducted on eight species of Sesiidae, Melittia satyriniformis, Paranthrene simulans, Pennisetia marginata, Synanthedon pictipes, S. exitiosa, S. scitula, S. rileyana, and Vitacea polistiformis, using two restriction enzymes, Dra I and Hinf I. PCR-RFLP provides an accurate method to differentiate eight species of adult clearwing moths commonly found in traps baited with commercially available pheromone lures. A molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA 16S rRNA gene was undertaken to determine the extent of genetic variation and distribution of Reticulitermes, Zootermopsis, and Insistitermes termites from Oregon and Washington. Eighty seven samples were sequenced and evaluated. From 17 Oregon and Washington counties, a total of one I. minor, 15 Z. angusticollis, one R. flavipes, one R. hageni, 65 R. hesperus, two R. okanaganensis, and two undescribed Reticulitermes were identified. Glacial refugia theories about the distribution of termites occupying both states provide possible clues into the possible mechanisms which have fostered genetic variability in R. hesperus haplotypes and restricted gene flow in Z. angusticollis. They also provide some understanding to the driving forces behind the identification of newly identified termite groups from the region. Molecular identification of Africanized honey bees, Apis mellifera scutellata (AHB) in the United States often involves the use of polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) on a portion of the mitochondrial DNA (mtDNA) cytochrome b gene. One problem with this technique is that the restriction enzyme site is only found in European honey bee subspecies (EHB) and not AHB. To enhance this technique, we developed a multiplex PCR-RFLP assay that can discriminate Africanized bees from European honey bees. Based on DNA sequence variation, a PCR primer specific for AHB was designed. Multiplex PCR-RFLP provides a more robust identification of maternally Africanized bees than PCR-RFLP alone. The ability to identify individual human hosts based on analyses of blood recovered from blood feeding insects is beneficial for both medical and forensic entomology. Bed bugs, Cimex lectularius L., may have several advantages over other blood-feeding arthropods for forensics since they do not remain on the host after their blood-feeding activity and remain in close proximity to a crime scene. Successful isolation, amplification, and sequencing of human DNA obtained from adult bed bugs, is reported for the first time from this study. These results demonstrate that DNA isolated from bed bugs is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals for forensic analysis.

Impacts
PCR-RFLP provides an accurate method to differentiate eight species of adult clearwing moths commonly found in traps baited with commercially available pheromone lures. Multiplex PCR-RFLP provides a more robust identification of maternally Africanized bees than PCR-RFLP alone.DNA isolated from bed bugs is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals for forensic analysis.Glacial refugia theories about the distribution of termites occupying Oregon and Washington provide possible clues into the possible mechanisms which have fostered genetic variability in R. hesperus haplotypes and restricted gene flow in Z. angusticollis.

Publications

• McKern, J.A., and A.L. Szalanski. 2007. Molecular diagnostics of economically important clearwing moths (Lepidoptera: Sesiidae). Florida Entomologist 90: 475-479.
• Szalanski, A.L., and J.A. McKern. 2007. Multiplex PCR-RFLP diagnostics of the Africanized honey bee (Hymenoptera: Apidae). Sociobiology 50: 939-945.
• McKern, J.A., A.L. Szalanski, J.W. Austin, M.T. Messenger, J. Mahn, and R.E. Gold. 2007. Phylogeography of termites (Isoptera) from Oregon and Washington. Sociobiology 50: 607-622.
• King, S.W., J.W. Austin, and A.L. Szalanski. 2007. Use of soldier pronotal width and mitochondrial DNA sequencing to distinguish the subterranean termites, Reticulitermes flavipes (Kollar) and R. virginicus (Banks) (Isoptera: Rhinotermitidae), on the Delmarva peninsula. Entomological News 118: 41-48.
• Szalanski, A.L., J.W. Austin, J.A. McKern, D. Miller, C.D. Steelman, and R.E. Gold. 2006. Isolation and characterization of human DNA from the bed bug, Cimex lectularius L. (Heteroptera: Cimicidae). Journal of Agricultural and Urban Entomology 23: 189-194.
• Austin, J.W., A.-G. Bagneres, A.L. Szalanski, R.H. Scheffrahn, B.P. Heintschel., J.-L. Clement, M.T. Messenger, and R.E. Gold. 2007. Reticulitermes malletei (Isoptera: Rhinotermitidae): a valid Nearctic subterranean termite from eastern North America. Zootaxa 1554: 1-26.
• Austin, J.W., G.J. Glenn, A.L. Szalanski, J.A. McKern, and R.E. Gold. 2007. First record of Asian cockroach Blatella asahinai Mizukubo (Blattodea: Blattelidae) from Texas. Florida Entomologist 90: 574-576.

