TY - JOUR
AU - Haynes, Kenneth F.
AB - Abstract Control of bed bugs is problematic, balancing among efficacy, safety, and cost. In this study, ultralow oxygen (ULO) and vacuum treatments were tested on bed bugs to develop a safer, effective, and environmentally friendly solution to kill bed bugs on infested items. ULO treatments were established by flushing sealed enclosures with nitrogen. All life stages of bed bugs were found to be susceptible to ULO and vacuum treatments, and efficacy of the treatments increased with reduced oxygen levels, increased treatment time, and temperature. In the ULO treatments, 100% mortality of bed bug nymphs and adults and >98% mortality of bed bug eggs were achieved in the 8-h treatment under 0.1% O 2 atmosphere at 30°C. Different levels of vacuum that yielded different oxygen levels were tested on all life stages of bed bugs. The susceptibility of different stages to vacuum treatments increased from nymphs to adults to eggs. Complete control of all life stages was achieved in 12 h under −982 mbar (−29.0 inHg) vacuum at 30°C. This study demonstrated that bed bugs were very susceptible to low oxygen stresses and ULO and vacuum treatments have potential to be used as effective and safe treatments to decontaminate bed bug-infested removable objects. ultralow oxygen, vacuum, bed bug control, pest, insect The bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), has re-emerged as an important pest around the world in recent years ( Doggett and Russell 2007 , Potter et al. 2008 ). Insecticides are commonly used to control bed bugs. But bed bug resistance to pyrethroids is widespread, and there is a need for new tools for managing infestations ( Romero et al. 2007 , Potter et al. 2008 , Zhu et al. 2010 , Wang and Cooper 2011 ). As bed bugs are nocturnally active and difficult to find because they seek out hidden cracks and crevices on and around the bed, they can be difficult to kill with insecticide sprays. The use of some insecticides is restricted because of potential health hazards in bedrooms and other human living spaces. Sulfuryl fluoride fumigation is also effective against bed bugs ( Wang and Cooper 2011 , Phillips et al. 2014 ), but its utility is limited by expensive setup and the need to prevent human exposure. Steaming, freezing, heating, and hot washing are used to eliminate bed bugs from infested household goods ( Pinto et al. 2007 , Taisey and Neltner 2010 , Wang and Cooper 2011 ). However, all of these treatment methods have their limitations. Heat treatment may not be suitable for treating certain items and can also be a fire hazard when used improperly. Controlled atmosphere (CA) with high carbon dioxide was also found to be effective in killing bed bugs ( Wang and Cooper 2011 , Wang et al. 2012 ). Complete elimination of bed bug active stages and eggs in 7 and 10 h, respectively, in 100% CO 2 at 24–26°C ( Wang and Cooper 2011 ) was achieved. CO 2 fumigation at ≥94%, however, takes at least 24 h to control bed bug eggs ( Wang et al. 2012 ). Additional effective, economical, and environmental friendly treatment options are needed for bed bug management. Oxygen levels below 1% are typically considered as ultralow oxygen (ULO). CA and MA (modified atmosphere) with ULO and vacuum have been studied or used to control pests on museum artifacts, stored products as well as fresh products ( Calderon et al. 1966 ; Selwitz and Maekawa 1998 ; Mitcham et al. 2001 ; Navarro et al. 2001 , 2007 ; Liu 2010 ). However, its effectiveness against bed bugs has not been investigated. CA treatment has been studied over 30 years for postharvest pest control on fresh commodities with various outcomes and no sustained practical applications. Both poor efficacy and low tolerance by fresh commodities are the reasons for the lack of effective and safe CA treatments against pests on fresh commodities ( Mitcham et al. 2001 ). More recently, CA with ultralow oxygen (ULO treatment) has been found safe and effective to control several pests on fresh products, and there are great variations among different pest species and life stages in susceptibility to ULO treatment ( Liu 2010 ). Effectiveness of ULO treatment is directly related to oxygen level, temperature, and treatment time, with lower oxygen levels, higher temperatures, and longer treatment durations increasing effectiveness of ULO treatment ( Liu 2010 ). Mobile life stages are more susceptible to ULO treatment than stationary life stages including both pupae and eggs, and eggs appear to be the most tolerant life stage to ULO treatment. At a low temperature used for cold storage of fresh commodities, it takes as long as 10 d to control grape mealybug ( Pseudococcus maritimus (Ehrhorn)) eggs using ULO treatment with <0.01 µl/liter O 2 ( Liu 2013a ). Some CA treatments use both reduced oxygen and increased CO 2 to achieve effective control of insects ( Mitcham et al. 2001 ). Even with combinations of low oxygen and high CO 2 , examples of successful CA treatments for insect control, especially eggs, are very few, and the treatments are often too harmful to host materials for practical use. CA with 0.5–2% O 2 and 30% CO 2 controls eggs of Caribbean fruit fly ( Anastrepha suspensa (Loew)) in 10 d at 15.6°C ( Benshoter 1987 ). Eggs of light brown apple moth ( Epiphyas postvittana (Walker)) are killed using CA with 0.3% O 2 and 4.6% CO 2 in 16 h at 20°C ( Chervin et al. 1997 ). These results reflect great variations among insect species in response to CA treatments. ULO treatments (also called anoxic treatment or nitrogen fumigation) were also studied for controlling pests on stored products and in museum objects ( Daniel et al. 1993 , Gilberg and Roach 1993 , Selwitz and Maekawa 1998 , Bergh et al. 2003 ). Daniel et al. (1993) detailed pest control treatments for museum objects using a sequential combination of nitrogen flush and oxygen absorbent in a sealed bag or by flushing fumigation chambers with nitrogen to maintain a <0.1% O 2 for pest control. Selwitz and Maekawa (1998) provided a comprehensive guide for treating museum objects using CA including ULO treatments. Vacuum can also be effective in controlling insects ( Aharoni et al. 1986 ; Navarro and Calderon 1979 ; Navarro et al. 2001 , 2007 ; Finkelman et al. 2003 ; Liu 2003 ; Mbata et al. 2004 ). The main mechanism for vacuum effects is considered to be the lack of oxygen ( Navarro and Calderon 1979 ). In this paper, we reported responses of bed bug eggs, nymphs, and adults to ultralow and low oxygen treatments and vacuum treatments. Materials and Methods Insects Bed bugs were maintained at the University of Kentucky using established procedures ( Montes et al. 2002 , Romero et al. 2010 ). The colony progenitors were collected from an apartment in Los Angeles, CA, in 2007. To feed, bed bugs would insert their mouthparts through an organza-lined lid to their plastic confinement jar (60 ml) and through a parafilm membrane into a pool of defibrinated rabbit blood contained in a custom made glass “mosquito” feeder (Kimble-Chase, Vineland, NJ). The blood was maintained at 39°C using a circulating water bath. Bed bug colonies were housed in environmental chambers at 26–28°C, 65% relative humidity, and a photoperiod of 14:10 (L:D) h. After feeding, ∼10 mated females were confined in a small plastic vial with screened lids (3 cm in diameter by 7 cm in height) and a piece of blotter paper (5 by 5 cm 2 folded in half into a tent). They were allowed to oviposit for the next 3–5 d. After this time, adults were removed. Ten third-instar nymphs or ten 7-d-old males and females (five of each sex) were similarly confined. The vials with eggs, nymphs, and adults were express-shipped with ice packs to USDA-ARS in Salinas, CA, for treatments. Low Oxygen Treatments ULO and low oxygen treatments were conducted in 1.9-liter glass jars at different temperatures in an environmental chamber. The lids for the jars were modified to have two outlets with valves on each lid. Plastic vials with bed bugs were sealed in glass jars. In each test, one to three vials containing bed bugs were sealed in each jar to be treated. Nitrogen gas from a compressed cylinder was released passing through a flow meter at about 1.0 liter/min to flush out oxygen in individual jars to establish desired low oxygen levels. An oxygen analyzer (Series 800, Illinois Instruments, Inc., Johnsburg, IL) was used to monitor oxygen levels in the jars. The jars were then kept at defined temperatures in the environmental chambers for specific time periods to complete specific treatments. Bed bug eggs, nymphs, and adults (males and females) in separate vials were subjected to ULO and low oxygen treatment with different combinations of oxygen level, temperature, and treatment time