Identification of Reticulitermes Subterranean Termites (Blattodea: Rhinotermitidae) in the Eastern United States Using Inter-Simple Sequence Repeats.

In the eastern United States, there are nine species of subterranean termites in three genera: Reticulitermes (six species), Coptotermes (two species), and Prorhinotermes (one species). These species serve as important ecological players by decomposing cellulose material, and some are important structural pests. Many of these species are difficult to discriminate morphologically and require examining the reproductive or soldier castes, which can be difficult to collect. While some genetic tools have been developed for species identification, they are often expensive and time-consuming. To help facilitate identification, we developed a more cost-effective and rapid genetic method to identify Reticulitermes species by screening 10 PCR primers that amplified inter-simple sequence repeats (ISSRs) in other termite species. From these, one primer was amplified in all five focal Reticulitermes species and contained conserved, species-specific fragments. We further screened this identification method on samples of each species covering a diversity of mitochondrial DNA haplotypes and localities. This identification method utilizing ISSRs can be used to quickly identify five species of Reticulitermes subterranean termites in the eastern United States in a matter of hours, providing a useful technique for pest management as well as future ecological research.

Molecular Detection of Rickettsia Species Within Ticks (Acari: Ixodidae) Collected from Arkansas United States.

Rocky Mountain spotted fever (RMSF), caused by the etiological agent Rickettsia rickettsii, is the most severe and frequently reported rickettsial illness in the United States, and is commonly diagnosed throughout the southeast. With the discoveries of Rickettsia parkeri and other spotted fever group rickettsiae (SFGR) in ticks, it remains inconclusive if the cases reported as RMSF are truly caused by R. rickettsii or other SFGR. Arkansas reports one of the highest incidence rates of RMSF in the country; consequently, to identify the rickettsiae in Arkansas, 1,731 ticks, 250 white-tailed deer, and 189 canines were screened by polymerase chain reaction (PCR) for the rickettsial genes gltA, rompB, and ompA. None of the white-tailed deer were positive, while two of the canines (1.1%) and 502 (29.0%) of the ticks were PCR positive. Five different tick species were PCR positive: 244 (37%) Amblyomma americanum L., 130 (38%) Ixodes scapularis Say, 65 (39%) Amblyomma maculatum (Koch), 30 (9%) Rhipicephalus sanguineus Latreille, 7 (4%) Dermacentor variabilis Say, and 26 (44%) unidentified Amblyomma ticks. None of the sequenced products were homologous to R. rickettsii. The most common Rickettsia via rompB amplification was Rickettsia montanensis and nonpathogenic Candidatus Rickettsia amblyommii, whereas with ompA amplification the most common Rickettsia was Ca. R. amblyommii. Many tick specimens collected in northwest Arkansas were PCR positive and these were commonly A. americanum harboring Ca. R. amblyommii, a currently nonpathogenic Rickettsia. Data reported here indicate that pathogenic R. rickettsii was absent from these ticks and suggest by extension that other SFGR are likely the causative agents for Arkansas diagnosed RMSF cases.

Molecular diagnostics of Drosophila suzukii (Diptera: Drosophilidae) Using PCR-RFLP.

The invasive spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has become a serious pest in the United States. Identification of immature and poorly preserved specimens can be difficult. A molecular diagnostic method for distinguishing D. suzukii from other Drosophila spp. associated with fruit in the United States was developed. A 709-bp region of the mitochondrial DNA cytochrome oxidase I gene was amplified from D. suzukii collections in the United States and compared with sequences of other Drosophila taxa from GenBank. Based on DNA sequence polymorphisms, a polymerase chain reaction-restriction fragment length polymorphism analysis using the restriction enzyme Msp-I was found to differentiate D. suzukii from other Drosophila spp. in the United States. This technique can identify field-collected specimens from various sources and specimens regardless of life stage. This molecular diagnostic method will be useful for monitoring the spread of this economically important invasive insect.

Phylogenetics and population genetics of the louse fly, Lipoptena mazamae, from Arkansas, USA.

