HOUSEHOLD AND STRUCTURAL INSECTS Effect of Feeding Status on Mortality Response of Adult Bed Bugs (Hemiptera: Cimicidae) to Some Insecticide Products

1 DONG-HWAN CHOE AND KATHLEEN CAMPBELL

Department of Entomology, University of California, Riverside, CA 92521

J. Econ. Entomol. 107(3): 000Ð000 (2014); DOI: http://dx.doi.org/10.1603/EC13478 ABSTRACT Fresh and aged residual deposits of several insecticide products were tested against bed bug adults to determine if a recent bloodmeal affected their mortality response to the residues. The bed bugs with a recent bloodmeal survived signiÞcantly longer compared with the unfed ones on their exposure to fresh or aged residual deposits of chlorfenapyr and aged residual deposits of deltamethrin on a wooden substrate. Even though the survival time of fed bed bugs was signiÞcantly longer than that of unfed ones on their exposure to fresh residue of deltamethrin and aged residue of desiccant pyrethrin dust, these treatments resulted in similarly high Þnal mortalities regardless of feeding status of the insects. Mortality responses of fed and unfed bed bugs were similar to fresh or aged residual deposits of imidacloprid ϩ cyßuthrin combination and fresh residual deposits of desiccant pyrethrin dust. Topical application assays indicated that a recent bloodmeal signiÞcantly increased the bed bugÕs survival time for chlorfenapyr, but not for deltamethrin. Pyrethroid-resistant bed bugs also showed a similar increase in their survival time for chlorfenapyr after a bloodmeal. The comparison of mortality responses between fed and unfed bed bugs treated with similar amount of chlorfenapyr per fresh body weight indicated that increased body mass was not the primary cause for this bloodmeal-induced tolerance increase for chlorfenapyr. Because the surviving bed bugs can continue ovipositing, the effectiveness of chlorfenapyr residual deposits in bed bug harborages could be signiÞcantly affected by the feeding status of the adult bed bug populations.

KEY WORDS deltamethrin, chlorfenapyr, imidacloprid, ␤-cyßuthrin, Cimex lectularius

Several insecticides currently available for bed bug efÞcacy to treat bed bug harborages. Romero et al. control include pyrethroids, a pyrrole, neonicotinoids, (2009a) reported that bed bugs avoided deltamethrin and natural oils that are derived from various botanical residues on clean Þlter paper substrate, but natural sources. Even though pyrethroids typically provide harborages, containing fecal materials and eggs, re- fast knockdown and kill of bed bugs, the use of alter- mained attractive to bed bugs even after being treated native insecticides with different modes of action is with a deltamethrin-based product. While being rel- also important because of the widespread pyrethroid atively slow-acting, residual deposits of chlorfenapyr resistance in bed bug populations (Romero et al. were also found to be effective against four different 2007a,b; Yoon et al. 2008). Mixtures of pyrethroids and bed bug strains, including two strains that were highly neonicotinoids have been recently introduced to com- resistant to pyrethroids without apparent avoidance bat resistance. Several different types of desiccant behavior (Romero et al. 2009a, 2010). agents such as silica gel dust and diatomaceous earth Previous evaluations of insecticides applied to har- are also available for bed bug management as a resid- borage sites have been conducted with unfed adult ual treatment option. bed bugs (i.e., no bloodmeal for 7Ð12 d after adult Because of the practical difÞculty of Þnding every emergence) because their responses to insecticides bed bugÕs location in infested items or structures, the were more consistent than those that had recently use of effective residual insecticides for potential bed molted or fed (Romero et al. 2009a, 2010). However, bug harborages (i.e., cracks and crevices in the fur- both adult and immature stages of bed bugs typically niture or infested room) has been suggested (Meyers take regular and frequent bloodmeals for their normal 2012). This type of treatment might potentially im- growth, longevity, and reproduction (Usinger 1966). prove bed bug control by targeting bed bugs moving For example, female bed bugs in a highly infested into the harborages after feeding (Romero et al. room kept at a constant temperature (26ЊC) were 2009a). In particular, deltamethrin and chlorfenapyr found to take a bloodmeal every 2.5 d (Reinhardt et al. have been recently evaluated for their repellency and 2010). Because bed bugs typically return to their har- borages immediately after obtaining a bloodmeal (i.e., 1 Corresponding author, e-mail: [email protected]. Ͻ30 min; Reis and Miller 2011), it is likely that at least

0022-0493/14/0000Ð0000$04.00/0 ᭧ 2014 Entomological Society of America 2JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3 some individuals that would contact the residual de- posits in the harborages would be the ones that have taken a recent bloodmeal. In several species of mosquitoes, a recent bloodmeal appears to be one of the factors inßuencing insecticide tolerance levels (Brown and Pal 1971). Hadaway and Barlow (1956) reported that tolerance of female Anopheles stephensi Liston and Aedes aegypti (L.) to dichlorodiphenyltrichloroethane and dieldrin in- creased about twofold at 24 h after a bloodmeal. Hal- liday and Feyereisen (1987) and Spillings et al. (2008) also reported similar phenomena in female Culex pipi- ens L. and a pyrethroid-resistant strain of Anopheles funestus Giles. Thus, it was of interest to determine if bed bugs that are recently fed would show