https://orcid.org/0000-0003-1979-5985
Abstract
Steam application is an effective and environmentally friendly method for controlling bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae). While a few studies documented the bed bug control efficacy of steam treatment, the sublethal effect of steam treatment on bed bug behavior and female fecundity is unknown. In this study, we evaluated the effect of steam treatment on the movement, feeding, mating behavior, and fecundity of female bed bugs in the laboratory. Bed bug adults received a calibrated steam treatment that caused approximately 28% mortality. The surviving bed bugs were observed for their feeding and mating behavior at 1 d posttreatment, female fecundity during a 7-d observation period, and offspring hatching. Steam-treated bed bugs were less active based on the percentage of bed bugs with movement, moving distance during a 10-min observation period, and feeding rate. However, steam treatment had no significant effect on blood intake (amount of blood taken per meal) among the fed bed bugs. After blood feeding, the steam-treated bed bugs had similar mating events and egg production as the control bed bugs. Furthermore, parental steam exposure had no significant effect on the offspring hatching. In conclusion, steam treatment could temporarily decrease bed bug activity levels and feeding rate, but had no significant impact on bed bug mating behavior and female fecundity.
Bed bugs (Cimex lectularius L. and Cimex hemipterus (F.)) are obligate blood-sucking insects that feed upon the blood of human and other animals (Usinger 1966). In recent years, these pests have undergone a major resurgence and spread in communities around the world (Potter et al. 2010, Eddy and Jones 2011, Doggett et al. 2012); and they have been considered as one of the most difficult urban pests to control (Romero et al. 2007, Zhu et al. 2010, Doggett et al. 2012). A large number of methods for controlling bed bugs have been developed, with the increase of public concern over this pest’s resurgence (Doggett et al. 2012, Koganemaru and Miller 2013). Nonchemical treatments are considered an important component in bed bug management practices and are highly effective and advantageous over the traditional chemical treatment through avoiding the potential health risks from insecticide applications.
Bed bugs are sensitive to high temperatures; thus, heat treatment can be used as one of the nonchemical control methods of bed bug control (Doggett et al. 2012). Pereira et al. (2009) reported that mortality of bed bug adults started at 41°C with an exposure time of 100 min, and this time gradually decreased as temperature increased, to about 1 min at 49°C. Benoit et al. (2009) studied the bed bug response to heat stress, and found that nearly none of the bed bugs survived a 1-h exposure to 48°C. Benoit (2011) reported that the upper thermal limit for short-term exposure of bed bug eggs, nymphs, and adults was between 40 and 45°C. DeVries et al. (2016) reported that the critical thermal maximum of bed bugs was related to their feeding status, bed bugs starved 9 d having the greatest thermal tolerance, followed by bed bugs starved 1 d, and finally bed bugs starved 21 d.
The use of steam is another effective nonchemical control method, which capitalizes on the delivery of lethal temperatures for bed bug treatment (Potter et al. 2008; Wang et al. 2009, 2013; Doggett 2013). In laboratory studies, steam treatment at the rate of 10 s/30.50 cm can rise the temperature of the treatment arena surface to 73.91°C, and cause a >80% mortality rate of bed bug nymphs and males, and a 100% mortality rate for eggs (Puckett et al. 2013). Our previous study has demonstrated that steamers with different grades (two affordable consumer-grade commercial steamers and one commonly used professional-grade steamer) achieved similar bed bug control efficacy (Wang et al. 2018b).
The boiling point of water at 1 atm of pressure (sea level) is 100°C, which is much higher than the maximum lethal temperature of bed bugs (52°C) (Kells 2006, Pinto et al. 2007, Kells and Goblirsch 2011). In theory, bed bugs would be killed when exposed to steam directly. However, steam treatment sometimes does not kill all bed bugs (Puckett et al. 2013, Wang et al. 2018b). Reasons for control failure include: 1) during steam treatment period, bed bugs (nymphs and adults) move away from the treated area or drop to the floor immediately after being exposed to a sublethal or nonlethal temperature; 2) the steamer head is moved too quickly to induce the minimum effective temperature on the treated surface; 3) bed bugs underneath covers (such as bed sheet, sofa fabric, or sofa leather) or in cracks are not exposed to lethal temperatures from the steam treatment. Generally, only mortality was considered when evaluating the effect of the steam treatment on bed bugs. Whether there is a sublethal effect to bed bugs that survived steam exposure is not clear. The sublethal effect of steam treatment may encompass bed bug physiology, reproduction, and behavior, which all can be important factors when assessing the effect of steam treatment for protecting humans from bed bugs.