Louse flies, also known as deer keds (Lipoptena mazamae Rondani), infest cervids such as white-tailed deer, Odocoileus virginianus and vector pathogens such as Anaplasma and Bartonella schoenbuchensis to cattle and humans, respectively. The population genetic structure of 30 L. mazamae collected from white-tailed deer in four regions of Arkansas, U.S.A., designated by county boundaries, was examined using DNA sequences of a 259-bp region of the mitochondrial DNA rRNA 16S gene. Of the 259 nucleotide characters, 33 were variable and 6 haplotypes were identified. Two haplotypes occurred only once (haplotype 3 and 4), whereas two other haplotypes occurred in 43% (haplotype 1 in two regions) and 40% (haplotype 6 in three regions) of the samples. Phylogenetic relationships of the six L. mazamae haplotypes were constructed with other Hippoboscid and Glossinid samples and two clades resulted. Clade 1 was located in the north and western Ozarks whereas clade 2 was found in the northern and eastern Ozarks. Results from the present study indicate that Lipoptena may be a polyphyletic genus; consequently, more research into genetic variation within this genus is necessary.

Population genetics of Amblyomma americanum (Acari: Ixodidae) collected from Arkansas.

Lone star ticks, Amblyomma americanum L. (Acari: Ixodidae),infest multiple hosts such as birds, and mammals of various sizes (rodents to white-tailed deer) and can harbor human pathogens such as Borrelia lonestari and Ehrlichiosis chaffeensis. The population structure of 251 A. americanum ticks, collected from canines and two white-tailed deer in six Arkansas ecoregions, was examined using DNA sequences of a 247-bp region of the mitochondrial DNA ribosomal RNA 16S gene. Of the 247 nucleotide characters, 26 were variable. Thirty-three haplotypes were identified of which 25 haplotypes occurred once (10%). The most common haplotype was Aa25, occurring in 60% of the samples and found in all six ecoregions. The excess of low frequency haplotypes combined with the overall negative Tajima's D and Fu and Li statistics suggests population expansion. Phylogenetic relationships of the 33 A. americanum haplotypes were constructed with other Amblyomma species and identified A. americanum as a monophyletic species with two groups. The patterns of high nucleotide and haplotype diversity found in this study suggests that the A. americanum population is expanding perhaps due to its ability to survive in a variety of habitats and feed on multiple hosts. Given the gene flow in Arkansas, the spread of acaricide resistance and pathogens may be rapid.

Secondary structure models of D2-D3 expansion segments of 28S rRNA for Hoplolaiminae species.

The D2-D3 expansion segments of the 28S ribosomal RNA (rRNA) were sequenced and compared to predict secondary structures for Hoplolaiminae species based on free energy minimization and comparative sequence analysis. The free energy based prediction method provides putative stem regions within primary structure and these base pairings in stems were confirmed manually by compensatory base changes among closely and distantly related species. Sequence differences ranged from identical between Hoplolaimus columbus and H. seinhorsti to 20.8% between Scutellonema brachyurum and H. concaudajuvencus. The comparative sequence analysis and energy minimization method yielded 9 stems in the D2 and 6 stems in the D3 which showed complete or partial compensatory base changes. At least 75% of nucleotides in the D2 and 68% of nucleotides in the D3 were related with formation of base pairings to maintain secondary structure. GC contents in stems ranged from 61 to 73% for the D2 and from 64 to 71% for the D3 region. These ranges are higher than G-C contents in loops which ranged from 37 to 48% in the D2 and 33-45% in the D3. In stems, G-C/C-G base pairings were the most common in the D2 and the D3 and also non-canonical base pairs including A•A and U•U, C•U/U•C, and G•A/A•G occurred in stems. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments provide useful information to assign positional nucleotide homology and reconstruction of more reliable phylogenetic trees.

Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA.

DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.

Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP analysis of nuclear rDNA ITS regions.

