Ralph B. Narain

Genetic Variability and Geographic Diversity of the Common Bed Bug (Hemiptera: Cimicidae) Populations from the Midwest Using Microsatellite Markers

ABSTRACT With the recent global resurgence of the bed bugs (Cimex lectularius L.), there is a need to better understand its biology, ecology, and ability to establish populations. Bed bugs are domestic pests that feed mainly on mammalian blood. Although bed bugs have not been implicated as vectors of pathogens, their biting activity inflicts severe insomnia and allergic reactions. Moreover, they have recently developed resistance to various insecticides, which requires further molecular research to determine genetic variation and appropriate interventions. Population dynamics, including genetic differentiation and genetic distance of 10 populations from the Midwest were analyzed in this study. The bed bug samples collected by pest control companies were genotyped using eight species-specific microsatellite markers. Results showed all eight markers were polymorphic, with 8–16 alleles per locus, suggesting high genetic diversity. The FST values were >0.25, signifying pronounced genetic differentiation. The G-test results also indicated high genetic differentiation among populations. The frequency of the most common allele across all eight loci was 0.42. The coefficient of relatedness between each of the populations was >0.5, indicative of sibling or parent—offspring relationships, while the FIS and its confidence interval values were statistically insignificant within the populations tested. The populations departed from Hardy—Weinberg equilibrium, possibly because of high heterozygosity. The genetic distance analysis using a neighbor-joining tree showed that the populations from Kansas City, MO, were genetically separate from most of those from Nebraska, indicating a geographic pattern of genetic structure. Our study demonstrated the effectiveness of using microsatellite markers to study bed bugs population structure, thereby improving our understanding of bed bug population dynamics in the Midwest. Overall, this study showed a high genetic diversity and identified several new alleles in the bed bug populations in the Midwest.

Effects of Ibuprofen and Caffeine Concentrations on the Common Bed Bug (Cimex lectularius L.) Feeding and Fecundity

Bed bugs are ectoparasites of humans and require a blood meal for their growth and reproduction. Since humans consume Ibuprofen as pain medication and drink coffee (caffeine), bed bugs are likely to acquire these drugs through blood feeding. In this study, we determined the biological effects of Ibuprofen and caffeine on bed bug feeding, fecundity and egg hatch. Five concentrations of Ibuprofen and caffeine were incorporated into reconstituted human blood (RHB). Control treatment had no Ibuprofen or caffeine. Each treatment had six replications. Groups of 20 adult bed bugs (10 males: 10 females)/treatment/replication were pre-weighed, allowed to feed for 45 minutes, and then reweighed. After feeding, bed bugs were transferred into a glass jar equipped with harborage and all the jars were placed in an environmental chamber undisturbed for three 7-day intervals to determine fecundity and nymph emergence. Ibuprofen data showed that the mean mass of the 20 adult bed bugs increased by 0.1074 g (125.65%) in the control but by 0.1336 g (157.30%) at 200 ppm after feeding. After 7 days, 306 and 146 eggs were laid by the 60 female bed bugs fed on 0 and 200 ppm Ibuprofen, respectively. After 2 weeks, 94% eggs hatched with no significant differences amongst treatments. In the caffeine experiments, the bed bug mean mass increases were 0.1219 g (163.90%) in control and 0.0790 g (104.62%) at 50 ppm caffeine. After 7 days, 264 eggs were laid by 60 female bed bugs in control but only 81 eggs were laid at the 50 ppm. Nymph emergence was >80% for all caffeine concentrations after 2 weeks. These results demonstrated that increasing Ibuprofen concentrations had positive effects on mass gain but negative effects on egg laying capacity of bed bugs. Caffeine concentrations had negative effects on bed bug feeding, fecundity and egg hatch.

Differential Gene Expression Profiling in Bed Bug ( Cimex Lectularius L.) Fed on Ibuprofen and Caffeine in Reconstituted Human Blood

The recent resurgence of the common bed bug (Cimex lectularius L.) infestations worldwide has created a need for renewed research on biology, behavior, population genetics and management practices. Humans serve as exclusive hosts to bed bugs in urban environments. Since a majority of humans consume Ibuprofen (as pain medication) and caffeine (in coffee and other soft drinks) so bug bugs subsequently acquire Ibuprofen and caffeine through blood feeding. However, the effect of these chemicals at genetic level in bed bug is unknown. Therefore, this research was conducted to determine differential gene expression in bed bugs using RNA-Seq analysis at dosages of 200 ppm Ibuprofen and 40 ppm caffeine incorporated into reconstituted human blood and compared against the control. Total RNA was extracted from a single bed bug per replication per treatment and sequenced. Read counts obtained were analyzed using Bioconductor software programs to identify differentially expressed genes, which were then searched against the non-redundant (nr) protein database of National Center for Biotechnology Information (NCBI). Data on comparison of differentially expressed genes between control and Ibuprofen treatments revealed that 659 genes were significantly differentially regulated and 95% of them returned BLAST hits. Heat stress proteins were among the top significantly differentially down regulated genes. Comparison of the control vs caffeine treatments revealed that 2,161 genes were significantly differently regulated (Padj <0.05). Heat shock proteins were among the top ten down regulated genes in both treatments. Finally, using RNAi to identify the exact function of these highly differentially expressed genes and regulating these genes may offer potential for managing bed bug populations.

DNA Barcode of Thief Ant Complex (Hymenoptera: Formicidae)

Abstract The thief ant, Solenopsis molesta (Say), a common nuisance species found throughout the United States is genetically related to red imported fire ants, S. invicta Buren. Therefore, its identification at the molecular level is very important. The deoxyribonucleic acid (DNA) barcoding, a recent technique was used to identify thief ant complex at species and subspecies levels using a short DNA sequence from the cytochrome oxidase subunit 1 (COI) mitochondrial region. The DNA from thief ants collected from 9 states was extracted using Qiagens Gentra PUREGENE® DNA Isolation Kit. The polymerase chain reactions (PCR) were run on the extracted DNA to amplify partial sequence of COI using primers Lep-F1 (forward) and Lep-R1 (reverse). The resulting DNA products were concentrated, purified and sequenced. The 600 bp sequences of the COI generated were submitted to GenBank that issued accessions numbers from HM179641 to HM179653. The sequences associated with these accession numbers were used as DNA barcodes for distinguishing species and subspecies. Based on this molecular analysis, thief ants collected from New York, Indiana and 1 location in Nebraska were separated in 1 group as S. molesta validiuscula (Emery) and another with ants from Louisiana identified as S. carolinensis (Forel). The third group was comprised of ants from South Dakota, Washington, New Jersey, Tennessee, Kansas and 2 other locations in Nebraska was identified as S. molesta molesta (Say).