depending on life stages. Eggs were subjected to treatments under ULO levels of 0.1 and 0.5% and low oxygen levels of 1, 2, and 3%. The 0.1 and 0.5% ULO treatments were conducted at 25 and 30°C and had treatment times of 6 to 24 h. The 1, 2, and 3% low oxygen treatments were conducted at 20, 25, and 30°C and had treatment times of 24 and 48 h depending on oxygen levels. Nymphs and adults were subjected to 0.1 and 0.5% O 2 atmosphere storage treatments for 6 to 24 h at 30°C. They were stored for 48 h under 1% O 2 at 30°C and 3% O 2 at 2°C. Controls were maintained at 25°C during treatments. Each treatment was replicated two to four times. Short ULO treatments of 3 and 4 h were also conducted at 35°C against bed bug nymphs, adults, and eggs. ULO treatments with 0.1% O 2 and 3 and 4 h treatment times were conducted against all life stages. ULO treatments with 0.2 and 0.3% O 2 and 4 h treatment time were also conducted against all life stages. Each treatment was replicated five times. Controls were maintained at 35°C during treatments. After treatment, treated eggs and untreated control eggs were incubated in an environmental chamber at 25°C for at least 10 d before being examined under a magnifying glass to count the numbers of bed bug nymphs, hatched eggs, and unhatched eggs. Treated nymphs and adults were held at room temperature overnight before being scored for mortality. Dead bed bugs invariably fell to the bottom and were motionless. Bed bugs hanging on the cardboard substrates even after vigorous shaking were examined under a microscope. Vacuum Treatments Eggs, nymphs, and adults of bed bugs were also exposed to low oxygen conditions created by different levels of vacuum. The treatment apparatus consists of a vacuum chamber of 7.6 liter with ports, a vacuum test gauge (Ashcroft, Stratford, CT), a digital vacuum controller (Adsens Technology Inc., City of Industry, CA), a vacuum pump (model E2M1.5, Edwards, Wilmington, MA), treatment jars with ports, and a 562-liter (107 by 74 by 71 cm 3 ) temperature chamber modified from a large plastic container. The temperature chamber was lined inside with bubble foil insulation on all surfaces and had a ceramic electric heating panel (ECO-heater Inc., http://www.eco-heater.com ) suspended about 5 cm above the bottom with wood blocks. An air circulation fan connected to a short plastic conduit was directed at the underside of the heating plate to circulate air in the temperature chamber. An insulated platform was suspended about 5 cm above the upper surface of the heating plate. The treatment jars were placed on the platform when subjected to vacuum treatments. A digital thermostat (Hydrofarm, Petaluma, CA) was connected to the heating panel, and its temperature probe was held above the platform to control the temperature in the temperature chamber to ±1°C of a set temperature. To conduct a vacuum treatment, bed bugs in small screen-sealed vials were placed in 1.9-liter glass jars, and the jars were sealed with lids that had ports equipped with stop valves. The jars were placed on the platform in the temperature chamber and linked with tubing to the vacuum chamber. Temperature in the chamber was monitored using a Hobo temperature logger (Onset, Bourne, MA). Vacuum level was set and maintained using a vacuum controller. As all treatment jars in the temperature chamber were linked to the vacuum chamber, they all had the same vacuum level as in the vacuum chamber. The treatment jars were held under a set vacuum level at a set temperature in the temperature chamber for a specific time period to complete a treatment. They were then disconnected and removed from the temperature chamber. Bed bugs at different life stages were subjected to treatments at three vacuum levels: −800, −900, and −982 mbar, which were equivalent to 4.2, 2.1, and 0.38% O 2 , respectively, based on 1 bar normal atmospheric pressure and 20.9% oxygen in the air. Bed bug eggs were tested under −800 and −900 mbar vacuum for 48 h at 15, 20, 25, and 30°C and tested under −900 mbar vacuum for 24 h at 20, 25, and 30°C. Each treatment was replicated two to four times. Eggs, nymphs, and adults were subjected to −982 mbar vacuum treatments for 4 to 24 h at 25, 30, and 35°C. Each treatment was replicated 4 to 10 times. For all treatments, untreated bed bugs were also held at the same temperatures under the normal atmosphere as