Dini M. Miller

Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

Diversity of gut bacteria of Reticulitermes flavipes as examined by 16S rRNA gene sequencing and amplified rDNA restriction analysis.

The phylogenetic species richness of the bacteria in the gut of the termite Reticulitermes flavipes was examined using near full-length 16S rRNA gene sequencing and amplified rDNA restriction analysis (ARDRA). We amplified the genes by polymerase chain reaction (PCR) directly from a mixed population of termite gut bacteria and isolated them using cloning techniques. Sequence analysis of 42 clones identified a broad taxonomic range of ribotypes from six phyla within the domain Bacteria: Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the recently proposed “Endomicrobia.” Analysis of the sequence data suggested the presence of a termite specific bacterial lineage within Bacteroidetes. The ARDRA data included 261 different ARDRA profiles of 512 clones analyzed. These data suggest the gut flora in R. flavipes is extremely diverse.

Population Growth Potential of the Bed Bug, Cimex lectularius L.: A Life Table Analysis

Experimental life tables were constructed and analyzed for three strains of the common bed bug: a pyrethroid-susceptible laboratory strain (HS), a highly resistant field strain (RR), and a field strain with a declining level of resistance (KR). Egg to adult survival in the RR strain was 94% compared with 79% and 69% in the HS and KR strains, respectively. The RR strain also developed significantly faster from egg to adult (∼35 days) than the other two strains (∼40 days). Analysis of a survivorship and fecundity life table for the RR strain produced the following results. The average life expectancy for a newly laid egg was ∼143 days, and that of a newly molted adult was ∼127 days. Females produced an average of 0.64 daughter eggs/day with the highest weekly production during the fifth week of adult life. Analysis of daily reproductive parity showed that females produced 1–3 and 4–6 eggs on 79 and 21% of the days, respectively, when egg laying occurred. The net reproductive rate (R0) of the RR strain was ∼35, which represents a 35-fold increase in the population per generation (∼92 days). The intrinsic rate of increase, r, was 0.054 indicating that the population multiplies 1.1 times/female/day (λ) and doubles in size every 13 days. The stable age distribution (cx) was dominated by nymphs (54%), followed by eggs (34%) and adults (12%). Reproductive values (vx) for the strain increased from egg to the adult stage.

Host Searching and Aggregation Activity of Recently Fed and Unfed Bed Bugs (Cimex lectularius L.)

Groups of starved, virgin adult male or female bed bugs were stimulated to search for a host by the presence of a heated artificial feeder. Some of the bed bug groups were allowed to obtain a blood meal and some were not. After the removal of the feeder, bed bugs were observed throughout the scotophase to record their searching and aggregation behavior. Groups of male and female bed bugs that were unable to obtain a blood meal continued to search in the arena for the majority of the scotophase. Bed bugs that were able to obtain a blood meal returned to their shelter to aggregate 30 min after feeding. Overall, the proportion of bed bugs aggregating in shelters during the scotophase was significantly greater for those that had fed successfully than those that had not. However, all bed bugs, regardless of feeding status, began to return to shelters to aggregate 2 h prior to the photophase.

Survivorship During Starvation for Cimex lectularius L.

Four bed bug strains (Cimex lectularius) with different levels of pyrethroid resistance were evaluated to determine their ability to survive extended periods of starvation. First instar bed bugs of all strains were the most vulnerable to starvation (13.8–36.3 days mean survival time). Fifth instars and adults survived the longest during starvation (41.5–142.6 days). Significant differences in survivorship during starvation were observed between resistant and susceptible strains of bed bugs. Overall, all immature and adult stages of the resistant bed bug strains had significantly shorter survival times than those of the susceptible strains (P < 0.05).

Trail-Following Behavior in the German Cockroach (Dictyoptera: Blattellidae)

Abstract The trail-following behavior of the German cockroach, Blattella germanica (L.), was evaluated by comparing the distance between cockroach movement paths and preapplied “trails” of fecal extract. For each cockroach group tested (adult males, females, gravid females, and late instars), the mean perpendicular distance of the cockroach from the trail was significantly less than the distance from a control trail. The results indicated that the German cockroaches did exhibit trail-following behavior. Trail- following accuracy varied among the cockroach groups. The mean distance from the fecal trail ranged from 18.45 to 110.05 cm with adult males ≤ adult females ≤ late instars < gravid females. Very dilute fecal extract could still induce trail-following behavior in adult male cockroaches. A 5.6% concentration of fecal extract in methanol was able to induce trail-following behavior in 50% of the cockroaches. Although German cockroaches have demonstrated trail-following behavior, fecal trails are still not thought to be actively deposited. Rather, the passive distribution of fecal material within the home range results in the accumulation of trails along frequently traveled routes (i.e., between resources and the cockroach harborage).

