Comparative Insecticide Uptake Characteristics of Chlorfenapyr Suggests an Additional Reason Why Bed Bugs (Hemiptera: Cimicidae) are so Difficult to Control.

Insecticides interact with biochemical sites, disrupting homeostasis, leading to moribundity and death. Considering this process of intoxication, investigating insecticide uptake ensures an understanding of exposure profile. This study evaluated insecticide uptake by bed bugs, Cimex lectularius L., as affected by time and formulation, and a comparative uptake by German cockroaches, Blattella germanica (L.). Insects exposed to chlorfenapyr residues from two Phantom insecticide formulations (i.e., Aerosol and Suspended Concentrate (SC)) were solvent extracted and analyzed by GC-ECD. Chlorfenapyr uptake by bed bugs significantly increased with time, but the rate of external chlorfenapyr adsorption between the two formulations was not significantly different. However, initial exposure and uptake to the aerosol-treated versus SC-treated surfaces were significantly greater by 2.7x. Absorption-rates of chlorfenapyr residues inside the insect, associated with internal insect tissues or exoskeleton-bound, were significantly lower compared with external adsorption, with aerosol and SC formulations accumulating at 4.8x and 14.4x less, respectively. An unexpected formulation effect occurred with aerosol-treated papers and aging of residues. A reformulation of the aerosol provided a greater adsorption of chlorfenapyr at 19.1x that of the original formula after 2 hr. External versus internal uptake of chlorfenapyr by cockroaches was inverse to that seen in bed bugs, with greater chlorfenapyr absorption. Obstructing cockroach mouthparts prevented an internal uptake of chlorfenapyr and provided a similar exposure profile to bed bugs. This work demonstrates a need to evaluate insecticide uptake with formulation and insect morphology to maximize dose, especially in such a morphologically and environmentally isolated insect as the bed bug.

Two compounds in bed bug feces are sufficient to elicit off-host aggregation by bed bugs, Cimex lectularius.

BACKGROUND: After feeding, bed bugs aggregate in cracks and crevices near a host. Aggregation and arrestment are mediated by tactile and chemical stimuli associated with the bugs' feces and exuviae. Volatiles derived from fecally stained filter papers were analyzed by solid-phase microextraction (SPME) and evaluated using a multichoice behavioral assay to determine their impact on bed bug aggregation. In addition, crude fecal extracts were collected in methanol, analyzed by gas chromatography coupled with electroantennogram detection (GC-EAD) and mass spectrometry (GC-MS) and evaluated in open-air multichoice behavioral assays. RESULTS: The SPME method was used to detect (E)-2-hexenal and (E)-2-octenal in heated bed bug feces. The presence of these two volatile components did not affect aggregation. Analysis of the crude fecal extracts revealed several semi-volatile nitrogenous compounds, a carboxylic acid and a sulfur-based compound. Adult antennae responded to compounds eluted from three regions of the crude extract using GC-EAD. A combination of two compounds, dimethyl trisulfide and methyldiethanolamine, resulted in aggregation responses equivalent to the original crude extract. CONCLUSION: Bed bug aggregation is mediated by semi-volatile compounds derived from fecal extracts, and two compounds are sufficient to elicit aggregation. The two compounds identified here could be used to enhance the effectiveness of insecticidal applications or improve monitoring techniques. © 2016 Society of Chemical Industry.

The influence of time and distance traveled by bed bugs, Cimex lectularius, on permethrin uptake from treated mattress liners.

BACKGROUND: Residual insecticides interrupt the dispersal of bed bugs (Cimex lectularius, L.), but one of the issues encountered with residual applications is understanding the uptake of active residues by the insect. This study determined permethrin uptake by bed bugs walking on the ActiveGuard® Mattress Liner product, via a combination of video recording in arenas and gas chromatography analyses. RESULTS: The best model for estimating permethrin uptake utilized a covariance model (r2 = 0.469) with two factors: time of exposure (F1,55 = 2.44, P < 0.001) and distance traveled (F1,55 = 0.30, P = 0.0460). Bed bug permethrin uptake was 15.1 (95% CI: 10.3-22.1) ng insect-1 within 1 min exposure, 21.0 (15.0, 31.0) ng insect-1 within 10 min and ≈ 42 (29.8, 60.6) ng insect-1 within ≥50 min exposure. Correcting for percentage recovery, these values would be increased by a factor of 1.21. CONCLUSION: This permethrin-treated fabric provides a surface from which bed bugs begin rapidly to absorb permethrin on contact and within the first 1 cm of travel. Variability in uptake was likely a result of grooming and thigmotaxis, and future work should use quantitative methods to study behaviors and formulations that increase exposure to the toxicant. © 2016 Society of Chemical Industry.