controls. Bed bug mortality was scored using the procedures described above. Data Analyses Mortality data were transformed by arcsine√x before statistical analyses. One-way analysis of variance (ANOVA) was used to compare treatments under different treatment conditions, and Tukey’s HSD multiple range tests were used to compare mean mortalities at an α value of 0.05 using Fit model of JMP statistical discovery software ( SAS Institute 2012 ). Results All life stages of bed bugs were susceptible to ULO treatment. Higher efficacy of ULO treatments correlated with lower oxygen levels, longer treatment times, and higher temperatures ( Table 1 ). Under 0.1% O 2 , 8-h treatment caused 36.1% egg mortality and mortality increased dramatically to 85.7% when treatment was extended to 12 h at 25°C. There were no significant differences among the 12-h treatment at 25°C and the 8 and 12-h treatments at 30°C. Under 0.5% O 2 , egg mortality increased significantly with treatment time and temperature increases. The 12-h treatment at 25°C only caused 50.2% mortality of eggs. However, the 18-h treatment at 25°C and 12-h treatment at 30°C caused 94.3% and 98.4% egg mortalities, respectively. Also at 30°C, an increase of treatment time from 6 to 8 h resulted in a dramatic increase of egg mortality from 29.1 to 93.3% ( Table 1 ). Table 1. Mortalities of bed bug eggs in response to low oxygen storage treatments at different temperatures Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 25 8 83 36.1 ± 6.8b df = 3, 4 F  = 38.7797 12 116 85.7 ± 2.3a P  = 0.0020 30 8 108 99.1 ± 0.9a 12 91 98.8 ± 1.3a 0.5 25 12 87 50.2 ± 1.7bc df = 6, 8 F  = 15.028 18 55 94.3 ± 0.6a P  = 0.0006 24 59 97.6 ± 2.4a 30 6 73 29.1 ± 16.6c 8 75 93.3 ± 0.0ab 12 97 98.4 ± 1.6a 18 115 98.1 ± 1.9a 1.0 20 24 173 30.7 ± 13.4b df = 2, 11 F  = 26.4999 25 24 162 97.8 ± 1.0a P  < 0.0001 30 24 157 98.9 ± 0.7a 2.0 20 48 135 79.1 ± 7.2ab df = 4, 11 F  = 6.6931 25 24 176 47.9 ± 21.2b P  = 0.0055 48 137 98.5 ± 0.7a 30 24 187 98.7 ± 0.9a 48 133 94.7 ± 3.4a 3.0 20 48 176 64.8 ± 6.7b df = 2, 7 F  = 7.7136 25 48 120 89.0 ± 6.3a P  = 0.0170 30 48 128 96.0 ± 0.3a Control 25 881 12.8 ± 2.5 Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 25 8 83 36.1 ± 6.8b df = 3, 4 F  = 38.7797 12 116 85.7 ± 2.3a P  = 0.0020 30 8 108 99.1 ± 0.9a 12 91 98.8 ± 1.3a 0.5 25 12 87 50.2 ± 1.7bc df = 6, 8 F  = 15.028 18 55 94.3 ± 0.6a P  = 0.0006 24 59 97.6 ± 2.4a 30 6 73 29.1 ± 16.6c 8 75 93.3 ± 0.0ab 12 97 98.4 ± 1.6a 18 115 98.1 ± 1.9a 1.0 20 24 173 30.7 ± 13.4b df = 2, 11 F  = 26.4999 25 24 162 97.8 ± 1.0a P  < 0.0001 30 24 157 98.9 ± 0.7a 2.0 20 48 135 79.1 ± 7.2ab df = 4, 11 F  = 6.6931 25 24 176 47.9 ± 21.2b P  = 0.0055 48 137 98.5 ± 0.7a 30 24 187 98.7 ± 0.9a 48 133 94.7 ± 3.4a 3.0 20 48 176 64.8 ± 6.7b df = 2, 7 F  = 7.7136 25 48 120 89.0 ± 6.3a P  = 0.0170 30 48 128 96.0 ± 0.3a Control 25 881 12.8 ± 2.5 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab Table 1. Mortalities of bed bug eggs in response to low oxygen storage treatments at different temperatures Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 25 8 83 36.1 ± 6.8b df = 3, 4 F  = 38.7797 12 116 85.7 ± 2.3a P  = 0.0020 30 8 108 99.1 ± 0.9a 12 91 98.8 ± 1.3a 0.5 25 12 87 50.2 ± 1.7bc df = 6, 8 F  = 15.028 18 55 94.3 ± 0.6a P  = 0.0006 24 59 97.6 ± 2.4a 30 6 73 29.1 ± 16.6c 8 75 93.3 ± 0.0ab 12 97 98.4 ± 1.6a 18 115 98.1 ± 1.9a 1.0 20 24 173 30.7 ± 13.4b df = 2, 11 F  = 26.4999 25 24 162 97.8 ± 1.0a P  < 0.0001 30 24 157 98.9 ± 0.7a 2.0 20 48 135 79.1 ± 7.2ab df = 4, 11 F  = 6.6931 25 24 176 47.9 ± 21.2b P  = 0.0055 48 137 98.5 ± 0.7a 30 24 187 98.7 ± 0.9a 48 133 94.7 ± 3.4a 3.0 20 48 176 64.8 ± 6.7b df = 2, 7 F  = 7.7136 25 48 120 89.0 ± 6.3a P  = 0.0170 30 48 128 96.0 ± 0.3a Control 25 881 12.8 ± 2.5 Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 25 8 83 36.1 ± 6.8b df = 3, 4 F  = 38.7797 12 116 85.7 ± 2.3a P  = 0.0020 30 8 108 99.1 ± 0.9a 12 91 98.8 ± 1.3a 0.5 25 12 87 50.2 ± 1.7bc df = 6, 8 F  = 15.028 18 55 94.3 ± 0.6a P  = 0.0006 24 59 97.6 ± 2.4a 30 6 73 29.1 ± 16.6c 8 75 93.3 ± 0.0ab 12 97 98.4 ± 1.6a 18 115 98.1 ± 1.9a 1.0 20 24 173 30.7 ± 13.4b df = 2, 11 F  = 26.4999 25 24 162 97.8 ± 1.0a P  < 0.0001 30 24 157 98.9 ± 0.7a 2.0 20 48 135 79.1 ± 7.2ab df = 4, 11 F  = 6.6931 25 24 176 47.9 ± 21.2b P  = 0.0055 48 137 98.5 ± 0.7a 30 24 187 98.7 ± 0.9a 48 133 94.7 ± 3.4a 3.0 20 48 176 64.8 ± 6.7b df = 2, 7 F  = 7.7136 25 48 