Thermal stress can result in a physiological effect on the insects (Silbermann and Tatar 2000, Cui et al. 2008, Xie et al. 2008, Roux et al. 2010, Wang et al. 2014), but also may modify their behavior (Liao et al. 2014; Bodlah et al. 2016, 2017). Rukke et al. (2015) demonstrated that prolonged exposure to high temperature could shorten the longevity and decrease the fecundity of adult bed bugs, as well as lower their offspring’s feeding and molting ability. However, no data are available on the effect of instant high temperature from steam treatment on the physiology and behavior of bed bugs.
The objective of this study was to evaluate the short-term effect of steam treatment on the movement, feeding, mating behavior, and fecundity of C. lectularius. The results may have important implications on the use of steam treatment for bed bug management.
Materials and Methods
Insects
Cimex lectularius bed bugs were collected in 2012 from multiple infested apartments in Irvington, NJ. They were maintained in plastic containers (5 cm diameter and 4.7 cm height; Consolidated Plastics, Stow, OH) with folded red construction paper (4 cm length and 3 cm width) as harborages, and held at 26 ± 1°C, 40 ± 10% RH, with a photoperiod of 12:12 (L:D) h. Bed bugs were fed weekly on defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA) using a Hemotek membrane-feeding system (Discovery Workshops, Accrington, United Kingdom).
Only virgin adults with a similar age (<7 d post adult emergence) were used in our study. To obtain the virgin adults, fifth instar nymphs were individually placed into plastic Petri dishes (3.5 cm diameter and 1 cm height; Fisher Scientific, Pittston, PA; one nymph per dish) after feeding. Each dish contained a small piece of white paper (2 cm length and 2 cm width). After emergence, males and females were sexed and held in the male or female group.
All experiments were conducted in the laboratory at 24–26°C. In Experiment I and III, bed bug behaviors were recorded in a walk-in chamber illuminated with two 25-W red bulbs.
Steam Treatment
ClimbUp HD Black insect interceptors (10 cm diameter and 2.2 cm height; Susan McKnight, Inc., Memphis, TN) were used as experimental arenas. One piece of filter paper (5.5 cm diameter) was glued onto the bottom of the interceptor; the inner wall of the interceptor was coated with a thin film of fluoropolymer resin (BioQuip Products, Rancho Dominguez, CA) to prevent bed bugs from escaping. A Steamax Commercial Steam Cleaner (Model: STM-BASIC; AmeriVap Systems, Inc., Dawsonville, GA) was used to treat the bed bugs. During treatment, the steamer working pressure was set at ‘medium’ (379 kPa) and the steamer attachment was positioned 15 cm above the experimental arena. The maximum temperature of the steam where it comes out of the device and where it reaches the bed bugs was measured using a thermocouple thermometer (Cole-Parmer Instrument Company, Vernon Hills, IL), and the temperature was 85.6 and 51.7°C, respectively. The interceptors with bed bugs were moved at a speed of approximately 3 cm/s through the steam treatment area (10.5 cm length and 10.5 cm width) by estimation. The moving speed and treatment area were measured in our preliminary experiment, and this steam treatment caused approximately 28% mortality for both male and female bed bugs in preliminary experiments. During the following experiment, the same steam treatment method was selected.
For evaluating the effect of steam treatment on bed bugs, 36 or 37 bed bugs were introduced into each interceptor 10 min prior to the experiment. Males and females were treated separately. In order to make bed bugs expose to steam evenly, bed bugs were restricted to staying in one layer in the interceptor during steam treatment. Each treatment (control or steam treatment) was replicated twice. The total number of bed bugs used for control males, control females, treated males, and treated females were 74, 74, 73, and 74, respectively. After steam treatment, bed bugs of each group (control males, control females, treated males, or treated females) were housed separately in clean Petri dishes (6 cm diameter and 1.5 cm height) and were held in an incubator at 26 ± 1°C. The bottom of each dish was covered with a piece of filter paper (3.5 cm diameter). Interceptors with bed bugs in the control did not receive a steam treatment. One hour after treatment, the survival rates of control males, control females, treated males, and treated females were 100, 100, 72.6, and 71.6%, respectively; and there was no additional mortality at 1, 3, and 7 d after treatment. The surviving bugs were used in the following experiments.
