Amber D. Tripodi

Multiplex Polymerase Chain Reaction Diagnostics of Bed Bug (Hemiptera: Cimicidae)

ABSTRACT Cimex lectularius L. (Hemiptera: Cimicidae) 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 polymerase chain reaction (PCR) was developed to distinguish bed bug eggs, leg fragments, and degraded samples from other arthropods that 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 bug samples from the United States, Canada, and Australia, along with sequences of other Cimicidae and arthropods that 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 410-428-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.

Molecular Detection of Nosema apis and N. Ceranae from Southwestern and South Central USA Feral Africanized and European Honey Bees, Apis mellifera (Hymenoptera: Apidae)

Abstract A Polymerase Chain Reaction (PCR) molecular diagnostic survey for the honey bee pathogens Nosema apis Zander and N. ceranae Fries was conducted on feral Africanized honey bee (AHB) and European honey bee (EHB), Apis mellifera L., populations sampled from Mississippi, Arkansas, Texas, Utah, Oklahoma, and New Mexico. Polymerase Chain Reaction — Restriction Fragment Length polymorphism (PCR-RFLP) analysis of a 220 bp small subunit (SSU) marker was conducted on 517 samples, consisting of 245 AHB and 272 EHB individuals. A total of 43 samples (8.3%) were positive for Nosema; of these, 82.1% were N. ceranae, and the remainder were N. apis. No mixed samples were observed. For the AHB samples, Nosema was detected in 9.0% of the samples with 89.5% of the Nosema identified as N. ceranae, and 10.5% as N. apis. With the EHB samples, 7.7% had Nosema; 75.0% of these with N. ceranae, and 25.0% with N. apis. No significant difference was observed between AHB and EHB feral samples for occurrence of each Nosema species and prevalence of Nosema infection. Among the AHB samples, Nosema was more common in Utah and Texas than in New Mexico and Oklahoma. Nosema infection rates for feral honey bees was considerably lower than levels observed with managed honey bees in studies from New York, South Dakota, and Virginia.

Novel multiplex PCR reveals multiple trypanosomatid species infecting North American bumble bees (Hymenoptera: Apidae: Bombus )

Crithidia bombi and Crithidia expoeki (Trypanosomatidae) are common parasites of bumble bees (Bombus spp.). Crithidia bombi was described in the 1980s, and C. expoeki was recently discovered using molecular tools. Both species have cosmopolitan distributions among their bumble bee hosts, but there have been few bumble bee studies that have identified infections to species since the original description of C. expoeki in 2010. Morphological identification of species is difficult due to variability within each stage of their complex lifecycles, although they can be easily differentiated through DNA sequencing. However, DNA sequencing can be expensive, particularly with many samples to diagnose. In order to reliably and inexpensively distinguish Crithidia species for a large-scale survey, we developed a multiplex PCR protocol using species-specific primers with a universal trypanosomatid primer set to detect unexpected relatives. We applied this method to 356 trypanosomatid-positive bumble bees from North America as a first-look at the distribution and host range of each parasite in the region. Crithidia bombi was more common (90.2%) than C. expoeki (21.3%), with most C. expoeki-positive samples existing as co-infections with C. bombi (13.8%). This two-step detection method also revealed that 2.2% samples were positive for trypanosmatids that were neither C. bombi nor C. expoeki. Sequencing revealed that two individuals were positive for C. mellificae, one for Lotmaria passim, and three for two unclassified trypanosomatids. This two-step method is effective in diagnosing known bumble bee infecting Crithidia species, and allowing for the discovery of unknown potential symbionts.

New records of Orussus minutus Middlekauff, 1983 (Hymenoptera: Orussidae) represent a significant western range expansion.

Abstract Background Orussus minutus is an uncommonly collected parasitoid sawfly known from the eastern United States. New information We report specimens Orussus minutus Middlekauff, 1983, from Arkansas, Iowa, Minnesota, and Manitoba, which represent new state and province records and significantly expand the known range of the species west from previous records; provide collection information for unpublished specimens housed in the United States National Museum collection, which includes new state records for West Virginia and Michigan; and report two specimens housed in the Biological Museum at Lund University that represent new state records for Connecticut.

Prevalence of Nosema from Managed Honey Bee Colonies in South Dakota and New York1

Abstract Despite a number of studies on the prevalence of Nosema across the United States, limited information exists regarding comparative rates of N. ceranae and N. apis infection within individual states. We conducted a study to detect N. ceranae and N. apis using molecular diagnostics on managed honey bee colonies from New York and South Dakota. A total of 1200 apiaries were visited by New York state bee inspectors in 2009, and samples were collected from 4300 honey bee colonies. A total of 1023 apiaries were sampled from South Dakota in 2009. Microscopy was used to detect Nosema spore presence and to estimate the intensity of infection by estimating spore counts in positive samples. Nosema was detected in 528 (44%) of the New York and 300 (29%) of the South Dakota sampled apiaries. A total of 464 New York and 290 South Dakota Nosema-positive apiary samples were subjected to molecular diagnostic polymerase chain reaction (PCR) analysis. For the New York samples, 250 (54%) were PCR-positive for Nosema, with 96.8% being N. ceranae, 0.8% N. apis, and 2.4% both N. ceranae and N. apis. For the South Dakota samples, 122 (42%) were PCR-positive for Nosema, all of which were N. ceranae.

