Silencing of Four Genes Involved in Chromatin Remodeling by RNA Interference Adversely Affects Fecundity of Bed Bugs (Hemiptera: Cimicidae).

DNA, the blue print of life, is densely wrapped around histone proteins to form chromatin. Chromatin remodeling ATPases unwind histone-DNA interactions to facilitate DNA transcription, modification, and repair. Four genes involved in chromatin remodeling, namely, imitation SWI (iswi), chromodomain-helicase-DNA-binding protein 1 (chd-1), DNA helicase INO80 (ino80) and mi-2 were silenced through the injection of dsRNA, and phenotypes were assessed in bed bugs. Bed bugs were injected with 0.2 µg dsRNA per insect between the last thoracic segment and first abdominal segment using a fine capillary tube fitted to a nanoinjector. We observed a significant reduction in reproductive potential with all the genes tested, suggesting the essential function of chromatin remodeling ATPases in many cellular processes including egg-laying and egg-hatching. Knockdown of mi-2 and iswi completely inhibited oviposition over time. Real-time quantitative polymerase chain reaction confirmed significant knockdown of targeted mRNAs for at least 30 d, which supports persistence of RNAi in bed bugs. In addition, we observed a significant depletion of targeted transcripts in eggs laid by bed bugs injected with dsRNAs specific to chromatin remodeling ATPases. This study demonstrates the importance of chromatin remodeling ATPase in bed bug reproduction.

RNA Interference of the Muscle Actin Gene in Bed Bugs: Exploring Injection Versus Topical Application for dsRNA Delivery.

Bed bugs are one the most troublesome household pests that feed primarily on human blood. RNA interference (RNAi) is currently being pursued as a potential tool for insect population management and has shown efficacy against some phytophagous insects. We evaluated the different techniques to deliver dsRNA specific to bed bug muscle actin (dsactin) into bed bugs. Initially, stability of dsRNA in human blood was studied to evaluate the feasibility of feeding method. Adult bed bugs were injected with dsRNA between last thoracic segment and first abdominal segment on the ventral side, with a dose of 0.2 µg dsactin per insect. In addition to injection, dsactin was mixed in acetone and treated topically in the abdomens of fifth stage nymphs. We found the quick degradation of dsRNA in blood. Injection of dsactin caused significant depletion of actin transcripts and substantial reduction in oviposition and lethality in female adults. Topically treated dsRNA in fifth stage nymphs had no effect on actin mRNA expression and survival. Our results demonstrated that injection is a reliable method of dsRNA delivery into bed bugs while topical treatment was not successful. This research provides an understanding on effective delivery methods of dsRNA into bed bugs for functional genomics research and feasibility of the RNAi based molecules for pest management purposes.

RNAi-Mediated Knockdown of vATPase Subunits Affects Survival and Reproduction of Bed Bugs (Hemiptera: Cimicidae).

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has resurged as one of the most troublesome household pests affecting people across the globe. Bed bug infestations have increased in recent years primarily due to the evolution of insecticide resistance and the insect's ability to hitchhike with travelers. vATPases are one of the most evolutionarily conserved holoenzymes in eukaryotes, which are mainly involved in proton transport across the plasma membranes and intracellular organelles. RNA interference (RNAi) has been developed as a promising tool for insect control. In this study, we used RNAi as an approach to knock down subunits A and E of the vATPase gene of bed bugs. Delivery of 0.2 µg/insect of dsRNA specific to vATPase-A and vATPase-E into female bed bugs dramatically impaired the laying and viability of eggs over time. Injection of the vATPase-E dsRNA decreased survival of the bed bugs over 30 d. Our results also showed that the knockdown of mRNA is highly effective and persistent up to 30 d post injection. This research demonstrated that silencing of the two vATPase subunits A and E offers a potential strategy to suppress bed bug populations.

Knockdown of the Chromatin Remodeling Gene Brahma by RNA Interference Reduces Reproductive Fitness and Lifespan in Common Bed Bug (Hemiptera: Cimicidae).