Estimating the critical thermal maximum (CTmax) of bed bugs, Cimex lectularius: Comparing thermolimit respirometry with traditional visual methods.

Evaluating the critical thermal maximum (CTmax) in insects has provided a number of challenges. Visual observations of endpoints (onset of spasms, loss of righting response, etc.) can be difficult to measure consistently, especially with smaller insects. To resolve this problem, Lighton and Turner (2004) developed a new technique: thermolimit respirometry (TLR). TLR combines real time measurements of both metabolism (V·CO2) and activity to provide two independent, objective measures of CTmax. However, several questions still remain regarding the precision of TLR and how accurate it is in relation to traditional methods. Therefore, we evaluated CTmax of bed bugs using both traditional (visual) methods and TLR at three important metabolic periods following feeding (1d, 9d, and 21d). Both methods provided similar estimates of CTmax, although traditional methods produced consistently lower values (0.7-1°C lower than TLR). Despite similar levels of precision, TLR provided a more complete profile of thermal tolerance, describing changes in metabolism and activity leading up to the CTmax, not available through traditional methods. In addition, feeding status had a significant effect on bed bug CTmax, with bed bugs starved 9d (45.19[±0.20]°C) having the greatest thermal tolerance, followed by bed bugs starved 1d (44.64[±0.28]°C), and finally bed bugs starved 21d (44.12[±0.28]°C). Accuracy of traditional visual methods in relation to TLR is highly dependent on the selected endpoint; however, when performed correctly, both methods provide precise, accurate, and reliable estimations of CTmax.

Effects of Starvation on Deltamethrin Tolerance in Bed Bugs, Cimex lectularius L. (Hemiptera: Cimicidae).

Bed bugs, Cimex lectularius L., are a major pest in the urban environment. Their presence often results in physical, psychological, and financial distress of homeowners and apartment dwellers. Although many insecticide bioassays have been performed on this pest, little attention has been paid to bed bug feeding status, which is closely linked to metabolism, molting, and mass. Therefore, we evaluated the toxicity of topically applied deltamethrin on insecticide susceptible adult male bed bugs fed 2 d, 9 d, and 21 d prior to testing. When toxicity was evaluated on a "per-bug" basis, there was no difference between 2 d [LD50 = 0.498 (0.316 - 0.692) ng·bug(-1)] and 9 d [LD50 = 0.572 (0.436 - 0.724) ng·bug(-1)] starved bugs, while 21 d starved bugs had a significantly lower LD50 [0.221 (0.075 - 0.386) ng·bug(-1)]. When toxicity was evaluated in terms of body mass, 9 d starved bugs had the highest LD50 values [0.138 (0.102 - 0.176) ng·mg(-1)], followed by 2 d starved bugs [0.095 (0.060 - 0.134) ng·mg(-1)], and then 21 d starved bugs [0.058 (0.019-0.102) ng·mg(-)¹]; the LD50 values of 2 d and 9 d starved bugs were significantly different from 21 d starved bugs. These results indicate that feeding status plays an important role in the toxicity of deltamethrin. In addition, the lack of differences between 2 d and 9 d starved bugs indicate that the blood meal itself has little impact on tolerance, but rather it is some physiological change following feeding that confers increased tolerance to bed bugs.

Effects of starvation and molting on the metabolic rate of the bed bug (Cimex lectularius L.).