Progress 01/01/06 to 12/31/06

Outputs
A multiplex PCR-RFLP technique was developed for molecular diagnostics of European honey bee from Africanized honey bee. This diagnostic technique is ideal for samples that are in poor morphological condition, and for samples that consist of fewer than five bees. Honey bee samples were collected by the Arkansas Plant Board, Oklahoma State University, or by county extension personnel from various locations in Arkansas, Oklahoma, and New Mexico. DNA sequences were examined for nucleotide variation between EHB and AHB, and a PCR primer specific for AHB was designed. Multiplex PCR with the AHB specific primer (AHB-F) along with two A. mellifera primers (Apis-F, Apis-R) was tested and revealed that the multiplex PCR-RFLP results matched those previously obtained using PCR-RFLP. A molecular diagnostic techinque using PCR-RFLP was developed to distinguish eight economically important species of Sesiid moths. We found the first evidence for Reticulitermes hageni and R. flavipes in Oregon. The phylogeography of Reticulitermes from the American great plains, California, and the Middle East was updated. We found genetic evidence for a new species of Reticulitermes, R. okanaganensis, in western United States and Canada. Genetic evidence of two introductions of the Formosan subterranean termite to the United States was also published.

Impacts
Multiplex PCR combined with RFLP provides a more robust method to identify Africanized honey bee samples than just PCR-RFLP, by adding a marker that is specific for AHB. This should be an important new method for identification of Africanized honey bees for regulatory purposes. Our PCR-RFLP sesiid diagnostic study is the first molecular diagnostic technique designed to distinguish sesiid pests commonly found in pheromone traps in Arkansas. This technique is reliable, cost efficient and most importantly useful for identification of degraded or battered specimens with portions of scales or limbs missing. Other advantages to the PCR-RFLP technique include reliability and time efficiency. Knowledge of subterranean termite phylogeography will provide insight into the distribution of species and gene flow across the United States.

Publications

• McKern, J.A., A.L. Szalanski, and J.W. Austin. 2006. First record of Reticulitermes flavipes and Reticulitermes hageni in Oregon (Isoptera: Rhinotermitidae). Florida Entomologist 89: 541-542.
• Taylor, D.B., R. Moon, G. Gibson, and A. Szalanski. 2006. Genetic and morphological comparisons of New and Old World populations of Spalangia species (Hymenoptera: Pteromalidae). Annals of the Entomological Society of America. 99: 799-808.
• Austin, J.W., A.L. Szalanski, R. Ghayourfar, A. Kence, and R.E. Gold. 2006. Phylogeny and genetic variation of Reticulitermes (Isoptera: Rhinotermitidae) from the eastern Mediterranean and Middle East. Sociobiology 47: 873-890.
• Szalanski, A.L., J.W. Austin, J. McKern, and M.T. Messenger. 2006. Genetic evidence for a new subterranean termite species (Isoptera: Rhinotermitidae) from western United States and Canada. Florida Entomologist. 89: 299-304.
• Austin, J.W., A.L. Szalanski, M.T. Messenger, J.A. McKern, and R.E. Gold. 2006. Genetic variation and phylogenetics of Reticulitermes (Isoptera: Rhinotermitidae) from the American great plains. Sociobiology 48: 427-445.
• Tripodi, A.D., J.W. Austin, A.L. Szalanski, J. McKern, M.K. Carroll, R.K. Saran, and M.T. Messenger. 2006. Phylogeography of Reticulitermes termites (Isoptera: Rhinotermitidae) in California inferred from mitochondrial DNA sequences. Annals of the Entomological Society of America 99:697-705.
• Szalanski, A.L., C.B. Owens, J.A. Lewter, and A.B. Broce. 2006. Genetic structure of Aedes vexans (Diptera: Culicidae) populations from central United States based on mitochondrial ND5 sequences. Annals of the Entomological Society of America 99: 157-163.
• Kelley, M.B., S.W. Wingard, A.L. Szalanski, and F.M. Stephen. 2006. Molecular diagnostics of Enaphalodes rufulus (Coleoptera: Cerambycidae). Florida Entomologist 89: 251-256.
• Austin, J.W., A.L. Szalanski, R.H. Scheffrahn, M.T. Messenger, J.A. McKern, and R.E. Gold. 2006. Genetic evidence for two introductions of the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae), th the United States. Florida Entomologist 89: 183-193.
• Lewter, J.A., A.L. Szalanski, R.N. Nagoshi, R.L. Meagher, Jr., C.B. Owens, and R.G. Luttrell. 2006. Genetic variation within and between strains of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Florida Entomologist 89: 63-68.