different mortality response to insecticide residual deposits compared with starved ones. Fig. 1. Wooden shelter (75 by 40 by 6 mm) built with In this study, we examined the effect of a recent balsa wood panels and sticks for the residual activity assay. bloodmeal on the survival time of adult bed bugs on their exposure to fresh and aged residual deposits of of the feeding groups) were determined immediately several insecticide products that are currently regis- before insecticide treatment (see Topical application tered for bed bug control. Topical application studies assay) on an analytical scale (AE 240, Mettler-Toledo, were also conducted on the insecticide products to LLC, Columbus, OH) to the nearest 0.1 mg after which the fed and unfed bed bugs showed different anesthetization with carbon dioxide. mortality responses. The potential implications for Wooden Shelter. Small wooden shelters (75 by 40 bed bug management are discussed. by 6 mm) were built with balsa wood panels and sticks (Revell Inc., Elk Grove Village, IL) to simulate po- tential harborages in structures. A rectangular wooden Materials and Methods panel (75 by 40 by 3 mm) was attached with hot glue Insects. Two strains of bed bugs, “Earl” and “Jersey,” on the bottom center of a plastic petri dish (14 cm in were purchased from Sierra Research Laboratories diameter, 2.5 cm in height) with two ßat legs (40 by (Modesto, CA). The Earl strain was originally col- 7 by 3 mm) at the shorter sides. The narrow space (7.3 lected in Modesto, CA, in 2007. Bioassays with tech- cm3) formed between the petri dish bottom and nical-grade insecticide residues on a glass surface wooden panel served as a harborage for bed bugs, and indicated that this strain was susceptible to technical- bed bugs were able to climb in and out through the grade permethrin and deltamethrin (i.e., 90 and 100% openings along the longer sides (Fig. 1). Bed bugs knockdown in2honaglass surface treated with placed on the top of the wooden shelter typically technical-grade permethrin and deltamethrin, respec- moved to the underside of the wooden shelter to avoid tively; B. Donahue, unpublished data). Jersey strain is the bright light and open environment. The bed bugs a relatively pyrethroid-resistant strain collected in Jer- contacted the insecticide residues on the underside of sey City, NJ, in 2010 (i.e., 30 and 50% knockdown in the top panel as they moved under the shelter, but 2 h on a glass surface treated with technical-grade they also could avoid the treatment by remaining on permethrin and deltamethrin, respectively; B. Dona- the untreated surfaces of the shelter (e.g., top of the hue, unpublished data). The bed bugs were kept in shelter or outside of the legs). screened containers, and fed with a grafting tape Residual Activity Assay. The residual activity assay membrane (Aglis & Co., Ltd., Yame City, Fukuoka, was conducted with the Earl strain to determine if fed Japan) feeder containing rabbit blood plus sodium and unfed bed bugs differed in their survival time citrate (Hema Resource and Supply, Inc., Aurora, OR) when they contacted insecticides applied in their har- once a week. All colonies were kept at 26ЊC, 35Ð38% borage sites. The top inner surface of the wooden RH, and a photoperiod of 12:12 (L:D) h. shelter was treated with one of four different insec- To examine the effect of recent bloodmeals on the ticides: deltamethrin 0.06% (D-Force HPX pressur- survival time of adult bed bugs on their exposure to ized aerosol, Waterbury Companies, Inc., Waterbury, various insecticide residual deposits, we prepared two CT), chlorfenapyr 0.5% (Phantom pressurized aero- groups of bed bug that differed in the periods since sol, BASF Corporation, Research Triangle Park, NC), their most recent bloodmeal: 1 and 9 d postfeeding. imidacloprid 21% and ␤-cyßuthrin 10.5% combination Bed bugs fed the night before the experiment were (Temprid SC, Bayer Environmental Science, Re- considered as the 1 d postfeeding or “fed” bed bugs. search Triangle Park, NC), and silica gel dust with The “unfed” bed bugs were obtained by feeding a pyrethrins 1% and piperonyl butoxide (PBO) 10% group of bed bugs until they reached their engorge- (Drione, Bayer Environmental Science). For the pres- ment and keeping them in the laboratory for an ad- surized aerosol formulations (deltamethrin and chlo- ditional 9 d without bloodmeal. Weights of fed and rfenapyr), aliquots of the liquids were dispensed from unfed bed bugs (10 random samples selected for each pressurized containers into glass vials, and all the vol- June 2014 CHOE AND CAMPBELL:INSECTICIDE TOXICITY TESTS WITH BED BUGS 3 atile propellant gases were removed from the liquids Aliquots of deltamethrin and chlorfenapyr aerosols by shaking the vial rigorously and releasing the pres- were dispensed from pressurized containers into glass sure by opening the cap. For the suspension concen- vials, and subsequently dissolved in acetone so that 0.5 trate formulation (imidacloprid ϩ cyßuthrin combi- ␮l of the acetone preparation applied to the insect nation), a 0.075% (label rate for indoor bed bug provided the required quantity of AIs. The concen- infestation based on the combined active ingredient trations tested were 1, 2, and 5 ng (AI) per insect for [AI]) aqueous preparation was used. An aliquot of 700 deltamethrin, and 100, 200, and 500 ng (AI) per insect ␮l liquid was evenly applied on the top inner surface for chlorfenapyr. These concentrations were selected 2 of the wooden shelter (24.4 cm ), providing approx- based on LD50 values reported for a highly pyrethroid- imately 0.01, 0.6, 