The bed bug (Cimex lectularius L.) is a common hematophagous pest in the urban environment and is capable of surviving extended periods of starvation. However, the relationship between starvation and metabolism in bed bugs is not well understood. To better understand this relationship, we measured the metabolism of all life stages for >900 h after feeding (starvation) using closed-system respirometry. Measurements were made around molting for the immature life stages, which occurs only after a blood meal. In addition, both mated and unmated adults were measured. Starvation and molting had significant effects on the metabolism of the bed bug. Mass-specific metabolic rate (V(O2); mL g(-1) h(-1)) declined in a curvilinear fashion with the period of starvation for adults and with the postmolting period for immature bed bugs (used to standardize all immature life stages). A standard curve was developed to depict the generalized pattern of metabolic decline observed in all life stages that molted. Individual metabolic comparisons among life stages that molted revealed some differences in metabolic rate between unmated males and females. In addition, the mass scaling coefficient was found to decline with starvation time (postmolting time) for all life stages that molted. In most life stages, the ratio of V(CO2) to V(O2) (respiratory exchange ratio) declined over time, indicating a change in metabolic substrate with starvation. Finally, daily percent loss in body mass declined in a pattern similar to that of V(O2). The observed patterns in metabolic decline are evaluated in relation to the life history of bed bugs. In addition, the evolutionary development of these patterns is discussed. The metabolic pattern after feeding was also found to share several similarities with that of other ectothermic species.

Morphology, ultrastructure and functional role of antennal sensilla in off-host aggregation by the bed bug, Cimex lectularius.

After blood feeding on a host, bed bugs, Cimex lectularius, assemble in aggregation sites away from the host. Off-host aggregation is mediated by a combination of mechanical and chemical stimuli associated with bug feces. Partial antennectomies indicated removal of flagellomeres did not affect aggregation, but removal of the whole pedicel or its distal half significantly reduced (P < 0.01) aggregation, suggesting that sensilla related to off-host aggregation occur on the distal half of the pedicel. Scanning electron microscopy (SEM) revealed that serrated hairs were distributed throughout the pedicel, but newly described smooth hairs were present mainly on the distal half, and a distinct patch of grooved pegs, smooth pegs and immersed cones was present on the posterior edge of the distal half of the pedicel in adults, but not in nymphs. Numbers of different types of sensilla increased significantly during metamorphosis from first instar to adult (P < 0.05), but were similar between genders (P = 0.11) and between females from a laboratory and field strain of bugs (P = 0.19). Transmission electron microscopy (TEM) revealed that cuticular pores were present in the two types of pegs, indicating that the pegs have an olfactory function. The smooth hairs resembled gustatory sensilla previously described in Cimex hemipterus F. The existence of both olfactory and gustatory sensilla on the distal half of the pedicel suggests those sensilla may be the sensory basis of off-host aggregation behavior.

Standard metabolic rate of the bed bug, Cimex lectularius: effects of temperature, mass, and life stage.

Metabolic rates provide important information about the biology of organisms. For ectothermic species such as insects, factors such as temperature and mass heavily influence metabolism, but these effects differ considerably between species. In this study we examined the standard metabolic rate of the bed bug, Cimex lectularius L. We used closed system respirometry and measured both O2 consumption and CO2 production across a range of temperatures (10, 20, 25, 30, 35°C) and life stages, while also accounting for activity. Temperature had a stronger effect on the mass specific .VO2 (mlg(-1)h(-1)) of mated males (Q10=3.29), mated females (Q10=3.19), unmated males (Q10=3.09), and nymphs that hatched (first instars, Q10=3.05) than on unmated females (Q10=2.77) and nymphs that molted (second through fifth instars, Q10=2.78). First instars had significantly lower respiratory quotients (RQ) than all other life stages. RQ of all stages was not affected by temperature. .VO2 (mlh(-1)) scaled more with mass than values previously reported for other arthropods or that would be predicted by the 3/4-power law. The results are used to understand the biology and ecology of the bed bug.

Efficacy and application considerations of selected residual acaricides against the mold mite Tyrophagus putrescentiae (Acari: Acaridae) in simulated retail habitats.

The mold mite, Tyrophagus putrescentiae (Schrank), is a stored product pest of economic significance that commonly infests many types of food and animal feed products. There is limited information regarding pest management tactics, including residual acaricides for managing this pest in retail store habitats. The purpose of this study was to assess the potential of commercially available acaricides that could provide a protective barrier near susceptible food packages destined for consumer use. Mite mortality at 24 and 48 h was measured after exposing mites to acaricide residues applied to a nonporous surface. Two additional factors included in the study were humidity and the presence of an adjuvant to facilitate spread of acaricide on nonporous surfaces. Increasing humidity levels generally decreased acaricide efficacy. Use of the adjuvant itself did not appear to affect efficacy, but the type of application (wet or slurry, or dry) of dust acaricides significantly affected efficacy. The potential for residual acaricides to act as a barrier (within 24-48 h) against dispersal of this mite was discussed.

