Clinal variation in colony breeding structure and level of inbreeding in the subterranean termites Reticulitermes flavipes and R. grassei.

Social insects exhibit remarkable variation in their colony breeding structures, both within and among species. Ecological factors are believed to be important in shaping reproductive traits of social insect colonies, yet there is little information linking specific environmental variables with differences in breeding structure. Subterranean termites (Rhinotermitidae) show exceptional variation in colony breeding structure, differing in the number of reproductives and degree of inbreeding; colonies can be simple families headed by a single pair of monogamous reproductives (king and queen) or they can be extended families headed by multiple inbreeding neotenic reproductives (wingless individuals). Using microsatellite markers, we characterized colony breeding structure and levels of inbreeding in populations over large parts of the range of the subterranean termites Reticulitermes flavipes in the USA and R. grassei in Europe. Combining these new data with previous results on populations of both species, we found that latitude had a strong effect on the proportion of extended-family colonies in R. flavipes and on levels of inbreeding in both species. We examined the effect of several environmental variables that vary latitudinally; while the degree of inbreeding was greatest in cool, moist habitats in both species, seasonality affected the species differently. Inbreeding in R. flavipes was most strongly associated with climatic variables (mean annual temperature and seasonality), whereas nonclimatic variables, including the availability of wood substrate and soil composition, were important predictors of inbreeding in R. grassei. These results are the first showing that termite breeding structure is shaped by local environmental factors and that species can vary in their responses to these factors.

The Efficacy of a Pyrethroid-impregnated Mattress Liner on Multiple International Strains of Cimex lectularius (Hemiptera: Cimicidae) and Cimex hemipterus (Hemiptera: Cimicidae).

Modern bed bugs are resistant to multiple insecticide classes, particularly the pyrethroids. The efficacy of pyrethroid-impregnated mattress liners marketed for bed bug management has been variable. This study evaluated the efficacy of a permethrin-impregnated mattress liner, ActiveGuard, against 24 bed bug strains, consisting of both Cimex hemipterus (F.) and Cimex lectularius L. A 'mat assay', employing an allethrin-impregnated mat, was used to establish the pyrethroid resistance profile of all strains. Three experiments were conducted to evaluate the effect of ActiveGuard exposure on bed bug knockdown: 1) exposing the bed bugs continuously on the liner for up to 24 d, 2) holding the bed bugs on the liner for either 4 or 6 h, and 3) placing a noninsecticide treated fabric above the liner with the bed bugs held continuously on top. Our results indicated that all modern strains (collected within the last 15 years during the current resurgence) were pyrethroid-resistant, although the magnitude of resistance was highly variable between strains. In the continuous exposure study, an incomplete knockdown was recorded for most modern bed bug strains, with some having no knockdown even up to 7 d of constant exposure. In the 4 or 6 h exposure study, the level of knockdown was reduced even further, and very few bed bugs were knocked down in the double fabric study. The results of this study indicate that pyrethroid-impregnated mattress liners are not likely to be effective in the management of most modern bed bug infestations involving either C. hemipterus or C. lectularius.

Quantification of Fecal Spot Production as a Measure of Environmental Contamination Based on Common Bed Bug (Hemiptera: Cimicidae: Cimex lectularius L.) Population Size.

The presence of fecal spots has often been used to verify the existence of a bed bug (Cimex lectularius) infestation. However, no research has been conducted to determine how much fecal material that a bed bug population produces over time. In this study, the number of fecal spots that each nymphal life stage was capable of producing after a bloodmeal was quantified. Adult fecal spots were also quantified to determine if there was consistent production between feedings. During this study, it was discovered that bed bugs produced visible fecal spots and clear spots that were only visible under ultraviolet light. Therefore, three types of fecal spots were quantified: dark feces, light feces, and clear spots. Clear spots were produced in greater amounts (38.5-55.5%) than either dark spots (27.3-40.7%) or light spots (17.3-21.9%). For example, 5th instar bed bugs were thought to produce an average of 21 spots (dark and light) after a single bloodmeal. However, using the ultraviolet light, it was found that the 5th instars actually produced an average of 44.7 spots. Using the total fecal spot data collected during this study, researchers could project contamination potential for an infestation starting with a single gravid female over 30, 60, and 90 d. In addition, the amount of area covered by these spots was projected to be over 12 m2 in just 3 mo, which could greatly reduce the environmental and aesthetic quality of a home.

Quantifying the Efficacy of an Assessment-Based Pest Management (APM) Program for German Cockroach (L.) (Blattodea: Blattellidae) Control in Low-Income Public Housing Units.