0.015, and 0.007 mg AI/cm2 treatment susceptible strain of bed bug (0.03 ng deltamethrin/ rates for deltamethrin, chlorfenapyr, imidacloprid, insect for the Harlan strain of bed bug; Adelman et al. and cyßuthrin, respectively. For the desiccant dust 2011) and another insect species of similar size (29.98 with pyrethrins and PBO (Drione), 8 mg of dust was ng chlorfenapyr/insect for subterranean termite; Rust evenly spread on the top inner surface of the wooden and Saran 2006). A microapplicator equipped with a shelter with a cotton-tip applicator, providing 0.003 glass syringe (Micro Metric Instrument Co., Cleve- and 0.03 mg/cm2 treatment rates for pyrethrins and land, OH) was used to apply the acetone preparation PBO, respectively. Control shelters were treated with containing insecticide to the dorsal abdominal surface 700 ␮l of water. of the insects. A control group was treated with an Treated wooden shelters were allowed to dry in identical amount of acetone only. Ten or twenty adult ambient conditions (26ЊC, 35Ð38% RH) for 6 h (re- bed bugs (1:1 sex ratio) were treated and placed in ferred to as fresh residual deposit hereafter), and at- individual cells (16 by 19 mm) of 24-well cell culture tached on the bottom of the petri dishes using hot glue plates (Corning Inc., Corning, NY) with the bottoms (thermoplastic adhesive). The insecticides were also lined with small disks of Þlter paper. The treated bed tested as a 34-d-old residue. For the aging process, the bugs were kept at 26ЊC. The number of dead or treated wooden shelters were kept in ambient condi- knocked down bed bugs was recorded every 24 h for tions (referred to as aged residual deposit hereafter). 10 d. The bed bugs were considered to be dead or Approximately 10 adult bed bugs (10Ð12 individuals, knocked down if the bed bugs could not cling onto the 1:1 sex ratio) were introduced onto the top of the Þlter paper lining and respond to the mechanical stim- wooden shelter, and the number of dead or knocked uli provided by gentle touch with a wooden probe. down bed bugs was recorded every 24 h for 10 d. The Statistical Analysis. The survival time data in the 10-d observation time was selected based on a study by residual and topical studies were analyzed with sur-

Romero et al. (2010), in which LT50 and LT90 values vival analysis. The distribution of survival times was of four different bed bug strains for dry residues of described using the survivorship function S (t), the chlorfenapyr (fresh and aged) were within the range probability that an individual survives longer than of 3.5 and 8.9 d. Other insecticides containing pyre- time t. PROC LIFETEST statement was used to com- throids and pyrethrins were considered to be quicker pute nonparametric estimates of the survivor func- in their action compared with the chlorfenapyr insec- tions, to compare survival curves, and to compute ticide. If live bed bugs were found at the bottom of the log-rank test for association of the failure time vari- petri dish away from the wooden shelter, the bed bugs able (i.e., time until kill) with covariates (i.e., insec- were moved to the top of the shelter. The bed bugs ticide treatment; SAS Institute 2009). Survival curves were considered to be dead or knocked down if were generated using the Statistix 9 program (Ana- they were not able to cling to the wooden shelter with lytical Software 2008). Day 10 cumulative mortalities their legs. Each trial was replicated Þve times, result- were compared between fed and unfed bed bugs using ing in Ϸ50 bed bugs being tested per feeding status per the one-tailed Fisher exact test to determine if the insecticide treatment. Control was replicated 10 times ability to tolerate the insecticide treatment is depen- per feeding status. dent on the feeding status of bed bugs (Analytical Topical Application Assay. The topical application Software 2008). assay was conducted to determine if fed and unfed bed bugs differed in their survival time when known amounts of AIs were topically applied to their cuticle. Results First the Earl strain was tested with a subset of insec- ticides that were selected based on the residual ac- Residual Activity Assay. Upon their release on the tivity assay results. Deltamethrin and chlorfenapyr top of the treated wooden shelter, most (Ͼ50%) of the were chosen because the residual activity assay result bed bugs (Earl strain) readily moved into the struc- indicated that the fed and unfed bed bugs differed in ture in Ͻ30 min, contacting the insecticide deposits on their survival time when they contacted the fresh its inner surface. Because overall control mortalities residues of the insecticides. The Jersey strain was were low and identical between fed and unfed bed subsequently tested with chlorfenapyr that was cho- bugs (e.g., 3.5% for both fed and unfed bed bugs at day sen based on the results of the Þrst topical application 10), all statistical comparisons were made between fed assay with the Earl strain. and unfed bed bugs from the insecticide treatments The topical application assay was conducted with without correction based on corresponding control the methods developed by Romero et al. (2009b). mortalities. 4JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3

Fig. 2. KaplanÐMeier survival analyses for Earl strain bed bugs (fed and unfed) when they were exposed to the fresh residual deposits of deltamethrin, chlorfenapyr, imidacloprid ϩ cyßuthrin, and pyrethrin dust with PBO on a wooden substrate. Survivorship function S (t) was deÞned as the probability that an individual survives longer than time “t.” (Online Þgure in color.)

