Carrie B. Owens

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

Abstract.  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.

GENETIC VARIATION OF THE SOUTHERN CORN ROOTWORM, (COLEOPTERA: CHRYSOMELIDAE)

Abstract Corn rootworms of the genus Diabrotica (Coleoptera: Chrysomelidae) are among the most important insect pest of crops in the United States. The southern corn rootworm, Diabrotica undecimpunctata howardi Barber is an economically important pest of corn, cucurbits and peanuts. 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. The lack of genetic distinction observed in southern corn rootworm populations suggests either high levels of dispersal or a recent geographical expansion from a relatively small base.

GENETIC VARIATION WITHIN AND BETWEEN STRAINS OF THE FALL ARMYWORM, SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE)

Abstract Limited information exists on molecular genetic variation and distribution of the corn and rice strains of the fall armyworm, Spodoptera frugiperda (J.E. Smith). 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 was conducted on the resulting mtDNA haplotypes from the Arkansas and Florida populations and as a hierarchical analysis between populations in the two states. Results indicate a significant overall ΦST for all populations with the hierarchical analysis revealing that this significant ΦST is due to structuring of the populations between states. The observed genetic structure is possibly due to the distribution of fall armyworm strains.

Genetic Structure of Aedes vexans (Diptera: Culicidae) Populations from Central United States Based on Mitochondrial ND5 Sequences

Abstract Aedes vexans (Meigen), the vexans mosquito, is a species that prefers mammalian hosts and is a vector of West Nile virus (family Flaviviridae, genus Flavivirus). It is one of the most widespread pest mosquitoes in the world and North America, and it is commonly found in southern Canada and continental United States. Population structure of this species in Kansas was examined using DNA sequences of a 423-bp region of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene, relative to three other states. 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 (0.953) from 11 Kansas populations indicated a high level of genetic diversity in Ae. vexans. Among the Kansas, South Dakota, Texas, and Louisiana samples, a total of 40 haplotypes were observed. Analysis of molecular variance was conducted on the resulting haplotypes for all populations and no geographical structure was observed among populations by using isolation-by-distance tests. This first genetic study of Ae. vexans provides evidence that there is a large amount of haplotype variation within and among populations, and gene flow occurs across broad geographical areas in this species.

Filter Paper for Preservation, Storage, and Distribution of Insect and Pathogen DNA Samples

Abstract Proper DNA storage is critical for studies involving genetic analysis of insects and for molecular diagnostics of pathogens carried by them. Molecular surveillance of pathogens carried by insects can involve screening of thousands of insect DNA samples. Problems with storage and degradation of these samples can arise. In this study, a simple filter paper-based method for storage and preservation of insect DNA was evaluated using polymerase chain reaction (PCR). DNA was isolated from individual house fly, Musca domestica L., adults by using a cell lysis technique. From 50 house flies known to carry Campylobacter spp., a portion of the DNA sample was stored frozen and another portion was pipetted onto filter paper. At monthly intervals for 7 mo, PCR was conducted using 1 μl of the frozen DNA sample and a 2.0-mm disk from the filter paper samples as the PCR template. Two markers were used, a 450-bp region of the insect mitochondrial DNA (mtDNA) ND5 gene and a 857-bp region of the Campylobacter spp. mtDNA 16S rDNA gene. PCR amplification was successful for all of the samples regardless of the storage method. The filter paper method is a simple and economical way to store, preserve, and distribute DNA samples for PCR analysis.

Molecular assay for the detection of Cochlosoma anatis in house flies and turkey specimens by polymerase chain reaction

Abstract A 1520bp region of Cochlosoma anatis mtDNA 16S gene was subjected to DNA sequencing and a 466bp portion was compared with other protozoan 16S sequences to develop PCR primers specific for C. anatis. This PCR diagnostic method allowed identification of C. anatis from house flies, Musca domestica L., turkey gut, and fecal samples within 6h after field-collected samples reached the laboratory. House flies detected carrying C. anatis using the diagnostic 374bp amplicons represented the first record of this protozoan in house flies.

Sequence change and phylogenetic signal in muscoid COII DNA sequences.

The complete DNA sequence of the mtDNA cytochrome oxidase II gene from house fly, Musca domestica, face fly, Musca autumnalis, stable fly, Stomoxys calcitrans, horn fly, Haematobia irritans, and black garbage fly, Hydrotaea aenescens, are reported. The nucleotide sequence codes for a 229 amino acid peptide. The COII sequence is A+T rich (74.1%), with up to 12.3% nucleotide and 8.4% amino acid divergence among the five taxa. Of the 688 nucleotides encoding for the gene, 135 nucleotide sites (19.6%) are variable, and 55 (8.0%) are phylogenetically informative. A phylogenetic analysis using three calliphorids as the outgroup taxa, indicates that the two haematophagus species, horn fly and stable fly, form a sister group.