Evaluating Spore Count and Sporophorous Vesicle Size in Ovavesicula popilliae (Microsporidia: Ovavesiculidae) in Adult Japanese Beetles (Coleoptera: Scarabaeidae)

Ovavesicula popilliae is a microsporidian pathogen used as a biological control agent of the Japanese beetle ( Popillia japonica Newman), and reportedly produces 32 spores within a sporophorous vesicle measuring 20.0-21.0 μm × 15.0-15.5 μm. We determined the sporophorous vesicles to have a mean size of 13.3 μm × 9.3 μm and each with a mean of 24.7 spores. Varying number of spores may be due to failed divisions of sporonts, death of spores after formation, host effects or different pathogen strains. Ovavesicula popilliae es una microsporidio patogeno utilizado como agente de control biologico del escarabajo japones ( Popillia japonica Newman), que segun reportes produce 32 esporas dentro de vesiculas con medidas de 20.0-21.0 μm × 15.0-15.5 μm. Determinamos que las vesiculas tienen un tamano de 13.3 μm × 9.3 μm y una media de 24.7 esporas. La variacion en el numero de esporas puede deberse a fallas en la division de los sporonts, muerte de las esporas despues de su formacion, efectos del hospedero, o diferencias en las cepas del patogeno. View this article in BioOne

Further Range Extension of Xylocopa micans Lepeletier (Hymenoptera: Apidae)

There are seven species in the genus Xylocopa Latreille that occur in North America north of Mexico, and only two of these are known to occur in the Eastern United States: Xylocopa virginica L. and X. micans Lepeletier. While the range of X. virginica extends through much of the Eastern United States, the distribution of X. micans is concentrated along the coastal areas to the far south and east. Until 2010, X. micans had only been recorded in Alabama, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina and Texas (Hurd, 1955). In 2006 and 2007, four specimens of X. micans were collected in southwestern Arkansas, representing the first records of X. micans in Arkansas, about 250 km northwest of its historical range (Warriner, 2010). Additionally, we report here that two males of X. micans were netted on May 10, 2010 north of Biscoe in Prairie County, AR (34.845000uN, 91.415833uW). This location is approximately 50 km northeast of the first specimens found in Arkansas three years prior. The specimens were determined with the keys of Hurd (1955) and Mitchell (1962). The voucher specimens have been deposited at the University of Arkansas’ Arthropod Museum, Department of Entomology in Fayetteville, Arkansas, USA. This is a new locality record for X. micans in the state of Arkansas and supports Warriner’s (2010) suggestion that this represents an extension of its historically recorded range, rather than an incidental introduction. Xylocopa micans is a member of the Neotropical subgenus Schoenherria Lepeletier, and its range may be restricted to warmer climates. In the Lower Rio Grande Valley, Porter (1981) found that X. micans was only collected in the warmer months from spring through summer, while other Xylocopa species were collected year round. The presence of X. micans in Prairie County, Arkansas may reflect a climate-driven northward range expansion as predicted by climate change and already evidenced by the range shifts of species such as the nymphalid Euphydryas editha (Boisduval) (Parmesan, 1996). Conversely, the specimens found in Prairie County, Arkansas may be human-mediated introductions and not representative of a natural expansion. The site at which they were collected is located along Interstate 40, which connects to the southwestern locations via Interstate 30. It is possible that these individuals were transported from their native range to Arkansas and have spread by means of highway traffic. Xylocopa species, including X. micans, nest in dead wood, and may inadvertently be transported long distances in shipments of lumber or other wood products. This was shown to be the case for Xylocopa brasilianorum L. which arrived in San Francisco in a balsa wood shipment from Hawaii (Hurd, 1955). Further sampling of bees in Arkansas may help determine how X. micans arrived in and spread throughout Arkansas. Large bees, such as Bombus and Xylocopa species, have the ability to sonicate the anthers of flowers with poricidal dehiscence, such as Solanum species (King and Buchmann, 2003). Due to this somewhat unique capacity, large bees such as Xylocopa micans are of particular interest as pollinators of many plant species with poricidal anthers. Out of 153 bee visitors, X. micans and two species of Melissodes were shown to be the only effective pollinators of Chamaecrista keyensis Pennell (Fabaceae), the big pine partridge pea, which is listed as an endangered species in the state of Florida (Liu and Koptur, 2003). Clearly, the role of X. micans as generalist pollinator with special abilities can be an important factor in plant communities. If the distribution of X. micans is changing, either as the result of climate change or through anthropogenic introductions, there may be additional ecological repercussions.