Experiment I: Effect of Steam Treatment on Bed Bug Movement
The movement of surviving bed bugs at 1 d posttreatment was measured using EthoVision XT (Version 10.1, Noldus Information Technology, Wageningen, The Netherlands). Uncapped Petri dishes (3.5 cm diameter and 1 cm internal height) were used as experimental arenas. In each dish, clean filter paper (3.5 cm diameter) was placed at the bottom to facilitate bed bug movement; the inner wall was coated with a thin film of fluoropolymer resin to prevent bed bugs from escaping, bed bugs’ movement were recorded for 10 min. During the recording period, CO2 was added into the chamber from a 5-lb cylinder (Airgas East Inc., Piscataway, NJ) at 200 ml/min, to help stimulate movement and host-seeking behavior. The CO2 release site was ~40 cm above the experimental arenas. ‘Static subtraction’ was selected as the detection method for tracking bed bugs. The sample rate (rate at which EthoVision analyzes images to find the subject) was 25 samples per second. The ‘distance moved’ was used as dependent variable. For each treatment (control males, control females, treated males, or treated females), 45 replications were conducted. Once the recording was finished, all bed bugs were used in Experiment II.
Experiment II: Effect of Steam Treatment on Bed Bug Feeding
Once Experiment I was completed, 45 bed bugs in each treatment (control males, control females, treated males, or treated females) were divided into three groups, each group contained 15 bed bugs of the same sex. The weight of each group of 15 bed bugs was measured using an electronic balance (Model: PB153-S/FACT; Mettler Toledo, Zurich, Switzerland). After weighing, each group of bed bugs was introduced into a container with a folded red paper (4 cm length and 3 cm width) as harborage, and was fed for 30 min with defibrinated rabbit blood using the Hemotek membrane feeding system. The weight of each bed bug group was measured again immediately after feeding and the number of fed bed bugs (partially engorged or fully engorged) in each group was recorded. In each group, the percentage of bed bug feeding was the number of fed bed bugs divide by 15. The blood intake in each group of bed bugs was based on weight gain of 15 bed bugs divided by the number of fed bed bugs in the group. In each treatment, the fed bed bugs were used in Experiment III.
Experiment III: Effect of Steam Treatment on Bed Bug Mating Behavior and Fecundity
After feeding, pairs of fed bed bugs were placed in plastic Petri dishes (3.5 cm diameter and 1 cm internal height), each pair represented a replication; and 13 replications were conducted for each treatment (control or treated bed bugs). In each dish, a clean filter paper (3.5 cm diameter) was placed at the bottom to facilitate bed bug movement; the inner wall was coated with a thin film of fluoropolymer resin to prevent bed bug from escaping.
A female was introduced first and a male was introduced immediately afterwards. Their behavior was video recorded with a digital camera (Model: SONY HDR-XR 550V; Sony Corporation, Japan). The recording started as soon as the male was introduced into the Petri dish and ended after 10 min. Copulation rate (the number of pairs able to copulate divided by the total number of pairs used), courtship latency (defined as the time until the start of the characteristic male mounting display), and total duration of copulation were recorded.
After finishing recording the mating behavior, the female and male bed bugs were separated. The females were transferred into individual clean plastic Petri dishes (3.5 cm diameter and 1 cm internal height; one female per dish). Each Petri dish contained a folded red paper (3 cm length and 2 cm width) as an oviposition substrate. The total number of eggs laid per female bed bug was recorded after 7 d and this number was used for comparing female fecundity. There was no mortality of females during the 7-d observation period. The eggs were incubated at 26 ± 1°C, 40 ± 10% RH, with a photoperiod of 12:12 (L:D) h. The hatching rate of F1 offspring was recorded at 14 d.