Over the last several decades, low-income public housing facilities have been known to be infested with particularly large German cockroach populations. These populations persist even though the housing pest control contracts often require, and pay for, IPM practices to be used in their facilities. When Virginia Tech researchers began reviewing public housing contracts in Virginia and North Carolina, it was easy to see why these 'IPM programs' were not successful. Many of these 'low-bidder' contracts do not allow the technician enough time in each apartment to assess the size of the pest population. In addition, these pest management contracts did not require German cockroach population monitoring, even though all IPM programs are based on assessments of the pest population. There was a clear need for an effective, easy to apply cockroach management program in U.S. public housing authorities. This study determined the long-term efficacy of an Assessment-based Pest Management (APM) program for German cockroach control in U.S. public housing facilities. Specifically, we evaluated an APM program where the residents were not asked to clean or prepare for treatment, and where overnight cockroach trap counts were used to determine the volume of gel bait that would be applied. The APM baiting program was conducted for 15 mo in three housing authorities. In all three housing authorities, cockroach populations in test units were typically reduced by >90%. German cockroach infestations were even eliminated from 49 of the 65 (75%) test units during this study.

Field evaluations of insecticide treatment regimens for control of the common bed bug, Cimex lectularius (L.).

BACKGROUND: Because bedbugs, Cimex lectularius L., have not been a problem in the USA for over 40 years, few insecticide products are labeled for their control. Most products that are labeled for bedbugs are pyrethroids. However, recent studies indicate that field-collected bedbugs may be resistant to pyrethroids. There are also non-pyrethroid products labeled for bedbugs, but, like the pyrethroids, none of these products has been evaluated for field efficacy. This study evaluated the efficacy of two insecticide treatment regimens for bedbugs in multi-unit housing. Both of the treatments included multiple products currently being used by the pest management industry. RESULTS: The 'traditional' treatment consisted of applications of pyrethroid products and an insect growth regulator. The 'novel' treatment consisted of applications of non-pyrethroid products. The traditional treatment significantly reduced the number of bedbugs from 39.8 +/- 10.1 per unit prior to treatment to 2.2 +/- 1.0 at the end of the test period (P = 0.02; 95% reduction). The number of live bedbugs in the 'novel' treatment was also significantly reduced from 71.4 +/- 25.3 bedbugs per unit to 10.2 +/- 4.4 after 8 weeks (86% population reduction). CONCLUSIONS: Although both treatment regimens reduced bedbug numbers, the fact that bedbugs were not eliminated after multiple applications suggests that the insecticides, applied at the current label rates, were inadequate. These results suggest that a more integrated approach to bedbug control is necessary in multi-unit housing situations.

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

Laboratory evaluations of insecticide product efficacy for control of Cimex lectularius.

A susceptible, laboratory strain of bed bug, Cimex lectularius L., was used to determine the efficacy of insecticide products labeled or possessing a site label for bed bug control. Field strain bed bugs also were used to evaluate one insecticide product. The lethal time (LT),, values calculated for the laboratory strain bed bugs indicated that all of the pyrethroid products killed significantly faster than chlorfenapyr (0.5% [AIl; Phantom: BASF; LT50 = 10 d and 9 h). lamda-Cyhalothrin (0.03%; Demand CS; Syngenta) was the fastest acting insecticide (LT50 = 20 min), followed by bifenthrin (0.02% [AI]; Talstar One, FMC; LT50 = 53 min), deltamethrin (0.06% [AI]; Suspend SC; Bayer; LT50 = 61 min), and permethrin (0.05%; Dragnet SFR; FMC; LT50 = 88 min). The field strain bed bugs exposed to deltamethrin had an LT50 value of 14 day 8 h, indicating that the field strain was significantly less susceptible to deltamethrin than the laboratory strain. Chlorfenapyr exposure did not prevent the laboratory strain bed bugs from mating and laying eggs, nor did it prevent the eggs from hatching during the 2-wk exposure period. Surprisingly, none of the insecticides tested, including the pyrethroids, were repellent to laboratory strain bed bugs. Bed bugs rested on pyrethroid-treated panels and remained in contact with the panels until they died (2 h). Chlorfenapyr was also not repellent to bed bugs, but it caused no mortality during the 2-h test period. This study suggests that although pyrethroids were effective for controlling laboratory strain bed bugs, there is the potential for significant resistance in field strains. This study also determined that pyrethroid products were not repellent to bed bugs and would not cause bed bug aggregations to scatter or avoid treated surfaces.

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/ß-cyfluthrin, and Transport: acetamiprid/ bifenthrin). The lethal concentration (LC50) for the two resistant egg strains exposed to imidacloprid/ß-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/ß-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.

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.

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.

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).

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.

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 LD(50) 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.