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) is a nuisance household pest causing significant medical and economic impacts. RNA interference (RNAi) of genes that are involved in vital physiological processes can serve as potential RNAi targets for insect control. Brahma is an ATPase subunit of a chromatin-remodeling complex involved in transcription of several genes for cellular processes, most importantly the homeotic genes. In this study, we used a microinjection technique to deliver double stranded RNA into female bed bugs. Delivery of 0.05 and 0.5 µg/insect of brahma dsRNA directly into hemocele resulted substantial reduction in oviposition. Eggs laid by bed bugs receiving both doses of brahma dsRNA exhibited significantly lower hatching percentage as compared to controls. In addition, brahma RNAi in female bed bugs caused significant mortality. Our results disclosed the potential of brahma RNAi to suppress bed bug population through injection of specific dsRNA, suggesting a critical function of this gene in bed bugs' reproduction and survival. Based on our data, brahma can be a promising RNAi target for suppression of bed bug population.

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

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.

Forms of Melanoplus bowditchi (Orthoptera: Acrididae) collected from different host plants are indistinguishable genetically and in aedeagal morphology.

The sagebrush grasshopper, Melanoplus bowditchi Scudder (Orthoptera: Acrididae), is a phytophilous species that is widely distributed in the western United States on sagebrush species. The geographical distribution of M. bowditchi is very similar to the range of its host plants and its feeding association varies in relation to sagebrush distribution. Melanoplus bowditchi bowditchi Scudder and M. bowditchi canus Hebard were described based on their feeding association with different sagebrush species, sand sagebrush and silver sagebrush, respectively. Recently, M. bowditchi have been observed feeding on other plant species in western Nebraska. We collected adult M. bowditchi feeding on four plant species, sand sagebrush, Artemisia filifolia, big sagebrush, A. tridentata, fringed sagebrush, A. frigidus, and winterfat, Krascheninnikovia lanata. We compared the specimens collected from the four plant species for their morphological and genetic differences. We observed no consistent differences among the aedeagal parameres or basal rings among the grasshoppers collected from different host plants. Amplified Fragment Length Polymorphism markers were used to test the genetic relationships among the grasshoppers. Analysis of Molecular Variance and distance-based Unweighted Pair Group Method with Arithmetic mean dendrogram failed to reveal significant differences. Although the forms showed behavioral and minor color and size differences, the genetic data suggest all forms under study likely interbreed, which indicates they are a single species instead of four species or subspecies. These results indicate that host plant use may influence melanopline phenotype and suggest the need of further genetic analysis of subspecies recognized based on morphology, distribution, and ecology.

Delayed toxicity of two chitinolytic enzyme inhibitors (psammaplin a and pentoxifylline) against eastern subterranean termites (Isoptera: Rhinotermitidae).

By using a no-choice feeding bioassay, delayed toxicity and concentration-dependent mortality of two chitinolytic enzyme inhibitors, pentoxifylline and psammaplin A, were evaluated by determining LT50, LT90, and LT99 (lethal time) against the economically important eastern subterranean termite, Reticulitermes flavipes (Kollar). Pentoxifylline- and psammaplin A-incorporated diets (filter paper) were assayed at 0.01, 0.02, 0.04, 0.08, and 0.21% and 0.0375, 0.075, 0.15, and 0.3% active ingredient (wt:wt), respectively. Acetone-only treated filter paper served as diet for the control treatments. Termite workers were allowed to feed on diet until 100% test population mortality occurred (80-95 d). Both chitinase inhibitors were shown to be toxic to R. flavipes. Concentration-dependent toxicity occurred within the pentoxifylline treatments over the range of 0.01-0.08%, with 0.08% treatments producing an LT50 of 32.2 d. However, mortality in response to psammaplin A treatments lacked concentration-dependent toxicity. Treatment with 0.3% psammaplin A produced an LT50 of 21.3 d. Mortality in response to lower psammaplin A treatments displayed no concentration-dependent trends. This study provides the first report on delayed toxicity of chitinolytic enzyme inhibitors against eastern subterranean termites (order Isoptera) and toxicological data on pentoxifylline and psammaplin A over a range of concentrations.