Statistical Analysis
Chi-square test was used to compare the difference between control and treated bed bugs in the movement rate (bed bugs with a moving distance more than 0 divided the total number of bed bugs in the test) in Experiment I, and the difference between control and treated males or females in the copulation rate in Experiment III. In Experiment I, for the bed bugs with movement, two-sample t-test was used to compare the difference between control and treated bed bugs in their moving distance. In addition, the differences between control and treated males or females in the measured variables (percentage of bed bug feeding, blood intake of each fed bed bug, courtship latency, total duration of copulation, female fecundity during the 7-d period, and hatching rate of F1 offspring) were also compared using two-sample t-test. The percentage data were arcsine square root transformed prior to the analyses, in order to satisfy the assumptions of normality. All data analyses were carried out using SPSS version 22.0 (International Business Machines Corp., Armonk, NY) (IBM Corporation 2013).
Results and Discussion
Effect of Steam Treatment on Bed Bug Movement
Significantly less male bed bugs in the steam-treated group moved (moving distance was > 0) than the control males (χ2 = 68.8, df = 1, P < 0.001; Fig. 1A); however, no significant difference was observed in the number of moved female bed bugs (moving distance was > 0) between control and treated groups (χ2 = 3.1, df = 1, P = 0.078; Fig. 1B). Furthermore, significantly shorter distances were moved by the steam-treated bed bugs, compared with the controls (males: t = 4.5, df = 82, P < 0.001; females: t = 4.6, df = 85, P < 0.001; Fig. 2). This reduction in movement may cause a decrease in bed bug host-finding success (Crawley et al. 2017a). In locust, heat shock can cause a failure of neurons to propagate action potentials, as well as an inability of the muscle tissue itself to contract (Barclay and Robertson 2000, Rodgers et al. 2010). However, whether the reduced movement in bed bugs was due to the effect of steam treatment on their functional neuromuscular connections should be further studied.
Percentage of bed bugs with movement after steam treatment. Bed bug with a moving distance more than 0 during 10-min period was considered as the one with movement. (A) Nontreated (control) and steam-treated males. (B) Nontreated (control) and steam-treated females. Forty-five replications were conducted for each treatment. Chi-square test was used for comparing the difference in the movement rate between control and steam-treated bed bugs (***P < 0.001; nsP > 0.05).
Mean (±SEM) distance moved by bed bugs after steam treatment during 10-min period. (A) Nontreated (control) and steam-treated males. (B) Nontreated (control) and steam-treated females. Fourty-five replications were conducted for each treatment. Two-sample t-test was used for comparing the difference in the moving distance between control and steam-treated bed bugs (***P < 0.001).
Effect of Steam Treatment on Bed Bug Feeding
Bed bugs exposed to steam showed a significant decrease in their feeding rate than the controls (males: t = 8.0, df = 4, P = 0.001; females: t = 8.3, df = 4, P = 0.001; Fig. 3). This reduced feeding rate was likely due to their decreased movement. Among those fed bed bugs, there was no significant difference in the blood intake between the treated and nontreated bed bugs (males: t = 0.7, df = 4, P = 0.510; females: t = 2.7, df = 4, P = 0.055). The blood intake by control and treated males were 4.7 ± 0.1 and 4.5 ± 0.4 mg per male, respectively. The blood intake by control and treated females were 8.4 ± 0.1 and 7.2 ± 0.4 mg per female, respectively.
Mean (±SEM) feeding rate of bed bugs after steam treatment. (A) Nontreated (control) and steam-treated males. (B) Nontreated (control) and steam-treated females. Fifteen bed bugs were used in each treatment group, and each treatment was replicated three times. Two-sample t-test was used for comparing the difference in the feeding rate between control and steam-treated bed bugs (**P < 0.01).
Finding a host and feeding allows bed bug to develop from juvenile to adult, and allows adults to produce sperms and eggs (Reinhardt and Siva-Jothy 2007, Crawley et al. 2017a). In addition, blood feeding can help bed bug females increase their copulation success. Females exhibit decreased resistance to mating after feeding (Siva-Jothy 2006, Reinhardt et al. 2009, Wang et al. 2016). Thus, steam treatment could slow bed bug population growth by affecting their feeding rate. However, in our study, the bed bug feeding assay was only replicated for three times. In fact, more replicates should be conducted to evaluate the difference in bed bug blood intake.