120 89.0 ± 6.3a P  = 0.0170 30 48 128 96.0 ± 0.3a Control 25 881 12.8 ± 2.5 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab Under 1% O 2 , a temperature increase from 20 to 25°C for 24-h treatments resulted in a dramatic increase of egg mortality from 30.7 to 97.8%. There were also significant differences among treatments with different temperatures and treatment times under 2% O 2 . Over 98% mortalities were achieved in the 24-h treatment at 30°C and the 48-h treatment at 25°C ( Table 1 ). Under 3% O 2 , there were also significant differences among the three 48-h treatments at different temperatures and the highest mortality of 96.0% occurred at 30°C ( Table 1 ). For bed bug nymphs and adults, ULO treatment with 0.1% O 2 at 30°C achieved 73.3% mortality in 6 h and 100% mortality in 8 h ( Table 2 ). As oxygen level increased to 0.5 and 1% at 30°C, mortality rate declined dramatically from 65.0–76.7% range to 2.5–5.0% range regardless of treatment time. The treatments of 24 h at 25°C and 48 h at 20°C under low oxygen levels of 2 and 3%, respectively, were not effective against bed bug nymphs or adults ( Table 2 ). At 35°C, there was a significant increase in mortality from 64.0% in the 3-h treatment under 0.1% O 2 to 100% mortality in the 4-h treatment under the same low oxygen level of 0.1%. Increases of oxygen from 0.1 to 0.3% resulted in dramatic declines in mortality of nymphs and adults in the 4-h ULO treatments. There was also a dramatic increase in egg mortality from 45.8% in the 3-h treatment to 99.7% in the 4-h treatment under 0.1% O 2 . The 4-h ULO treatments with 0.2 and 0.3% O 2 had complete control of bed bug eggs ( Table 3 ). Table 2. Mortalities of bed bug nymphs and adults in response to low oxygen storage treatments at different temperatures Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 30 6 60 73.3 ± 15.8 df = 1, 12 F  = 4.7816 8 80 100 P  = 0.0493 0.5 30 6 60 65.0 ± 12.0a df = 2, 15 F  = 0.0236 12 60 66.7 ± 18.4a P  = 0.9767 24 60 76.7 ± 8.0a 1.0 30 48 60 5.0 ± 2.2 3.0 20 48 40 0 Control 25 40 0 Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 30 6 60 73.3 ± 15.8 df = 1, 12 F  = 4.7816 8 80 100 P  = 0.0493 0.5 30 6 60 65.0 ± 12.0a df = 2, 15 F  = 0.0236 12 60 66.7 ± 18.4a P  = 0.9767 24 60 76.7 ± 8.0a 1.0 30 48 60 5.0 ± 2.2 3.0 20 48 40 0 Control 25 40 0 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab Table 2. Mortalities of bed bug nymphs and adults in response to low oxygen storage treatments at different temperatures Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 30 6 60 73.3 ± 15.8 df = 1, 12 F  = 4.7816 8 80 100 P  = 0.0493 0.5 30 6 60 65.0 ± 12.0a df = 2, 15 F  = 0.0236 12 60 66.7 ± 18.4a P  = 0.9767 24 60 76.7 ± 8.0a 1.0 30 48 60 5.0 ± 2.2 3.0 20 48 40 0 Control 25 40 0 Oxygen (%) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . 0.1 30 6 60 73.3 ± 15.8 df = 1, 12 F  = 4.7816 8 80 100 P  = 0.0493 0.5 30 6 60 65.0 ± 12.0a df = 2, 15 F  = 0.0236 12 60 66.7 ± 18.4a P  = 0.9767 24 60 76.7 ± 8.0a 1.0 30 48 60 5.0 ± 2.2 3.0 20 48 40 0 Control 25 40 0 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab Table 3. Mortality of bed bugs in response to ULO treatments at 35°C Life stage . O 2 (%) . Time (h) . N . Alive . Mortality (%) (mean ± SE) . ANOVA . Nymph, adult 0.1 3 100 36 64.0 ± 6.0b df = 3, 16 F  = 20.6604 0.1 4 30 0 100a P  < 0.0001 0.2 4 50 5 90.0 ± 0.0a 0.3 4 20 15 25.0 ± 15.0c Control 50 50 0 Egg 0.1 3 529 257 45.8 ± 10.1b df = 3, 19 F  = 14.6893 0.1 4 291 1 99.7 ± 0.3a P  < 0.0001 0.2 4 75 0 100a 0.3 4 53 0 100a Control 286 270 5.3 ± 0.9 Life stage . O 2 (%) . Time (h) . N . Alive . Mortality (%) (mean ± SE) . ANOVA . Nymph, adult 0.1 3 100 36 64.0 ± 6.0b df = 3, 16 F  = 20.6604 0.1 4 30 0 100a P  < 0.0001 0.2 4 50 5 90.0 ± 0.0a 0.3 4 20 15 25.0 ± 15.0c Control 50 50 0 Egg 0.1 3 529 257 45.8 ± 10.1b df = 3, 19 F  = 14.6893 0.1 4 291 1 99.7 ± 0.3a P  < 0.0001 0.2 4 75 0 100a 0.3 4 53 0 100a Control 286 270 5.3 ± 0.9 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab Table 3. Mortality of bed bugs in response to ULO treatments at 35°C Life stage . O 2 (%) . Time (h) . N . Alive . Mortality (%) (mean ± SE) . ANOVA . Nymph, adult 0.1 3 100 36 64.0 ± 6.0b df = 3, 16 F  = 20.6604 0.1 4 30 0 100a P  < 0.0001 0.2 4 50 5 90.0 ± 0.0a 0.3 4 20 15 25.0 ± 15.0c Control 50 50 0 Egg 0.1 3 529 257 45.8 ± 10.1b df = 3, 19 F  = 14.6893 0.1 4 291 1 99.7 ± 0.3a P  < 0.0001 