Cold tolerance of bed bugs and practical recommendations for control.

Bed bugs were exposed to freezing temperatures for various exposure times to determine cold tolerance and mortality estimates for multiple life stages. The mean supercooling point for all bed bug life stages ranged from -21.3 degrees C to -30.3 degrees C, with the egg stage reporting the lowest value. A probit analysis provided a lower lethal temperature (LLT99) of -31.2 degrees C when estimates from all life stages were combined, demonstrating that all stages of bed bugs are not capable of surviving temperatures below body freezing and are therefore freeze intolerant. At conditions above the LLT99, bed bug mortality depended on temperature and exposure time at temperatures above LLT99. Based on our model estimates, survival was estimated for temperatures above -12 degrees C even after 1 wk of continuous exposure. However, exposure to temperatures below -13 degrees C will result in 100% mortality in d to ensure mortality of all life stages. Unfortunately, sublethal exposure to lower temperatures did not prevent subsequent feeding behavior in surviving stages. Practical recommendations for management of potentially infested items are discussed.

Freeze mortality characteristics of the mold mite Tyrophagus putrescentiae, a significant pest of stored products.

The mold mite Tyrophagus putrescentiae (Shrank) is a common pest of stored food products. Until recently, commodity and facility treatments have relied on acaricides and fumigants to control this mite. However, T. putrescentiae will cause infestations in areas where acaricide or fumigant use may be restricted, prohibited, or highly impractical. Because temperature is an essential factor that limits the survival of arthropod species, extreme temperatures can be exploited as an effective method of control. Making low-temperature treatments reliable requires better temperature-time mortality estimates for different stages of this mite. This was accomplished by exposing a representative culture (eggs, nymphs, and adults) of noncold-acclimated T. putrescentiae to subfreezing temperatures to determine their supercooling points (SCPs), lower lethal temperatures (LLTs) and lethal times (LTimes) at set temperatures. The results indicate that the adult and nymphal stages of T. putrescentiae are freeze intolerant; based on 95% CIs, the adult LLT90 of -22.5 degrees C is not significantly different from the SCP of -24.2 degrees C and the nymphal LLT90 of -28.7 degrees C is not significantly different from the SCP of -26.5 degrees C. The egg stage seems to be freeze tolerant, with an LLT90 of -48.1 degrees C, significantly colder by approximately 13.5 degrees C than its SCP of -35.6 degrees C. The LTime demonstrates that 90% of all mite stages of T. putrescentiae can be controlled within commodity or packaged product by freezing to -18 degrees C for 5 h. By achieving the recommended time and temperature exposures, freezing conditions can be an effective way of controlling mites and reducing chronic infestations.

Temperature and Time Requirements for Controlling Bed Bugs (Cimex lectularius) under Commercial Heat Treatment Conditions.

Developing effective alternative approaches for disinfesting bed bugs from residential spaces requires a balance between obtaining complete insect mortality, while minimizing costs and energy consumption. One method of disinfestation is the application of lethal high temperatures directly to rooms and contents within a structure (termed whole-room heat treatments). However, temperature and time parameters for efficacy in whole-room heat treatments are unknown given the slower rate of temperature increase and the probable variability of end-point temperatures within a treated room. The objective of these experiments was to explore requirements to produce maximum mortality from heat exposure using conditions that are more characteristic of whole-room heat treatments. Bed bugs were exposed in an acute lethal temperature (LTemp) trial, or time trials at sub-acute lethal temperatures (LTime). The lethal temperature (LTemp99) for adults was 48.3 °C, while LTemp99 for eggs was 54.8 °C. Adult bed bugs exposed to 45 °C had a LTime99 of 94.8 min, while eggs survived 7 h at 45 °C and only 71.5 min at 48 °C. We discuss differences in exposure methodologies, potential reasons why bed bugs can withstand higher temperatures and future directions for research.