When Earl strain bed bugs were tested on the fresh 0.0001 for deltamethrin; ␹2 ϭ 66.655, df ϭ 1, P Ͻ 0.0001 residual deposits, the survival times of the fed and for chlorfenapyr; ␹2 ϭ 19.757, df ϭ 1, P Ͻ 0.0001 for unfed bed bugs were signiÞcantly different for delta- desiccant pyrethrin dust; Fig. 3). By day 10, total methrin and chlorfenapyr (␹2 ϭ 5.784, df ϭ 1, P ϭ cumulative mortalities for fed and unfed bed bugs 0.016 for deltamethrin; ␹2 ϭ 10.94, df ϭ 1, P ϭ 0.001 for were 53% (26 of 49) and 98% (50 of 51) for deltame- chlorfenapyr; Fig. 2). For deltamethrin, 10-d cumula- thrin (Fisher exact test, P Ͻ 0.0001), 48% (24 of 50) tive mortalities for fed and unfed bed bugs were 92% and 98% (52 of 53) for chlorfenapyr (Fisher exact test, (46 of 50) and 100% (50 of 50), respectively (Fisher P Ͻ 0.0001), and 98% (49 of 50) and 100% (50 of 50) exact test, P ϭ 0.059). For chlorfenapyr, 10-d cumu- for desiccant pyrethrin dust with PBO (Fisher exact lative mortalities for fed and unfed bed bugs were 64% test, P ϭ 0.5), respectively. For aged residual deposits (32 of 50) and 88% (43 of 49), respectively (Fisher of imidacloprid ϩ cyßuthrin combination, the survival exact test, P ϭ 0.005). The survival times of the fed and times and 10-d cumulative mortalities were not sig- unfed bed bugs were not signiÞcantly different for the niÞcantly different between fed and unfed bed bugs fresh residual deposits of imidacloprid ϩ cyßuthrin (survival analysis: ␹2 ϭ 1.203, df ϭ 1, P ϭ 0.273; Þnal combination and desiccant pyrethrin dust with PBO mortality: Fisher exact test, P ϭ 0.393; Fig. 3). (␹2 ϭ 3.085, df ϭ 1, P ϭ 0.079 for imidacloprid ϩ Topical Application Assay. Because overall control cyßuthrin combination; ␹2 ϭ 0.075, df ϭ 1, P ϭ 0.785 mortalities were low and either similar or identical for desiccant pyrethrin dust; Fig. 2). The 10-d cumu- between fed and unfed bed bugs (e.g., 10 and 5% for lative mortalities of fed and unfed bed bugs were not fed and unfed Earl strain bed bugs, and 0% for both fed signiÞcantly different for these treatments (60% [31 of and unfed Jersey strain bed bugs at day 10, respec- 52] vs. 76% [38 of 50] for imidacloprid ϩ cyßuthrin tively; Fisher exact test, P Ͼ 0.05), all statistical com- combination; 100% [50 of 50] vs. 98% [49 of 50] for parisons were made between fed and unfed bed bugs desiccant pyrethrin dust) (Fisher exact test, P Ͼ 0.05). from the insecticide treatments without correction When Earl strain bed bugs were tested on the 34- based on corresponding control mortalities. d-old residual deposits, the survival times of the fed When three different doses of deltamethrin (i.e., 1, and unfed bed bugs were signiÞcantly different for 2, and 5 ng per insect) were topically tested on the Earl deltamethrin and chlorfenapyr aerosols and desiccant strain bed bugs, survival times of the fed and unfed bed pyrethrin dust with PBO (␹2 ϭ 42.226, df ϭ 1, P Ͻ bugs were not signiÞcantly different (␹2 ϭ 0.944, df ϭ June 2014 CHOE AND CAMPBELL:INSECTICIDE TOXICITY TESTS WITH BED BUGS 5

Fig. 3. KaplanÐMeier survival analyses for Earl strain bed bugs (fed and unfed) when they were exposed to the aged residual deposits (34-d-old) of deltamethrin, chlorfenapyr, imidacloprid ϩ cyßuthrin, and pyrethrin dust with PBO on a wooden substrate. Survivorship function S (t) was deÞned as the probability that an individual survives longer than time “t.” (Online Þgure in color.)