Hoplolaimus columbus is an important nematode pest which causes economic loss of crops including corn, cotton, and soybean in the Southeastern United States. DNA sequences of the ITS1-5.8S-ITS2 region of ribosomal DNA from H. columbus were aligned and analyzed to characterize intraspecific genetic variation between eleven populations collected from Georgia, Louisiana, North Carolina, and South Carolina. In comparative sequence analysis with clones from either one or two individuals obtained from the eleven populations, we found variability existed among clones from an individual and that clonal diversity observed from within individuals was verified by PCR-RFLP. PCR-RFLP analysis with Rsa I and Msp I restriction enzymes yielded several fragments on 3.0% agarose gel that corresponded to different haplotypes in all populations and the sum of digested products exceeded the length of undigested PCR products, which revealed that ITS heterogeneity existed in a genome of H. columbus. This indicates that heterogeneity may play a role in the evolution of this parthenogenetic species.

Detection of Campylobacter and Escherichia coli O157:H7 from filth flies by polymerase chain reaction.

Flies (Diptera: Muscidae) that breed in faeces and other organic refuse (filth flies) have been implicated as vectors of pathogenic bacteria including Escherichia coli O157:H7, which cause haemorrhagic colitis in humans, and Campylobacter, which is the principal causative agent of human enteritis. The potential role of filth flies in the epidemiology of these pathogens in the United States was investigated by examining the prevalence of Campylobacter spp. and E. coli O157:H7 from two Arkansas turkey facilities. Polymerase chain reaction was conducted on DNA extractions of individual Musca domestica Linnaeus, Stomoxys calcitrans (Linnaeus), Hydrotaea aenescens (Wiedemann), Adia cinerella Fallen and turkey faecal samples using primers specific for E. coli H7, O157 and Campylobacter spp. Culturing verified that the flies were carrying viable Campylobacter spp. and E. coli O157:H7. Results from this study indicated that M. domestica, S. calcitrans, H. aenescens and Anthomyids are capable of carrying Campylobacter in North American poultry facilities and that the E. coli O157:H7 is carried by house flies and black dump flies associated with poultry. This PCR method provided a rapid and effective method to identify Campylobacter spp. and E. coli O157:H7 directly from individual filth flies.

First Report of Columbia Root Knot Nematode (Meloidogyne chitwoodi) in Potato in Texas.

Columbia root-knot nematode, Meloidogyne chitwoodi Golden et al. (1) was identified from potatoes, Solanum tuberosum L., collected from Dallam County, Texas in October 2000. Seed potatoes are the most likely source for this introduction. This nematode is currently found infecting potatoes grown in California, Colorado, Idaho, New Mexico, Nevada, Oregon, Utah, and Washington. Some countries prohibit import of both seed and table stock potatoes originating in states known to harbor M. chitwoodi. Lesions on the potatoes had discrete brown coloration with white central spots in the outer 1 cm of the tuber flesh. Female nematode densities averaged 3 per square centimeter of a potato section beneath the lesions. Nematodes were morphologically identified as M. chitwoodi based on the perineal pattern of mature females and the tail shape of juveniles per Golden et al. (1). Using polymerase chain reaction-RFLP of the rDNA ITS1 region and the mtDNA COII-16S rRNA region (2), individual juveniles were identified as M. chitwoodi based on their restriction fragment patterns. This is the first report of Columbia root-knot nematode infecting potatoes in Texas. The distribution of this nematode in potato fields throughout central United States should be determined. References: (1) A. N. Golden et al. J. Nematol. 12:319, 1980. (2) T. O. Powers and T. S. Harris. J. Nematol. 25:1, 1993.

Identification of Seed Gall Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1.

A molecular analysis of eight described species of seed gall nematode, along with six undescribed isolates from different hosts, has revealed a strong association between nucleotide sequence polymorphism and host status. Each anguinid nematode associated with a unique host produced a unique PCR-RFLP pattern for the ITS1 region. Anguina species that had been synonymized in the past, Anguina agrostis, A. funesta, and A. wevelli (Afrina wevelli), were readily discriminated. Two undescribed species from northern New South Wales and southeastern South Australia, reported to be vectors of Rathyaibacter toxicus in the disease called ''floodplain staggers,'' were differentiated by a single restriction enzyme, and both could be separated easily from A. funesta, the vector of R. toxicus in annual ryegrass toxicity. Other species differentiated in this study include A. agropyronifloris, A. graminis, A. microlaenae, A. pacificae, and undescribed species from host species Dactylis glomerata, Agrostis avenacea, Polypogon monospeliensis, Stipa sp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITS1 region suggests that considerable anguinid genetic diversification has accompanied specialization on different host species.