Boring in response to bark and phloem extracts from North American trees does not explain host acceptance behavior of Orthotomicus erosus (Coleoptera: Scolytidae).

When invasive herbivorous insects encounter novel plant species, they must determine whether the novel plants are hosts. The Mediterranean pine engraver, Orthotomicus erosus (Wollaston), an exotic bark beetle poised to expand its range in North America, accepts hosts after contacting the bark. To test the hypothesis that O. erosus accepts hosts on the basis of gustatory cues, we prepared bark and phloem extracts from logs of four North American tree species that we had used in previous host acceptance experiments. Water, methanol, and hexane extracts of red pine, tamarack, balsam fir, and paper birch were presented alone and in combination on a neutral filter paper substrate in a section of a plastic drinking straw. Boring behavior in response to the three-extract combinations differed from the pattern of acceptance previously observed among species when the beetles were in contact with the bark surface. Only the aqueous extracts of tamarack, Larix laricina, increased the initiation and the extent of boring by O. erosus on the filter paper substrate. We conclude that the effects of extracted chemicals do not match the behavior of the beetles observed when penetrating excised bark and phloem discs, indicating that host selection by O. erosus may not be predictable from bark and phloem chemistry alone. Instead, host acceptance may be determined by nongustatory stimuli or by a combination of stimuli including gustatory and nongustatory cues.

Odorants that induce hygienic behavior in honeybees: identification of volatile compounds in chalkbrood-infected honeybee larvae.

Social insects that live in large colonies are vulnerable to disease transmission due to relatively high genetic relatedness among individuals and high rates of contact within and across generations. While individual insects rely on innate immune responses, groups of individuals also have evolved social immunity. Hygienic behavior, in which individual honeybees detect chemical stimuli from diseased larvae and subsequently remove the diseased brood from the nest, is one type of social immunity that reduces pathogen transmission. Three volatile compounds, collected from larvae infected with the fungal pathogen Ascosphaera apis and detected by adult honey bees, were identified by coupled gas chromatography-electroantennographic detection and gas chromatography-mass spectrometry. These three compounds, phenethyl acetate, 2-phenylethanol, and benzyl alcohol, were present in volatile collections from infected larvae but were absent from collections from healthy larvae. Two field bioassays revealed that one of the compounds, phenethyl acetate is a key compound associated with Ascosphaera apis-infected larvae that induces hygienic behavior.

Use of vapor pressure deficit to predict humidity and temperature effects on the mortality of mold mites, Tyrophagus putrescentiae.

The mold mite, Tyrophagus putrescentiae (Shrank), frequently infests a variety of stored food products in ideal, but rather limited conditions. Major factors limiting survival of this mite are the temperature and humidity imposed on T. putrescentiae as it develops within and disperses among sites. However, since relative humidity is dependent upon air temperature, determining survivability in a habitat can be difficult in the presence of structural temperature variations. Vapor pressure deficit (VPD) provides a method of combining both relative humidity and temperature into a single number that can be used to determine conditions detrimental to mite survival. This study utilized a bioassay format to measure mortality of T. putrescentiae when exposed to a range of seven temperatures (5-35 degrees C), 10 relative humidities (0-100% RH), 17 exposure times (0.5-240 h), with and without food. With these combinations of temperature and RH, mortality curves (mortality versus time) that displayed a sigmoidal relationship were used to calculate LT(50) and LT(90) estimates. These mortality estimates were then regressed on their associated VPD and the resulting regressions (LT(50) and LT(90)) were significant at P < 0.0001, and provided acceptable R(2) values >or=0.83, regardless of whether food was present or not. At room temperature, threshold of VPD for T. putrescentiae development was below 8.2 mbar, this estimate being initially calculated from published values. For mites exposed to drier conditions, above 8.2 mbar, survival time was curtailed dependant on the magnitude of VPD. As the VPD exceeded 12 mbar, mites experienced substantial (>90%) mortality within 58 (33, 101) h; and further increasing VPD decreased the time of exposure to achieve mortality. This study demonstrates that making subtle changes in humidity or temperature to reach a target VPD may provide control of mite outbreaks and reduce areas inhabitable for T. putrescentiae.