1, P ϭ 0.331 for 1 ng per insect; ␹2 ϭ 0.677, df ϭ 1, P ϭ with X amount of AI and the fed bed bugs treated with 0.411 for 2 ng per insect; ␹2 ϭ 0.057, df ϭ 1, P ϭ 0.811 2X the amount. The survival analysis and Þnal mor- for 5 ng per insect; Fig. 4, left). Total cumulative tality comparison indicated that the fed bed bugs that mortalities of the treated bed bugs at day 10 ranged received 27.4 ng chlorfenapyr per mg of fresh body from 50 to 90% depending on the dose, without sig- weight (data from 200 ng per insect dose) survived niÞcant difference between the fed and unfed bed signiÞcantly longer than did the unfed bed bugs bugs (Fisher exact test, P Ͼ 0.05). treated with 25 ng chlorfenapyr per mg of fresh body When three different doses of chlorfenapyr (i.e., weight (data from 100 ng per insect dose; survival 100, 200, and 500 ng per insect) were topically tested analysis: ␹2 ϭ 8.511, df ϭ 1, P ϭ 0.004; Þnal mortality: on the Earl strain bed bugs, survival times of the fed Fisher exact test, P ϭ 0.012). and unfed bed bugs were signiÞcantly different at all Chlorfenapyr was also topically tested on the Jersey rates tested (␹2 ϭ 11.497, df ϭ 1, P Ͻ 0.001 for 100 ng strain at three different rates (i.e., 100, 200, and 500 ng per insect; ␹2 ϭ 3.988, df ϭ 1, P ϭ 0.046 for 200 ng per per insect). The survival times of the fed bed bugs insect; ␹2 ϭ 7.959, df ϭ 1, P ϭ 0.005 for 500 ng per were signiÞcantly different from those of the unfed insect; Fig. 4, right). Total cumulative mortalities of bed bugs at all rates tested (Fig. 5). Total cumulative the fed and unfed bed bugs were 25% (5 of 20) versus mortalities of the fed and unfed bed bugs were 30% (3 75% (15 of 20) for 100 ng/insect dose (Fisher exact of 10) versus 100% (10 of 10) for 100 ng/insect dose test, P ϭ 0.002), 35% (7 of 20) versus 65% (13 of 20) (Fisher exact test, P ϭ 0.002), 40% (4 of 10) versus 90% for 200 ng/insect dose (Fisher exact test, P ϭ 0.056), (9 of 10) for 200 ng/insect dose (Fisher exact test, P ϭ and 70% (14 of 20) versus 100% (20 of 20) for 500 0.029), and 70% (7 of 10) versus 100% (10 of 10) for 500 ng/insect dose (Fisher exact test, P ϭ 0.01), respec- ng/insect dose (Fisher exact test, P ϭ 0.105), respec- tively. tively. The weights measured from fed and unfed bed Because the fed bed bugs were almost twice as bugs immediately before the assay were 7.5 Ϯ 0.5 and heavy as the unfed bed bugs (the weights of fed and 3.9 Ϯ 0.1 mg, respectively (mean Ϯ SEM, n ϭ 10 for unfed bed bugs were 7.3 Ϯ 0.6 and 4.0 Ϯ 0.2 mg, each). The survival analysis and Þnal mortality com- respectively [mean Ϯ SEM, n ϭ 10 for each]), it was parison indicated that the fed bed bugs that received of interest to compare the survival time and Þnal 26.7 ng chlorfenapyr per mg of fresh body weight mortality data between the unfed bed bugs treated (data from 200 ng per insect dose) survived signiÞ- 6JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3

Fig. 4. KaplanÐMeier survival analyses for Earl strain bed bugs (fed and unfed) when deltamethrin and chlorfenapyr insecticides were topically applied to the insects. Survivorship function S (t) was deÞned as the probability that an individual survives longer than time “t.” (Online Þgure in color.) cantly longer than did the unfed bed bugs treated with longer than did unfed ones when exposed to fresh or 25.6 ng chlorfenapyr per mg of fresh body weight aged residual deposits of chlorfenapyr and aged re- (data from 100 ng per insect dose; survival analysis: ␹2 sidual deposits of deltamethrin. Even though our anal- ϭ 14.791, df ϭ 1, P ϭ 0.0001; Þnal mortality: Fisher ysis indicated that survival times of fed and unfed bed exact test, P ϭ 0.005). bugs were different on their exposure to the fresh residue of deltamethrin and aged residue of desiccant pyrethrin dust with PBO, both feeding groups resulted Discussion in similarly high Þnal mortalities (92Ð100%) at day 10. The current study demonstrates that mortality re- For all the other treatments (i.e., fresh or aged residual sponses of fed and unfed bed bugs could be substan- deposits of imidacloprid ϩ cyßuthrin, and fresh re- tially different when they are exposed to some residual sidual deposits of desiccant pyrethrin dust with PBO), insecticide deposits. Based on the survival time and the fed and unfed bed bugs were similar in their Þnal mortality data at day 10 posttreatment, adult bed survival time and 10-d posttreatment mortality. Stud- bugs with a recent bloodmeal survived signiÞcantly ies with technical-grade insecticides at varying con- June 2014 CHOE AND CAMPBELL:INSECTICIDE TOXICITY TESTS WITH BED BUGS 7