Temporal changes in chlorantraniliprole and indoxacarb in four midwestern soils and bioefficacy against the eastern subterranean termite (Isoptera: Rhinotermitidae).

Temporal changes in indoxacarb and chlorantraniliprole were determined in four midwestern soils by simulating commercial field applications of termiticides. Indoxacarb (0.0625 and 0.125%) and chlorantraniliprole (0.05 and 0.10%) were applied to each soil type in a rotating cement mixer to ensure uniform distribution of active ingredient (AI). Temporal and spatial changes in termiticide concentrations were determined by sampling soil cores subdivided at different depths (0-20, 20-40, and 40-61 cm) at various intervals up to 705 d after application. Percentage loss of indoxacarb was initially greater (0-180 d) than losses after 180 d. The lowest indoxacarb extractable concentrations were detected in soils closest to the surface. Chlorantraniliprole losses with time from all soils were slower than indoxacarb, with no differences observed with soil type or depth. Bioefficacy was evaluated in laboratory glass tube bioassays that monitored the distance of termite penetration into treated soils and resulting eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), worker mortality. Bioassay data revealed that R. flavipes termites were unable to completely penetrate 50 mm of indoxacarb- and chlorantraniliprole treated soils at 0 d after treatment; however, termites were not deterred from foraging in these soils indicating no repellency to these termiticides. Termites completely penetrated (50 mm) soils treated with indoxacarb (0.0625%) by 360 d and complete penetration occurred in all soils treated with indoxacarb (0.0625 and 0.125%) by 705 d. Termites were unable to completely penetrate chlorantraniliprole-treated soils at 705 d. Mortality of termite workers was high in all chlorantraniliprole-treated soils at all sampling intervals. These data confirm that vertical differences in termiticide persistence occur in various soils.

Sorption and desorption of fipronil in midwestern soils.

Fipronil, {5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile} is commonly applied to soil to protect structures against termite infestations. The fate and bioavailability of fipronil in soil is dependent upon the variability of sorption processes and will differ from soil to soil. Adsorption of fipronil to three Nebraska soils with varying organic matter (OM) content was determined. At the concentrations tested (0.025, 0.1, 0.25, 0.5, 1.0, 1.5, and 2.0 mg L(-1)), adsorption curves showed constant partitioning of fipronil to the soil matrices (r (2) = 0.998 - 0.999). Calculated organic carbon partitioning coefficients (K (oc)) ranged from 244 to 628 with an average K(oc) of 396. Reported K(d) and K(f) values increased with increasing organic matter content. Desorption hysteresis was observed as fipronil has a propensity to stay in the adsorbed state. After five soil washes with 0.003 M CaCl2, approximately 30% of adsorbed fipronil residues were desorbed. Reported K (oc) values for fipronil suggests that it has intermediate mobility in the field collected soils utilized in this study.

Bioavailability of chlorantraniliprole and indoxacarb to eastern subterranean termites (Isoptera: Rhinotermitidae) in various soils.

A laboratory study was conducted to determine the toxicity of indoxacarb and chlorantraniliprole to Eastern subterranean termites, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) resulting from topical applications and exposure to treated soil. Soils with varying organic matter (0.57-3.64%) and chemical characteristics were used in termiticide bioassays. Lethal dose resulting from topical application indicated that chlorantraniliprole was two- to 11-fold more toxic than indoxacarb. Lethal concentration assays yielded opposite results where concentrations of indoxacarb in soil that caused either 50 or 90% mortality of R. flavipes workers at 48 and 144 h were two- to six-fold lower than chlorantraniliprole. The bioavailability of indoxacarb and chlorantraniliprole was negatively correlated with soil organic matter. Our results suggest that indoxacarb is more bioavailable to termites in soil than chlorantraniliprole based on calculated bioavailability ratios. However, how these laboratory results correlate to actual field application data and termite efficacy is unknown, and more research is needed. These compounds seem to have excellent activity on termites and have potential to provide new modes of action and new chemistry as liquid termiticides.

Concentration-dependent degradation of three termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (Isoptera: Rhinotermitidae).