Progress 01/01/05 to 12/31/05

Outputs
Fall Armyworm Genetics: This study was conducted to investigate the genetic structure of S. frugiperda across a part of its range in the United States. A 608 base pair portion of the mitochondrial cytochrome oxidase I and II genes was sequenced from 71 individuals resulting in three corn and four rice strain haplotypes. Genetic divergence between the two strains ranged from 0.66 to 0.99%. A 562 base pair region of the nuclear ITS-1 gene was also amplified and sequenced from 17 individuals representing both corn and rice strains. No variation was detected in any of the samples for the ITS-1 region. Analysis of molecular variance indicated a significant overall F for all populations with the hierarchical analysis revealing that this significant F is due to structuring of the populations between states. The observed genetic structure is possibly due to the distribution of fall armyworm strains. Termite genetics: We conducted a molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA 16S gene to determine the extent of genetic variation within Reticulitermes from California. A total of 89 samples were analyzed. Twenty-five nucleotide sites were variable in R. hesperus, and 19 mtDNA haplotypes were observed in the 428 bp mtDNA sequence. Fourteen haplotypes (37%) occurred only once, while the most common haplotypes, HE4 and HE9, each occurred in 18% of the samples. While some haplotypes were found to have a broad geographical range across the state, some were restricted to the southern region, as were all samples identified as R. tibialis. Twelve haplotypes of an undescribed new species, R. okanaganensis, were found, and its distribution throughout the state is discussed. Additionally, genetic evidence of an additional undescribed Reticulitermes species R. n. sp. from Arrowbear Lake, CA California is presented. Exotic introductions of Formosan Subterranean Termite, Coptotermes formosanus (FST) to the United States from Asia have had significant economic consequences. 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 US, 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 US populations. This is the first study which has documented 2 distinct lineages to continental United States and Hawaii. Mosquito Genetics: 99 adult Aedes vexans mosquitoes representing 11 sampled populations from 3 Louisiana parishes were subjected to genetic analysis using PCR to amplify and sequence a region of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene. High levels of heterozygosity were observed within the sampled populations, indicating high levels of gene exchange among populations. This is not typical of insects under selection pressure from insecticides and may be due to Aedes vexans breeding in marshes outside of New Orleans and migrating into the city.

Impacts
Fall Armyworm:Phylogeography of fall armyworm haplotypes across the southeast was conducted, providing insight into the dispersal of this species. A molecular diagnostic technique was developed to distinguish fall armyworm adults from other noctuids using PCR-RFLP, facilitating identication of samples from pheromone traps. Aedes vexans mosquito: This genetic study of A. vexans from New Orleans provides evidence that there is a large amount of haplotype variation within and among populations. This is not typical of insects under selection pressure from insecticides and may be due to Aedes vexans breeding in marshes outside of New Orleans and migrating into the city. Knowledge of genetic variation within medically important insect species is important for understanding vector transmission, disease epidemiology and disease control. A better understanding of these factors in A. vexans is important because of its status as a vector of SLE, EEE and West Nile virus.

Publications

• Austin, J.W. 2005. Genetic variation and molecular systematics of Reticulitermes termites from the south central United States. PhD Dissertation, University of Arkansas.
• Lewter, J.A. 2005. Genetic variation and molecular diagnostics of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae). MS Thesis, University of Arkansas.
• Austin, J.W., A.L. Szalanski, R.H. Scheffrahn, and M.T. Messenger. 2005. Genetic variation of Reticulitermes flavipes (Isoptera: Rhinotermitidae) in North America using the mitochondrial rRNA 16S gene. Annals of the Entomological Society of America 98: 980-988.
• Austin, J.W., A.L. Szalanski, R.H. Scheffrahn, M.T. Messenger, S. Dronnet, and A-G. Bagneres. 2005. Genetic evidendence for the synonymy of two Reticulitermes species: Reticulitermes flavipes and Reticulitermes santonensis. Annals of the Entomological Society of America 98: 395-401.
• Owens, C.B., and A.L. Szalanski. 2005. Filter Paper for Preservation, Storage, and Distribution of Insect and Pathogen DNA Samples. Journal of Medical Entomology 42: 709-711.