Fig. 5. KaplanÐMeier survival analyses for Jersey strain bed bugs (fed and unfed) when a chlorfenapyr insecticide was topically applied to the insects. Survivorship function S (t) was deÞned as the probability that an individual survives longer than time “t.” (Online Þgure in color.) centrations would be helpful to understand if the differences in day 10 survivorships between fed and phenomenon is speciÞc for certain types of AIs. unfed bed bugs were more pronounced when they For the treatments with deltamethrin and chlorfe- were tested on the 34-d-old residual deposits com- napyr aerosols and desiccant pyrethrin dust with PBO, pared with the fresh ones. For example, the Þnal mor- the degree of difference between fed and unfed tality of fed bugs was lower than that of unfed bed bugs groups in their mortality responses varied depending by 8 and 45% for the fresh versus aged residues of on the age of the insecticide residual deposits. For deltamethrin, respectively. For chlorfenapyr, the Þnal deltamethrin and chlorfenapyr aerosols, we found that mortality of fed bugs was lower than that of unfed bed 8JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3 bugs by 24 and 50% for the fresh versus aged residues, tected only in our residual activity assays. Other be- respectively. For desiccant pyrethrin dust with PBO, havioral differences between the fed and unfed bed survival times of the fed bed bugs were signiÞcantly bugs might be in part responsible for the different longer than that of the unfed ones only when they mortality responses observed for the deltamethrin re- were tested on the aged residual deposits. sidual activity assay. For example, the unfed bed bugs The aforementioned results could be explained by might have moved more frequently while searching possible differences between the fresh and aged re- for a bloodmeal compared with the fed bed bugs (How sidual deposits of these insecticide products. For del- and Lee 2010, Reis and Miller 2011), potentially con- tamethrin and desiccant pyrethrin dust with PBO, it is tacting the insecticide residues more often compared likely that the aged residues had relatively lower tox- with the fed bed bugs. icity compared with the fresh ones as a result of po- In contrast to the deltamethrin data, results from tential degradation or other unknown processes. An- topical application assays with chlorfenapyr were con- derson and Cowles (2012) reported that residual sistent with the residual activity assays, showing the deposits of D-Force HPX aerosol (deltamethrin) dis- survival times and 10-d cumulative mortalities of fed played reduced residual toxicity against bed bugs as and unfed bed bugs were signiÞcantly different for the deposits aged (2Ð24 wk). Ebeling (1971) reported most of the doses tested (except Þnal mortality data that the lethal action of pyrethrin in Drione was for 200 and 500 ng /insect dose for Earl and Jersey slightly slowed down when the dust deposits aged for stains, respectively). Because the fed bed bugs were 146 d inside of a wall void (i.e., aged dust required more tolerant to chlorfenapyr than unfed ones even longer time to achieve 100% kill of German cock- when they were treated with a similar dose per unit of roaches compared with the fresh material). For chlo- fresh body weight, it is unlikely that the increased rfenapyr, however, it is less clear what could be the body mass and subsequent dilution of internalized possible difference between the fresh and aged resid- insecticides (“vigor tolerance,” Hoskins and Gordon ual deposits. Romero et al. (2010) reported that aged 1956) are playing a central role in this bloodmeal- (4 mo) deposits of chlorfenapyr aqueous spray on induced tolerance increase for chlorfenapyr. Then, Þlter paper were as toxic as fresh deposits for bed bugs what could be possible explanations for this phenom- based on a continuous exposure study. However, the enon? First, it is possible that some kinds of detoxiÞ- persistency of residual toxicity and rate of degradation cation mechanisms stimulated by the intake and di- of AIs of the insecticide residue can vary depending on gestion of the bloodmeal inadvertently reduced the the substrates (Chadwick 1985). Arthur (2008) also toxicity of chlorfenapyr. In mosquitoes, it was sug- reported that residual efÞcacy of chlorfenapyr is sig- gested that a series of enzymatic detoxiÞcation path- niÞcantly inßuenced by the kinds of substrates ways activated by the ingestion of a bloodmeal and its treated. In addition to these potential toxicity differ- digestion were responsible for their elevated toler- ences, other chemical differences between the fresh ance level to insecticides (Spillings et al. 