Assessing nuclear and mitochondrial DNA sequence variation within steinernema (rhabditida: steinernematidae).

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.

Genetic variation in geographical populations of western and Mexican corn rootworm.

Genetic variation in the nuclear rDNA ITS1 region of western corn rootworm, Diabrotica virgifera virgifera (WCR), and Mexican corn rootworm, D. v. zeae (MCR) was studied. Two sites were detected which differentiated WCR and MCR in the 642-base sequence. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the first internal transcribed spacer region (ITS1) sequence revealed no variation within or among the twelve WCR and two MCR populations. PCR-RFLP of 75% of the mitochondrial DNA genome detected one significant polymorphic site out of the approximately 190 restriction sizes observed in WCR. The polymorphism did not differentiate geographical populations of WCR and is not diagnostic for the subspecies. The low levels of variation observed in WCR 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 programmes, such as insecticide resistance or behavioural modifications. The minimal genetic differentiation between WCR and MCR raises questions about the evolutionary history of these subspecies and how the distinct phenotypes are maintained.

The internal transcribed spacer region of belonolaimus (nemata: belonolaimidae).

Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1.

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes.

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.

Population genetics and gene variation of stable fly populations (Diptera:Muscidae) in Nebraska.

Genetic variation in stable fly, Stomoxys calcitrans (L.), populations from Nebraska, Canada, and Texas was sampled. Four of 12 allozyme loci were polymorphic, with an average of 1.7 alleles per locus. Observed and expected heterozygosities were 0.086 and 0.070, respectively. Nei's genetic distance between populations averaged 0.001 and ranged from 0.000 to 0.005. Wright's F statistics revealed greater variation within than among populations. Allele frequencies were homogeneous among temporal samples from a single population. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 6.4 kb of the mitochondrial DNA genome with 16 restriction enzymes revealed no variation in stable fly populations from Canada, Nebraska, and Texas. PCR-RFLP analysis of a 2.0-kb fragment of the nuclear ribosomal DNA internally transcribed spacer region also revealed no variation. The lack of genetic differentiation among stable fly populations indicates high levels of gene flow among populations. The low levels of variation observed with biochemical and molecular techniques are consistent with a genetic bottleneck during stable fly colonization of North America.

Mitochondrial DNA variation in screwworm.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to characterize mitochondrial DNA (mtDNA) variation in screwworms, Cochliomyia hominivorax, and secondary screwworm, C.macellaria, from the Caribbean, North America and South America. Four amplicons, totaling 7.1 kb, were analysed with sixteen restriction enzymes. A total of 133 restriction sites was observed in the two species, 104 in C.hominivorax, of which nineteen were variable, and ninety-five in C.macellaria, none of which was variable. Fourteen mtDNA haplotypes were observed among eighteen C.hominivorax examined. Mean divergence between C.hominivorax haplotypes (d) was 0.0064 substitutions per base-pair and genotypic diversity (G) was 0.97. Mean divergence between C.hominivorax and C.macellaria was 0.0824. Cochliomyia hominivorax haplotypes could be divided into three assemblages representing North America, South America and Jamaica, based on UPGMA clustering with d values. The assemblages did not exhibit complete geographic fidelity. These data were discordant with previously published allozyme data indicating little differentiation between screwworm populations. A scenario invoking historically isolated populations coming into contact with the introduction and movement of European livestock is proposed to explain the observed population structure of screwworm.

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism.

Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (F.). Five restriction fragment length patterns were polymorphic in C.hominivorax while all fragment patterns were fixed in C.macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than $2.50.