0.2 4 75 0 100a 0.3 4 53 0 100a Control 286 270 5.3 ± 0.9 Life stage . O 2 (%) . Time (h) . N . Alive . Mortality (%) (mean ± SE) . ANOVA . Nymph, adult 0.1 3 100 36 64.0 ± 6.0b df = 3, 16 F  = 20.6604 0.1 4 30 0 100a P  < 0.0001 0.2 4 50 5 90.0 ± 0.0a 0.3 4 20 15 25.0 ± 15.0c Control 50 50 0 Egg 0.1 3 529 257 45.8 ± 10.1b df = 3, 19 F  = 14.6893 0.1 4 291 1 99.7 ± 0.3a P  < 0.0001 0.2 4 75 0 100a 0.3 4 53 0 100a Control 286 270 5.3 ± 0.9 Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  < 0.05, SAS Institute 2012 ). Open in new tab In vacuum treatments, egg mortality increased as temperature increased from 15 to 30°C for both 24- and 48-h treatment times in the −800 and −900 mbar vacuum treatments. However, 100% mortality of eggs was not achieved in the −800 and −900 mbar vacuum treatments even in the longest treatments of 48 h at the highest temperature of 30°C ( Table 4 ). Table 4. Mortalities of bed bug eggs in response to the treatments of storing under low pressures at different temperatures Vacuum (mbar) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . −800 15 48 121 17.1 ± 0.5b df = 3, 7 F  = 26.6153 20 48 47 10.3 ± 10.3b P  = 0.0003 25 48 148 64.8 ± 3.9a 30 48 197 87.0 ± 2.8a −900 20 24 82 21.1 ± 1.1a df = 2, 3 F  = 8.1711 25 24 69 51.3 ± 22.1a P  = 0.0611 30 24 83 94.5 ± 5.5a −900 15 48 93 12.5 ± 4.7c df = 3, 5 F  = 34.9391 20 48 104 58.0 ± 8.0b P  = 0.0009 25 48 175 86.8 ± 5.4ab 30 48 93 97.8 ± 0.4a Control 25 40 0 Vacuum (mbar) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . −800 15 48 121 17.1 ± 0.5b df = 3, 7 F  = 26.6153 20 48 47 10.3 ± 10.3b P  = 0.0003 25 48 148 64.8 ± 3.9a 30 48 197 87.0 ± 2.8a −900 20 24 82 21.1 ± 1.1a df = 2, 3 F  = 8.1711 25 24 69 51.3 ± 22.1a P  = 0.0611 30 24 83 94.5 ± 5.5a −900 15 48 93 12.5 ± 4.7c df = 3, 5 F  = 34.9391 20 48 104 58.0 ± 8.0b P  = 0.0009 25 48 175 86.8 ± 5.4ab 30 48 93 97.8 ± 0.4a Control 25 40 0 Oxygen levels in the treatment chambers were calculated to be 4.18 and 2.09% for −800 and −900 mbar vacuum, respectively. Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  ≤ 0.05, SAS Institute 2012 ). Open in new tab Table 4. Mortalities of bed bug eggs in response to the treatments of storing under low pressures at different temperatures Vacuum (mbar) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . −800 15 48 121 17.1 ± 0.5b df = 3, 7 F  = 26.6153 20 48 47 10.3 ± 10.3b P  = 0.0003 25 48 148 64.8 ± 3.9a 30 48 197 87.0 ± 2.8a −900 20 24 82 21.1 ± 1.1a df = 2, 3 F  = 8.1711 25 24 69 51.3 ± 22.1a P  = 0.0611 30 24 83 94.5 ± 5.5a −900 15 48 93 12.5 ± 4.7c df = 3, 5 F  = 34.9391 20 48 104 58.0 ± 8.0b P  = 0.0009 25 48 175 86.8 ± 5.4ab 30 48 93 97.8 ± 0.4a Control 25 40 0 Vacuum (mbar) . Temp (°C) . Time (h) . N . Mortality (%) (mean ± SE) . ANOVA . −800 15 48 121 17.1 ± 0.5b df = 3, 7 F  = 26.6153 20 48 47 10.3 ± 10.3b P  = 0.0003 25 48 148 64.8 ± 3.9a 30 48 197 87.0 ± 2.8a −900 20 24 82 21.1 ± 1.1a df = 2, 3 F  = 8.1711 25 24 69 51.3 ± 22.1a P  = 0.0611 30 24 83 94.5 ± 5.5a −900 15 48 93 12.5 ± 4.7c df = 3, 5 F  = 34.9391 20 48 104 58.0 ± 8.0b P  = 0.0009 25 48 175 86.8 ± 5.4ab 30 48 93 97.8 ± 0.4a Control 25 40 0 Oxygen levels in the treatment chambers were calculated to be 4.18 and 2.09% for −800 and −900 mbar vacuum, respectively. Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  ≤ 0.05, SAS Institute 2012 ). Open in new tab In the −982 mbar vacuum treatments of all life stages of bed bugs, the 24-h treatment at 25°C achieved complete control of nymphs and had over 97% mortalities of adults and eggs ( Table 5 ). At 30°C, complete control was achieved in 8 h for nymphs, in 10 h for adults, and in 12 h for eggs. At 35°C, the 4-h treatment of −982 mbar vacuum resulted in 94% mortality for nymphs and adults and 75.2% mortality for eggs. The complete control of eggs was achieved in 6- and 8-h treatments ( Table 5 ). Table 5. Mortalities of different life stages of bed bugs in response to −982 mbar (−29.0 inHg) vacuum treatments of 4–24 h at different temperatures Temp (°C) . Time (h) . Life stage . N . Mortality (%) b (mean ± SE) . ANOVA . 25 24 Nymph 40 100 df = 2, 9 F  = 0.7150 Adult 40 97.5 ± 2.5 P  = 0.5150 Egg 319 98.8 ± 0.8 30 6 Nymph 80 98.8 ± 1.3a df = 2, 15 F  = 22.2076 Adult 80 95.0 ± 1.9a P  = < 0.0001 Egg 32 48.2 ± 9.7b 8 Nymph 150 100a df = 2, 25 F  = 6.2349 Adult 90 96.7 ± 1.7ab P  = 0.0064 Egg 33 86.1 ± 8.1b 10 Nymph 50 100 df = 2, 21 F  = 1.000 Adult 110 100 P  = 0.3847 Egg 156 99.3 ± 0.7 12 Egg 249 100a 35 4 Nymph 50 94.0 ± 6.0 df = 2, 9 F  = 2.4493 Adult 50 94.0 ± 4.0 P  = 0.1415 Egg 161 75.0 ± 3.0 6 Egg 196 100 8 Egg 179 100 Temp (°C) . Time (h) . Life stage . N . Mortality (%) b (mean ± SE) . ANOVA . 