Effect of Steam Treatment on Bed Bug Mating Behavior and Fecundity
There was no significant difference between control and steam-treated bed bugs in the copulation rate, male courtship latency, copulation frequency, or total copulation duration, during the 10-min observation period (Table 1). Sublethal effects of insecticides on insects have been demonstrated. This include increased activity (Romero et al. 2009), reduced mating success (Linn and Roelofs 1984, Clark and Haynes 1992, Crawley et al. 2017b, Wang et al. 2018a), altered mating behavior (Gu et al. 1995, Wang et al. 2018a), and disturbed sex pheromone communication (Dupont et al. 2010, Delpuech et al. 2012, Wang et al. 2018a). These sublethal effects are likely due to the influence of insecticide on the normal function of the insect nervous system (Wang et al. 2018a). In the current study, there was no significant effect of steam treatment on the mating success (copulation rate) and mating behavior (i.e., courtship latency and total copulation duration). It may be associated with the steam’s mechanism which affects the somatic function of insects, rather than the insect nervous systems.
Mating behavior and fertilization capacity of steam-treated bed bugs during a 10-min period
| Copulation rate (%) | Courtship latency (s) | Copulation frequency during the 10-min period | Total copulation duration (s) | Fecundity (no. eggs laid/adult female) | Hatch rate of F1 offspring | |
|---|---|---|---|---|---|---|
| Control | 100 | 25.2 ± 6.0 | 1.46 ± 0.18 | 58.2 ± 6.6 | 8.5 ± 0.8 | 90.4 ± 7.6 |
| Treated | 92.3 | 38.8 ± 12.2 | 1.50 ± 0.15 | 76.3 ± 13.7 | 6.8 ± 1.3 | 100 |
| Statistics | χ2 = 1.04 | t = −1.0 | t = −0.2 | t = −1.2 | t = 1.1 | t = −1.4 |
| df = 1 | df = 23 | df = 23 | df = 23 | df = 17.9 | df = 12 | |
| P = 0.308 | P = 0.317 | P = 0.874 | P = 0.231 | P = 0.295 | P = 0.173 |
| Copulation rate (%) | Courtship latency (s) | Copulation frequency during the 10-min period | Total copulation duration (s) | Fecundity (no. eggs laid/adult female) | Hatch rate of F1 offspring | |
|---|---|---|---|---|---|---|
| Control | 100 | 25.2 ± 6.0 | 1.46 ± 0.18 | 58.2 ± 6.6 | 8.5 ± 0.8 | 90.4 ± 7.6 |
| Treated | 92.3 | 38.8 ± 12.2 | 1.50 ± 0.15 | 76.3 ± 13.7 | 6.8 ± 1.3 | 100 |
| Statistics | χ2 = 1.04 | t = −1.0 | t = −0.2 | t = −1.2 | t = 1.1 | t = −1.4 |
| df = 1 | df = 23 | df = 23 | df = 23 | df = 17.9 | df = 12 | |
| P = 0.308 | P = 0.317 | P = 0.874 | P = 0.231 | P = 0.295 | P = 0.173 |
Mating behavior and fertilization capacity of steam-treated bed bugs during a 10-min period
| Copulation rate (%) | Courtship latency (s) | Copulation frequency during the 10-min period | Total copulation duration (s) | Fecundity (no. eggs laid/adult female) | Hatch rate of F1 offspring | |
|---|---|---|---|---|---|---|
| Control | 100 | 25.2 ± 6.0 | 1.46 ± 0.18 | 58.2 ± 6.6 | 8.5 ± 0.8 | 90.4 ± 7.6 |
| Treated | 92.3 | 38.8 ± 12.2 | 1.50 ± 0.15 | 76.3 ± 13.7 | 6.8 ± 1.3 | 100 |
| Statistics | χ2 = 1.04 | t = −1.0 | t = −0.2 | t = −1.2 | t = 1.1 | t = −1.4 |
| df = 1 | df = 23 | df = 23 | df = 23 | df = 17.9 | df = 12 | |
| P = 0.308 | P = 0.317 | P = 0.874 | P = 0.231 | P = 0.295 | P = 0.173 |
| Copulation rate (%) | Courtship latency (s) | Copulation frequency during the 10-min period | Total copulation duration (s) | Fecundity (no. eggs laid/adult female) | Hatch rate of F1 offspring | |
|---|---|---|---|---|---|---|
| Control | 100 | 25.2 ± 6.0 | 1.46 ± 0.18 | 58.2 ± 6.6 | 8.5 ± 0.8 | 90.4 ± 7.6 |
| Treated | 92.3 | 38.8 ± 12.2 | 1.50 ± 0.15 | 76.3 ± 13.7 | 6.8 ± 1.3 | 100 |
| Statistics | χ2 = 1.04 | t = −1.0 | t = −0.2 | t = −1.2 | t = 1.1 | t = −1.4 |
| df = 1 | df = 23 | df = 23 | df = 23 | df = 17.9 | df = 12 | |
| P = 0.308 | P = 0.317 | P = 0.874 | P = 0.231 | P = 0.295 | P = 0.173 |