Progress 01/01/04 to 12/30/04

Outputs
Termite genetics. 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 in North America. A total of 396 samples were analyzed from 31 US States, Ontario, Canada and Mexico. Twenty one nucleotide sites were variable in the 428 bp mtDNA sequence, and 45 mtDNA haplotypes were observed. Nine haplotypes (21.4%) occurred only once, while the most common haplotype, occurred in 15.6% of the samples. Three 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, while two of the three haplotypes found in Mexico are unique to that country. Based on this research there appears to be numerous R. flavipes haplotypes that are widespread due to anthropogenic interactions while other haplotypes are not and may represent locally adapted populations. We also discovered identical DNA sequence data from unidentified Reticulitermes specimens from South America and Easter Island. 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. The diagnostic assay consists of two independent PCR runs of the 16S primer pair along with the C. formosanus primer set. Aedes vexans genetics. Aedes vexans (Meigen), vexans mosquito, is a species that prefers mammalian hosts and is a vector of West Nile virus. It is one of the most widespread pest mosquitoes in the world and in North America, it is common in southern Canada and continental United States. Population structure of this species in Kansas, relative to three other states, was examined using DNA sequences of a 423 bp region of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene. From the 54 Kansas samples, a total of 39 nucleotide positions were polymorphic, with 34 haplotypes. Of the 34 haplotypes, 22 (79%) were not shared among populations. The average haplotype diversity of 0.953 from 11 Kansas populations indicated a high level of genetic diversity in Ae. vexans from Kansas. Analysis of molecular variance was conducted on the resulting haplotypes for all populations and as a hierarchial analysis between populations defied as eastern, central, and western geographical groups. For the geographical analysis, significant genetic structure was detected only within groups with 96.79 % of the variation occurring within groups and 3.21% among groups. Among all four states, a total of 40 haplotypes were observed and a FST value of 0.04 indicates that there is some level of genetic structure between states.

Impacts
The termite research is the first to integrate genetics and geographical distribution of Reticulitermes termites in south central United States. We have found genetic evidence of a new species of Reticulitermes in Mississippi and we are in the process of describing this species, which will be the first new Reticulitermes species to be described in North America since the 1930s. The molecular diagnostic technique for the Formosan subterranean termite provides a non-morphological method to identify field collected termites and may facilitate future quarantine programs for C. formosanus. This first genetic study of Ae. vexans provides evidence that there is a large amount of genetic variation within populations, locally adapted populations, and gene flow across broad geographical areas in this species.

Publications

• Austin, J.W., A.L. Szalanski, R.H. Scheffrahn, M.T. Messenger, S. Dronnet, and A-G. Bagneres. 2005. Genetic evidence for the synonymy of two Reticulitermes species: Reticulitermes flavipes (Kollar) and Reticulitermes santonensis (Feytaud). Annals of the Entomological Society of America (in press).
• Scheffrahn, R.H., J. Krecek, A.L. Szalanski, and J.W. Austin. 2005. Synonymy of the neotropical arboreal termites, Nasutitermes corniger and N. costalis (Isoptera: Termitidae), with evidence from morphology, genetics, and biogeography. Annals of the Entomological Society of America (in press).
• Szalanski, A.L., J.W. Austin, R.H. Scheffrahn, and M.T. Messenger. 2004. Molecular diagnostics of the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae). Florida Entomologist. 87:152-158.
• Scheffrahn, R.H., J. Krecek, B. Maharajh, N.-Y. Su, J.A. Chase, J.R. Mangold, A. L. Szalanski, J.W. Austin, and J. Nixon. 2004. Establishment of the African termite, Coptotermes sjostedti (Isoptera: Rhinotermitidae), on the Island of Guadeloupe, French West Indies. Annals of the Entomological Society of America. 97: 872-876.
• Scheffrahn, R.H., J. Krecek, A.L. Szalanski, J.W. Austin, and Y. Roisin. 2005. Synonymy of two arboreal termites (Isoptera: Termitidae: Nasutitermitinae): Nasutitermes corniger from the neotropics and N. polygynus from New Guinae. Florida Entomologist 88:(1) (in press).
• Scheffrahn, R.H., J. Krecek, A.L. Szalanski, and J.W. Austin. 2005. Synonymy of the neotropical arboreal termites, Nasutitermes corniger and N. costalis (Isoptera: Termitidae), with evidence from morphology, genetics, and biogeography. Annals of the Entomological Society of America (in press).

Progress 01/01/03 to 12/31/03

Outputs
Termite genetics: Research on genetic variation among and within species of Reticulitermes and other termite genera is ongoing. A molecular diagnostic technique for identifying Reticulitermes termites to species has been developed. A study on molecular phylogenetics of the termite family Rhinotermitidae has been completed. In collaboration with the University of Florida, a study on the phylogeography of Heterotermes in the Carribean and the likely origin of Heterotermes sp. to Florida has been completed. Three studies on genetic variation and geographic distribution of Reticulitermes termites in Texas, Oklahoma and Arkansas/Lousiana have been conducted. Results from this research has been presented at regional and national entomology meetings and has resulted in five publications. Fall armyworm genetics: Research on genetic variation of the fall armyworm is ongoing. In collaboration with USDA-ARS CMAVE we have detected three mtDNA haplotypes in the corn strain and two in the rice strain. Research will continue to determine the distribution of these haplotypes in Arkansas and southern States. We are also developing a molecular diagnostic technique to identify fall armyworm eggs, larvae and adults to species using PCR. Results from this research has been presented at regional and national entomology meetings.