25 24 Nymph 40 100 df = 2, 9 F  = 0.7150 Adult 40 97.5 ± 2.5 P  = 0.5150 Egg 319 98.8 ± 0.8 30 6 Nymph 80 98.8 ± 1.3a df = 2, 15 F  = 22.2076 Adult 80 95.0 ± 1.9a P  = < 0.0001 Egg 32 48.2 ± 9.7b 8 Nymph 150 100a df = 2, 25 F  = 6.2349 Adult 90 96.7 ± 1.7ab P  = 0.0064 Egg 33 86.1 ± 8.1b 10 Nymph 50 100 df = 2, 21 F  = 1.000 Adult 110 100 P  = 0.3847 Egg 156 99.3 ± 0.7 12 Egg 249 100a 35 4 Nymph 50 94.0 ± 6.0 df = 2, 9 F  = 2.4493 Adult 50 94.0 ± 4.0 P  = 0.1415 Egg 161 75.0 ± 3.0 6 Egg 196 100 8 Egg 179 100 The oxygen level was calculated to be 0.38% in all of the treatments based on the −982 mbar vacuum level in all treatment chambers. Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  ≤ 0.05, SAS Institute 2012 ). Controls for eggs, nymphs, and adults had no mortality and were not listed. Open in new tab Table 5. Mortalities of different life stages of bed bugs in response to −982 mbar (−29.0 inHg) vacuum treatments of 4–24 h at different temperatures Temp (°C) . Time (h) . Life stage . N . Mortality (%) b (mean ± SE) . ANOVA . 25 24 Nymph 40 100 df = 2, 9 F  = 0.7150 Adult 40 97.5 ± 2.5 P  = 0.5150 Egg 319 98.8 ± 0.8 30 6 Nymph 80 98.8 ± 1.3a df = 2, 15 F  = 22.2076 Adult 80 95.0 ± 1.9a P  = < 0.0001 Egg 32 48.2 ± 9.7b 8 Nymph 150 100a df = 2, 25 F  = 6.2349 Adult 90 96.7 ± 1.7ab P  = 0.0064 Egg 33 86.1 ± 8.1b 10 Nymph 50 100 df = 2, 21 F  = 1.000 Adult 110 100 P  = 0.3847 Egg 156 99.3 ± 0.7 12 Egg 249 100a 35 4 Nymph 50 94.0 ± 6.0 df = 2, 9 F  = 2.4493 Adult 50 94.0 ± 4.0 P  = 0.1415 Egg 161 75.0 ± 3.0 6 Egg 196 100 8 Egg 179 100 Temp (°C) . Time (h) . Life stage . N . Mortality (%) b (mean ± SE) . ANOVA . 25 24 Nymph 40 100 df = 2, 9 F  = 0.7150 Adult 40 97.5 ± 2.5 P  = 0.5150 Egg 319 98.8 ± 0.8 30 6 Nymph 80 98.8 ± 1.3a df = 2, 15 F  = 22.2076 Adult 80 95.0 ± 1.9a P  = < 0.0001 Egg 32 48.2 ± 9.7b 8 Nymph 150 100a df = 2, 25 F  = 6.2349 Adult 90 96.7 ± 1.7ab P  = 0.0064 Egg 33 86.1 ± 8.1b 10 Nymph 50 100 df = 2, 21 F  = 1.000 Adult 110 100 P  = 0.3847 Egg 156 99.3 ± 0.7 12 Egg 249 100a 35 4 Nymph 50 94.0 ± 6.0 df = 2, 9 F  = 2.4493 Adult 50 94.0 ± 4.0 P  = 0.1415 Egg 161 75.0 ± 3.0 6 Egg 196 100 8 Egg 179 100 The oxygen level was calculated to be 0.38% in all of the treatments based on the −982 mbar vacuum level in all treatment chambers. Mortality data were transformed by arcsin√x before analyses of variance. Mortalities within each group followed by the different letters were significantly different based on Tukey’s HSD multiple range test ( P  ≤ 0.05, SAS Institute 2012 ). Controls for eggs, nymphs, and adults had no mortality and were not listed. Open in new tab Discussion ULO and vacuum treatments were effective against all life stages of bed bugs. Based on mortality data, an ULO treatment with ≤0.5% O 2 either established through nitrogen gas flushing or vacuum will likely achieve complete or close to complete control of bed bug eggs, nymphs, and adults in 12 h at ≥30°C. The treatment can be completed overnight in commercial applications and has potential to serve as an integral part of bed bug management for decontamination of removable objects infested by bed bugs. Mortality data from the ULO treatments established by nitrogen flushing indicated that eggs were more susceptible to ULO treatments than nymphs and adults. At the lower oxygen levels of 0.1 to 0.5%, treatments of same duration at 30°C resulted in higher egg mortalities than those for nymphs and adults ( Tables 1 and 2 ). Especially, the 24- and 48-h treatments with respective higher oxygen levels of 1% at 30°C and 3% at 20°C resulted in egg mortalities of 98.9 and 64.8% as compared with 5% and zero mortality for nymphs and adults, respectively ( Tables 1 and 2 ). At 35°C and under very low oxygen levels of 0.1 to 0.3%, however, the difference between eggs and nymphs or adults was less obvious because most treatments had mortality rates at 100% or close to 100% and a few treatments had low sample numbers. Compared with previous CA treatments for bed bug control, ULO treatment and vacuum treatment of the current study were at least as effective as the CO 2 treatment ( Wang et