Degradation and bioavailability of imidacloprid, fipronil, and bifenthrin applied at label rates ([AI], wt:wt in soil) in the loamy soil of Nebraska were determined over a 6-mo duration. Based on the calculated half-lives of the three termiticides, it was concluded that the degradation rate was lowest when a termiticide was applied at the highest label rate. Bioassays of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) conducted at 8, 31, 65, 90, 135, 160, and 180 d posttreatment showed an inverse relationship between the LT90 values and the variable concentrations. At day 180, exposures to all the termiticide-treated soil samples (concentration x termiticide) resulted in 100% mortality of R. flavipes workers. However, lower LT90 values were observed for termites exposed to soils treated with highest label rates even when the treated soils were aged in the lab for 6 mo. This suggested a higher bioavailability of these three termiticides when applied at higher application rates. Termite mortality was fastest for bifenthrin followed by fipronil and imidacloprid.

Influence of temperature on rate of uptake and subsequent horizontal transfer of [14C]fipronil by eastern subterranean termites (Isoptera: Rhinotermitidae).

The effect of temperature on [14C]fipronil uptake and transfer from donor (D) to recipient (R) Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) workers was evaluated. Test chambers used in the fipronil uptake study were constructed from petri dishes containing autoclaved soil treated with 1 ppm [14C]fipronil (1.14 microCi of total radioactivity per petri dish), distilled water, and R. flavipes workers. Test chambers were held in environmental growth chambers preset at 12, 17, 22, 27, and 32 degrees C. For the fipronil transfer study, donor termites stained with Nile blue-A were exposed to soil treated with 1 ppm [14C]fipronil for 2 h. Donors were then combined with unexposed recipient termite workers at either 1D:5R, 1D:10R, or 1D:20R ratios. Test chambers consisted of a nest and feeding chamber connected by a piece of polyethylene tube and held in growth chambers at 12, 17, 22, 27, and 32 degrees C. Worker termites were sampled over time and the amount of [14C]fipronil present was measured by scintillation counting. Some degree of uptake and transfer occurred at all temperatures and ratios in this study. The highest level of uptake occurred by termites held at 22-32 degrees C, followed decreasingly by 17 and 12 degrees C. Maximum transfer of [14C]fipronil occurred at the higher ratios (1:5 > 1:10 > 1:20) of donors to recipients. Data presented in this study suggest that temperature is one of the key factors affecting the rate of uptake and subsequent horizontal transfer of [14C]fipronil in subterranean termites.

Temperature effect on kinetics of uptake, transfer, and clearance of [14C]noviflumuron in eastern subterranean termites (Isoptera: Rhinotermitidae).

[14C]Noviflumuron uptake, clearance, rate of excretion, and transfer from treated to untreated termite workers were evaluated at 15,19, 23, and 27 degrees C. Feeding units were constructed from plastic containers provisioned with washed sand, distilled water, [14C]noviflumuron-treated feeding discs (0.05 or 0.5% [AI]), and Reticulitermes flavipes (Kollar) workers. Feeding units were held in environmental growth chambers preset at 15, 19, 23, and 27 degrees C. The amount of [14C]noviflumuron present within R. flavipes was measured by scintillation counting and subsequently quantified. Uptake of noviflumuron by R. flavipes workers at 15 degrees C was approximately 2.8 times less than at 19 or 23 degrees C and approximately 4.4 times less than at 27 degrees ighest uptake of [14C]noviflumuron occurred at 27 degrees C and 144 h. Most transfer of [14C]noviflumuron from treated to untreated termite workers occurred between 19 and 27 degrees C. [14C]Noviflumuron had a half-life in R. flavipes workers of approximately 31-45 d, dependent on temperature. A higher amount of [14C]noviflumuron was lost through excretion at > or = 19 degrees C (approximately 15-22%) compared with 15 degrees C (0.27%). Results indicated that increased uptake, transfer, and clearance of noviflumuron by R. flavipes occurred at warmer temperatures (19-27 degrees C), and all of these processes were significantly lower at 15 degrees C.