Use of fecal extract trails to enhance trap catch in German cockroach (Dictyoptera: Blattellidae) monitoring stations.

Abstract An aqueous extract of German cockroach, Blattella germanica (L.), fecal material was evaluated for inducing trail-following behavior in German cockroaches. In arena tests the fecal extract was found to stimulate trail following in 74% of adult male cockroaches. Significantly fewer cockroaches (22%) followed water-treated (control) trails. Residual activity of the fecal extract trails was evaluated by bioassay after the trails had been stored in the refrigerator or in the open air. Although trails stored in the refrigerator showed no decline in activity after 14 d, those stored in the open air declined significantly after 3 d, inducing only 40% of adult male cockroaches to follow the trail. After 7 d the activity of trails stored in the open air was further reduced to 23%. The ability of fecal-extract trails to influence trap catch in monitoring stations was determined by bioassay. Paper trails treated with fecal extract or water were positioned between cockroach harborages and monitoring stations inside 122-cm2 arenas. The presence of the fecal extract-treated trails significantly enhanced trap catch. Mean catch in the traps with fecal extract trails was 28 cockroaches compared with a mean of 11 cockroaches in the control traps. The trap catch ratios of adults to nymphs in the treated and control treatments were not significantly different.

Insecticide Resistance in Eggs and First Instars of the Bed Bug, Cimex lectularius (Hemiptera: Cimicidae)

Two strains of the common bed bug, Cimex lectularius L., eggs and first instars collected from pyrethroid-resistant adults were evaluated for insecticide resistance and compared to a susceptible strain. Dose-response bioassays were conducted using two insecticide formulations (Temprid: imidacloprid/β-cyfluthrin, and Transport: acetamiprid/bifenthrin). The lethal concentration (LC50) for the two resistant egg strains exposed to imidacloprid/β-cyfluthrin ranged from 3 to 5-fold higher than susceptible strain eggs. Resistant strain eggs dipped into formulations of acetamiprid/bifenthrin had LC50 values which were significantly greater (39 to 1,080-fold) than susceptible strain eggs. Similar to eggs, resistant strain first instars exposed to residual applications of imidacloprid/β-cyfluthrin had LC50 values ranging from 121 to 493-fold greater than susceptible strain first instars. When resistant strain first instars were treated with acetamiprid/bifenthrin, they had LC50 values that were 99 to >1,900-fold greater than susceptible strain first instars. To determine differences between egg and first instar resistance, stage resistance ratios (SRR) were compared between the two stages. There was little difference between the egg and first instar stages, indicated by small SRR values ranging from 1.1 to 10.0. This study suggests that insecticide resistance is expressed early during bed bug development.

Reproductive Potential of Field-collected Populations of Cimex lectularius L. and the Cost of Traumatic Insemination

Egg production was compared among three field-collected bed bug strains over the course of 13 feeding/oviposition cycles, each of which lasted ∼10 days. No significant differences were found among bed bug strains in the mean number of eggs/female/day (∼1.0 egg). However, significant differences were found among strains in their patterns of egg production throughout the study period. Specifically, differences were observed in the timing of peak egg production and the rapidity of egg production decline among the three strains. Egg production was also quantified for female bed bugs that were subjected to single or multiple traumatic insemination events over a period of six feeding/oviposition cycles. Significant differences were found in egg production between females exposed to single and multiple inseminations. Females mated only once produced 83.8 ± 4.5 (mean ± SE) eggs over six feeding cycles. Females exposed to multiple inseminations produced 61.0 ± 3.1 eggs, indicating that multiple traumatic inseminations may reduce female fecundity by as much as 27%. This study is the first to suggest that, in a new infestation (first ∼6 weeks), a solitary, singly-mated female with access to regular blood meals is capable of producing greater numbers of offspring than the same female in the presence of a male.

Novel extraction of German cockroach (Dictyoptera: Blattellidae) fecal pellets enhances efficacy of spray formulation insecticides.

Abstract Methanol extracts of German cockroach, Blattella germanica (L.), fecal pellets have limited use as pheromone attractants in the urban environment because of their unpleasant color and odor. To eliminate these characteristics, a novel aqueous extract of German cockroach feces was formulated. Fecal material was extracted with methylene chloride and mixed with water. The aqueous phase of the extract was colorless and relatively odorless compared with methanol fecal extracts. Aqueous extract was bioassayed and compared with methanol extracts for aggregation activity. The efficacy of chlorpyrifos and boric acid formulations was tested with and without the addition of fecal extracts. In tests with chlorpyrifos, the addition of the aqueous extract produced significantly greater mortality in young nymphs than either chlorpyrifos alone or chlorpyrifos + methanol extract. There were no differences in mortality between the 2 extract + boric acid treatments during the test period, and both enhanced mortality.