al. 2012 ). Insects vary greatly in tolerance to oxygen deficiency ( Hoback and Stanley 2001 , Mitcham et al. 2001 , Liu 2010 ). In general, insect eggs are more tolerant of low oxygen conditions as compared with mobile life stages with a few exceptions. Eggs of the grape mealybug are significantly more tolerant to low oxygen than nymphs and adults ( Liu 2013a ). Storage of rice weevil ( Sitophilus oryzae (L.)) eggs in ≤0.005% O 2 atmosphere at 25°C for 48 h only resulted in 5.2% mortality ( Liu 2013b ). A 24-h storage treatment of Indian meal moth ( Plodia interpunctella (Hübner)) eggs in atmosphere with about 0.01% O 2 at 20°C resulted in a mortality of <80% (Liu unpublished). Treatments of larvae of six museum dermestids insect pests (Coleoptera) under ULO condition of 0.3% O 2 at 25°C resulted in LT 99 values of 32.2 to 88.1 h ( Bergh et al. 2003 ). A ULO atmosphere with 0.4% O 2 had complete controls of adults, eggs, larvae, and pupae of a powderpost beetle ( Lyctus brunneus (Stephens)), in 3, 6, 8, and 12 d, respectively, at 30°C ( Gilberg and Roach 1993 ). ULO atmospheres with <0.1% O 2 take 2 to 8 d at 25.5°C to control eggs of 10 museum insect pests ( Rust et al. 1996 ). In comparisons of these insects’ responses with ULO conditions, bed bug is highly susceptible to low oxygen stress especially at egg stage. The long ULO treatment times for fresh product insects at low temperatures ( Mitcham et al. 2001 , Liu 2010 ), however, could not be compared directly with ULO treatment for bed bugs in the current study because of the large differences in temperature. ULO treatments established either by nitrogen gas flushing or vacuum are environmentally friendly as compared with many other treatments. In addition to being toxic, sulfuryl fluoride fumigation for bed bug control takes longer (24 h) in structure or container fumigations ( Phillips et al. 2014 ) as compared with the 12-h ULO or vacuum treatment in the current study. Heat treatment, although effective eliminating bed bugs from infested rooms, may damage or degrade sensitive products such as electronic products and can be a fire hazard. The ability to control all life stages of bed bugs in 12 h at 30°C as in the current study makes the treatment convenient and practical to be used commercially, as it can be completed in the same day or during the night. The temperature of 30°C can be easily maintained in structures where treatments will be conducted or in treatment chambers. For ULO treatments using nitrogen flushing, a nitrogen source, an oxygen analyzer, and an airtight enclosure will be needed (Liu and Haynes unpublished). The enclosure can be a rigid chamber or a flexible bag. The treatment can be designed to maintain a desired ULO automatically. There are different nitrogen sources for bed bug ULO treatments depending on treatment scales and whether treatments will be conducted at fixed facilities or clients’ locations. Using compressed nitrogen gas cylinders as nitrogen source is likely the most expensive but more convenient for smaller scale treatments. Liquid nitrogen will likely be a more economical source for larger scale ULO treatments. For fixed treatment facilities, using a nitrogen generator is likely to be an ideal choice in comparison with compressed nitrogen cylinders or liquid nitrogen. Mobile nitrogen generators are also available commercially and can be adopted for commercial scale ULO treatments at clients’ locations. The vacuum treatment can be used economically to disinfest small objects such as clothing, beddings, towels, and suitcases in metal drums available on the market. The treatment has potential to be used by hotels, families, and pest control operators. Large vacuum chambers will likely be too expensive for practical use. Vacuum treatment can be maintained automatically using a vacuum switch. Most products can be treated safely under vacuum except items with airtight-sealed empty spaces such as light bulbs, which could burst under vacuum. 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TI - Effects of Ultralow Oxygen and Vacuum Treatments on Bed Bug (Heteroptera: Cimicidae) Survival
JF - Journal of Economic Entomology
DO - 10.1093/jee/tow034
DA - 2016-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/effects-of-ultralow-oxygen-and-vacuum-treatments-on-bed-bug-aHpMw84gGI
SP - 1310
EP - 1316
VL - 109
IS - 3
DP - DeepDyve
ER -