2008). For and aged residual deposits such as the amount of example, upregulation of cytochrome P450s in re- solvent left in the residues might have direct or indi- sponse to a bloodmeal has been shown in Cx. pipiens rect effects on the mortality responses by inßuencing and Ae. aegypti mosquitoes (Baldridge and Feyereisen insecticide transfer or bed bugsÕ behavior in the 1986, Sanders et al. 2003). Alternatively, it is possible treated shelter. According to their material safety data that the digestion and processing of the recent blood- sheet, all the three products (D-Force HPX and Phan- meal might inhibit the timely conversion of chlorfe- tom pressurized aerosols and Drione) contain petro- napyr to its toxic form. Chlorfenapyr is a pro-pesticide leum-based solvents as their ingredients. Chemical that needs to be converted to a toxic form by a target differences between fresh and aged residues of these organism via an oxidative process probably catalyzed pesticide products and their impacts on the mortality by P450s (Black et al. 1994). If most multifunction responses of fed and unfed bed bugs warrant further oxidases such as P450s are preoccupied for processing study. a recent bloodmeal, the conversion of chlorfenapyr to Even though the residual activity assay indicated the toxic form by the fed bed bugs could be substan- that the survival times of the fed and unfed bed bugs tially delayed. Investigation of chlorfenapyr insecti- were different when they were tested on the fresh and cidal action by elementary processes such as penetra- aged residual deposits of deltamethrin, the two feed- tion, distribution, metabolism, and interaction with ing groups did not differ in their survival time and Þnal target sites, along with careful examination of resis- mortality based on the topical application assays. This tance- or detoxiÞcation-associated gene products, apparent discrepancy could be explained by a differ- would help to understand how the feeding status in- ence in study design between the residual activity and ßuences the bed bugÕs response to this particular in- topical application assays. Unlike the topical applica- secticide. tion assays, the residual activity assay settings allowed Continued survival of blood-fed bed bugs after con- the bed bugs to move away from the treated surface tacting the insecticide residues would be problematic if the residual deposits of deltamethrin were repellent not only because the surviving bed bugs might con- to them. If prolonged or repetitive exposure of bed tinue feeding on humans, but also because the fed bugs to a lower rate of deltamethrin residue is nec- adult female bed bugs may continue producing eggs. essary to show the survival time difference between Moore and Miller (2006) reported that continuous fed and unfed bed bugs, the difference could be de- exposure to a chlorfenapyr residue on a hardboard June 2014 CHOE AND CAMPBELL:INSECTICIDE TOXICITY TESTS WITH BED BUGS 9 panel did not prevent bed bugs from mating and laying Arthur, F. H. 2008. EfÞcacy of chlorfenapyr against eggs. Similarly, most of the fed female bed bugs in the Tribolium castaneum and Tribolium confusum (Co- current study produced eggs even after being exposed leoptera: Tenebrionidae) adults exposed on concrete, to the chlorfenapyr insecticide. For example, in the vinyl tile, and plywood surfaces. J. Stored. Prod. Res. 44: chlorfenapyr residual activity assays with the Earl 145Ð151. strain, 9 of 10 replicates observed from the fresh and Baldridge, G. D., and R. Feyereisen. 1986. Blood meal and aged residue trials had eggs by day 10. Also, in the cytochrome P-450 monooxygenases in the northern chlorfenapyr topical application assays with the Earl house mosquito, Culex pipiens. Pestic. Biochem. Physiol. 25: 407Ð413. strain, 76.7% of the fed females observed (n ϭ 30) laid Black, B. C., R. M. Hollingworth, K. I. Ahammadsahib, C. D. eggs by day 10. In contrast, only 1 of 10 replicates from Kukel, and S. Donovan. 1994. Insecticidal action and the fresh and aged deltamethrin residue trials had eggs mitochondrial uncoupling activity of AC303,630 and re- by day 10. The topical application of deltamethrin also lated halogenated pyrroles. Pestic. Biochem. Physiol. 50: substantially inhibited the oviposition of the fed fe- 115Ð128. males, resulting in only 46.7% of the fed females (n ϭ Brown, A.W.A., and R. Pal. 1971. Insecticide resistance in 15) having laid eggs by day 10. Deltamethrin typically arthropods, The 2nd WHO Monograph Series 38, World knocks down the pyrethroid-susceptible bed bugs Health Organization, Geneva, Switzerland. within 1Ð2 h, so this fast neurotoxic action might be, Chadwick, R. R. 1985. Surfaces and other factors modifying at least in part, responsible for the oviposition inhibi- the effectiveness of pyrethroids against insects in public tion. health. Pestic. Sci. 16: 383Ð391. Based on the current study, we suspect that an Ebeling, W. 1971. Sorptive dusts for pest control. Annu. eradication effort using chlorfenapyr insecticide as a Rev. Entomol. 