We also found steam treatment had no significant effect on female bed bugs’ fecundity, or their F1 offspring’s hatching rate (Table 1). Previous studies reported that prolonged exposure to high temperature induced significant decrease in the reproductive rate of bed bugs (Chang 1974, 1975). Female fecundity was restored after high temperature exposure, and the time until fertility rebound was associated with the effective temperature summation experienced during high temperature exposure (Rukke et al. 2015). The lack of effect of steam treatment on female bed bugs’ fecundity and their progeny’s hatching rate might be due to the short exposure duration (only 3–4 s).
Similar to other insects (such as aphids, ants, weevils, and cockroaches), bed bugs also harbor symbiotic Wolbachia. These symbiotic bacteria have been demonstrated that were essential for bed bugs’ growth and reproduction through supplying of B vitamins (De Meillon and Golberg 1947, De Meillon et al. 1947, Moran et al. 2008, Hosokawa et al. 2010, Nikoh et al. 2014). The balance of such associations between insects and bacteria may be disrupted once the host is exposed to heat stress (Jia et al. 2009, Wernegreen 2012, Li et al. 2014). Thus, heat can be used as one of the methods to deliberately eliminate Wolbachia from the host. The loss of Wolbachia reduces bed bug viability due to the loss of nutrients. And the rebounding of bed bug fertility after prolonged high temperature exposure might due to the recovery of the microbial balance in the bacteriomes (Rukke et al. 2015). Studying changes in the microbial balance in bacteriomes after steam exposure would be beneficial to fully understand the sublethal effect of steam treatment on bed bugs.
Our study design used bed bugs that were treated in groups. During steam treatment, bed bugs stayed one layer in the interceptor. Due to gregarious behavior of bed bugs, the survived bed bugs might be due to uneven temperature exposed during treatment rather than physiological differences. A more precise measurement of the treatment would be treating individual bed bugs, so each individual receives more even temperature exposure. Nevertheless, our experiment design reflects what would happen in field bed bug treatment and thus the results are valuable for pest management professionals when choosing steam treatment.
Only adult bed bugs were evaluated for the sublethal effect of steam treatment in this study. Under field conditions, the majority of bed bugs would be in the nymphal or egg stage (Wang et al. 2010). Additional research on the sublethal effect to these stages would be beneficial to further understanding of the effect of steam treatment. The current results suggest that to achieve higher bed bug control efficacy, those bed bugs surviving steam exposure will continue to bite the human host and the infestation should be treated with steam again or in combination with other chemical or nonchemical methods to ensure bed bug elimination. Our study only determined the effect of steam treatment on bed bug movement, feeding and mating behavior on 1 and 2 d after steam exposure. In order to fully understand the effect of steam exposure on bed bugs, the parameters (such as the movement, feeding, mating behavior, and fecundity) should be measured for a longer time period (for instance, 1 or 2 wk following steam treatment).
Acknowledgments
We thank Qi Zhang for help in rearing bed bugs. This study was supported by the USDA National Institute of Food and Agriculture, Hatch project 1001098 through the New Jersey Agricultural Experiment Station, Hatch project NJ08127. The senior author also was supported by the State Scholarship Fund of China. This is New Jersey Experiment Station Publication No. D-08-08127-08-17.