Impacts
This research is the first to integrate genetics and geographical distribution of Reticulitermes termites in south central United States. We have found genetic evidence of a new species of Reticulitermes in Mississippi and we are in the process of describing this species, which will be the first new Reticulitermes species to be described in North America since the 1930s.

Publications

• Austin, J.W., A.L. Szalanski, and M.T. Messenger. 2004. Genetic variation and distribution of the subterranean termite genus Reticulitermes (Isoptera: Rhinotermitidae) in Arkansas and Louisiana. Florida Entomologist (accepted).
• Austin, J.W., A.L. Szalanski, and B.M. Kard. 2004. Distribution and genetic variation of Reticulitermes (Isoptera: Rhinotermitidae) in Oklahoma. Florida Entomologist (accepted).
• Austin, J.W., A.L. Szalanski, R.E. Gold, and B.T. Foster. 2004. Genetic variation and geographical distribution of the subterranean termite genus Reticulitermes in Texas. Southwestern Entomologist (accepted).
• Szalanski, A.L., R.H. Scheffrahn, J.W. Austin, J. Krecek, and N-Y Su. 2004. Molecular phylogeny and biogeography of Heterotermes (Isoptera: Rhinotermitidae) in the West Indies. Annals of the Entomological Society of America. 97: (in press).
• Austin, J.W., A.L. Szalanski, and B.J. Cabrera. 2004. A phylogenetic analysis of the subterranean termite family Rhinotermitidae (Isoptera). Annals of the Entomological Society of America 97: (in press).
• Roehrdanz, R.L., A.L. Szalanski, and E. Levine. 2003. Mitochondrial DNA and ITS1 differentiation in geographical populations of northern corn rootworm, Diabrotica barberi (Coleoptera: Chrysomelidae): Identification of distinct genetic populations. Annals of the Entomological Society of America. 96: 901-913.
• Szalanski, A.L., J.W. Austin, and C.B. Owens. 2003. Identification of Reticulitermes spp. (Isoptera: Rhinotermatidae) by polymerase chain reaction restriction fragment length polymorphism. Journal of Economic Entomology. 96: 1514-1519.
• Szalanski, A.L., and C.B. Owens. 2003. Genetic variation of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Florida Entomologist 86: 329-333.

Progress 01/01/02 to 12/31/02

Outputs
Termite Genetics: DNA sequencing analysis of the mitochondrial DNA cytochrome oxidase II region was used to examine genetic variation in the termite genus Reticulitermes. Twenty one species and subspecies from three continents were examined. Sequencing of a 677 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 formed a distinct clade along with R. santonensis. European R. lucifugus formed a distinct clade with R. banyulensis. Turkish R. lucifugus were distinct relative to European R. lucifugus, and along with R. clypeatus Lash from Israel formed a sister group with R. balkanensis from Greece. Southern Corn Rootworm: Genetic analysis of southern corn rootworms, collected from South Dakota, Nebraska, and Arkansas was undertaken using DNA sequences of the nuclear ribosomal first internal transcribed spacer region (ITS1), and a portion of the mitochondrial DNA (mtDNA) cytochrome oxidase I and II genes. Among the 22 beetles subjected to DNA sequencing analysis, no polymorphic nucleotide sites were observed for the ITS1 marker and one variable nucleotide site was observed for the mtDNA marker.

Impacts
Termite Genetics: This research provides support for the separation of Turkish R. lucifugus from European members of the species. This mitochondrial DNA marker was also able to identify unclassified Reticulitermes colonies to species. Southern Corn Rootworm: The lack of genetic distinction observed in southern corn rootworm populations suggests either high levels of gene flow or a recent geographical expansion from a relatively small base. Gene flow would facilitate the rapid spread of traits that could compromise control programs, such as insecticide resistance or behavioral modifications.

Publications

• Austin, J.W., A.L. Szalanski, P. Uva, A. Bagneres, and A. Kence. 2002. A comparative genetic analysis of the subterranean termite genus Reticulitermes (Isoptera: Rhinotermitidae). Annals of the Entomological Society of America. 95: 753-760.