16: 123Ð158. sole residual treatment option could be negatively Hadaway, A. B., and F. Barlow. 1956. Effects of age, sex and impacted if target bed bug populations can get fre- feeding on the susceptibility of mosquitoes to insecti- quent bloodmeals. For bed bug populations with re- cides. Ann. Trop. Med. Parasitol. 50: 438Ð443. Halliday, W. R., and R. Feyereisen. 1987. Why does DDT cent bloodmeals, longer exposure time on the insec- toxicity change after a blood meal in adult female Culex ticide residues or higher application rate might be pipiens? Pestic. Biochem. Physiol. 28: 172Ð181. necessary to achieve a similar level of control as for a Hoskins, W. M., and H. T. Gordon. 1956. Arthropod resis- relatively starved bed bug population. Frequent feed- tance to chemicals. Annu. Rev. Entomol. 1: 89Ð122. ing of a bed bug population could be partially pre- How, Y.-F., and C.-Y. Lee. 2010. Effects of life stages and vented by setting up barriers or pitfall traps (e.g., feeding regimes on active movement behavior of the interceptor devices) at the base of legs of couches or tropical bed bug, Cimex hemipterus (Hemiptera: Cimi- bed frames, where taking of the bloodmeal is most cidae). J. Med. Entomol. 47: 305Ð312. likely to occur. Where it is economically viable, an Meyers, J. 2012. Battling resistance. Pest Control Technol. evacuation of the infested structure for several days 40: 48Ð51. before the insecticide treatment would also decrease Moore, D. J., and D. M. Miller. 2006. Laboratory evaluations the proportion of bed bugs with a recent bloodmeal of insecticide product efÞcacy for control of Cimex lectu- within the population. Further study is still required to larius. J. Econ. Entomol. 99: 2080Ð2086. determine if the observed difference between fed and Reinhardt, K., D. Isaac, and R. Naylor. 2010. Estimating the unfed bed bugsÕ mortality responses to chlorfenapyr is feeding rate of the bedbug Cimex lectularius in an infested room: an inexpensive method and a case study. Med. Vet. a temporary phenomenon and, if so, how long it would Entomol. 24: 46Ð54. last after a bloodmeal. Reis, M. D., and D. M. Miller. 2011. Host searching and aggregation activity of recently fed and unfed bed bugs (Cimex lectularius L.). Insects 2: 186Ð194. Acknowledgments Romero, A., M. F. Potter, and K. F. Haynes. 2007a. Insecti- We thank Michael Rust for critical review of an earlier cide resistant bed bugs: implications for the industry. Pest draft of this manuscript. We also thank William Donahue, Jr., Control Technol. 35: 42Ð50. for providing insecticide toxicity data for two bed bug strains Romero, A., M. F. Potter, D. Potter, and K. F. Haynes. 2007b. used in the current study. Three anonymous reviews im- Insecticide resistance in the bed bug: a factor in the pestÕs proved the clarity of this manuscript. sudden resurgence? J. Med. Entomol. 44: 175Ð178. Romero, A., M. F. Potter, and K. F. Haynes. 2009a. Behav- ioral responses of the bed bug to insecticide residues. References Cited J. Med. Entomol. 46: 51Ð57. Adelman, Z. N., K. A. Kilcullen, R. Koganemaru, M.A.E. Romero, A., M. F. Potter, and K. F. Haynes. 2009b. Evalu- Anderson, T. D. Anderson, and D. M. Miller. 2011. Deep ation of piperonyl butoxide as a deltamethrin synergist for sequencing of pyrethroid-resistant bed bugs reveals mul- pyrethroid-resistant bed bugs. J. Econ. Entomol. 102: tiple mechanisms of resistance within a single population. 2310Ð2315. PLoS One 6: e26228. (doi:10.1371/journal.pone. 0026228) Romero, A., M. F. Potter, and K. F. Haynes. 2010. Evaluation Analytical Software. 2008. Statistix 9 userÕs manual. Analyt- of chlorfenapyr for control of the bed bug, Cimex lectu- ical Software, Tallahassee, FL. larius L. Pest Manag. Sci. 66: 1243Ð1248. Anderson, J. F., and R. S. Cowles. 2012. Susceptibility of Rust, M. K., and R. K. Saran. 2006. Toxicity, repellency, and Cimex lectularius (Hemiptera: Cimicidae) to pyrethroid transfer of chlorfenapyr against western subterranean insecticides and to insecticidal dusts with or without termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. pyrethroid insecticides. J. Econ. Entomol. 105: 1789Ð1795. 99: 864Ð872. 10 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3

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