Allen L. Szalanski

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

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

Genetic evidence for the synonymy of two Reticulitermes species : Reticulitermes flavipes and Reticulitermes santonensis

Abstract By applying the 16S rRNA mitochondrial marker to 434 populations of Reticulitermes termites from North, Central, and South America; France; and Germany with other locations around the world that we have analyzed, identical DNA sequence data were obtained from Reticulitermes flavipes (Kollar) in North America and Germany and for Reticulitermes santonensis Feytaud from undisturbed (nonurban) forested locations in western France. We also discovered identical DNA sequence data from previously unidentified Reticulitermes specimens from South America and Easter Island. Haplotypes F, M, and GG were observed in France; haplotype F in Germany; and haplotype GG was found on Easter Island, Santiago, Chile, and Montevideo, Uruguay. All of these haplotypes are found in numerous states within the continental United States. In light of their well documented morphological, chemical, and phylogenetic relationships, coupled with this new data that directly link these disjunct groups, R. flavipes and R. santonensis should be synonymized. Compared with other studies that largely suggest phyletic similarities, this is the first study that specifically matches haplotypes from North America (where populations of R. flavipes are endemic) with populations in Europe (where R. flavipes, described as R. santonensis, is presumed exotic).

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.

Population Genetics and Phylogenetics of the Endangered American Burying Beetle, Nicrophorus americanus (Coleoptera: Silphidae)

Abstract The burying beetle Nicrophorus americanus Olivier is an endangered species known to occur in disjunct populations in 6 states. Parsimony and maximum likelihood analysis of the nuclear ribosomal DNA first internal transcribed spacer (ITS1) sequences from 10 Nicrophorinae species revealed N. americanus to form a distinct clade with N. orbicollis Say. Genetic variation within and among 5 N. americanus populations, collected from South Dakota, Nebraska, Oklahoma, Arkansas, and Rhode Island, was studied. Ribosomal DNA ITS1 sequences from 14 beetles revealed 48 polymorphic and 20 informative nucleotide sites. N. americanus genetic divergence was between 0.16 and 4.76%. We found little evidence that these 5 populations have maintained unique genetic variation. No nucleotide sites were found that were diagnostic for any of the 5 populations examined, indicating that these populations may not be necessarily treated as separate, independent objects of conservation. However, further genetic investigation is warranted before translocations are attempted among the remaining populations of the American burying beetle.

Phylogenetic Analysis of the Subterranean Termite Family Rhinotermitidae (Isoptera) by Using the Mitochondrial Cytochrome Oxidase II Gene

Abstract Previous molecular phylogenetic studies have focused on either the relationship between isopteran families or among species within a given genus, but there are presently few studies focusing on individual families and no known molecular studies for Rhinotermitidae. We examined 38 rhinotermitid species representing 10 genera, relative to representatives of four other isopteran families. Sequencing of a 667-base pair region of the mitochondrial DNA cytochrome oxidase II gene revealed 343 polymorphic sites within the family. Tajima-Nei genetic distances ranged from 11 to 23% among rhinotermitid genera. Maximum parsimony and maximum likelihood analysis of DNA sequences support existing hypotheses that Mastotermitidae is the basal lineage among extant termites, and the family Rhinotermitidae is polyphyletic given the current familial status of Serritermitidae. DNA sequence data suggest that Serritermitidae should be relegated to the subfamily Serritermitinae, as proposed by Emerson in 1965.

Synonymy of Neotropical Arboreal Termites Nasutitermes corniger and N. costalis (Isoptera: Termitidae: Nasutitermitinae), with Evidence from Morphology, Genetics, and Biogeography

Abstract Morphological examination of soldiers and imagos assigned to Nasutitermes corniger or N. costalis from 13 Neotropical countries and 42 West Indian islands revealed congruent characters and biometric overlap. A portion of the mitochondrial DNA 16S rRNA gene was sequenced from nine N. costalis and N. corniger samples. Molecular phylogenetic analysis of the N. costalis/corniger DNA sequences relative to other Nasutitermes spp. supported the morphological evidence that these species are conspecific. Complementary biological, behavioral, biochemical, and reproductive ecology further support the presented synonymy. The senior synonym, N. corniger, is given nomenclatural precedence. The geographical distribution of N. corniger is revised.

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

Abstract. 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 (R). 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.

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

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

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

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

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

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