The Behavioral Response to Heat in the Common Bed Bug, Cimex lectularius (Hemiptera: Cimicidae)

Abstract

The bed bug, Cimex lectularius L., is a common ectoparasite found to live among its vertebrate hosts. Antennal segments in bugs are critical for sensing multiple cues in the environment for survival. To determine whether the thermo receptors of bed bugs are located on their antennae; innovative bioassays were created to observe the choice between heated and unheated stimuli and to characterize the response of bugs to a heat source. Additionally, the effect of complete antenectomized segments on heat detection were evaluated. Heat, carbon dioxide, and moisture are cues that are found to activate bed bug behavior; a temperature at 38°C was used to assess the direction/degree at which the insect reacts to the change in distance from said stimulus. Using a lightweight spherical ball suspended by air through a vacuum tube, bed bugs and other insects are able to move in 360° while on a stationary point. Noldus EthoVision XT was used to capture video images and to track the bed bugs during 5-min bioassays. A bioassay was created using four Petri dish arenas to observe bed bug attraction to heat based on antennae segments at 40°C. The purpose of this study was to evaluate the effects of heat on complete antenectomized segments of the antennae. The results in this experiment suggest that bed bugs detect and are attracted to heat modulated by nutritional status. Learning the involvement of antennae segments in heat detection will help identify the location and role of thermoreceptors for bed bug host interaction.

Bed bugs (Hemiptera: Cimicidae) are closely associated with humans but survive feeding on bats, birds, and rabbits (Usinger 1966, Reinhardt and Siva-Jothy 2007). This association dates back to 3,500 yr ago, when humans lived in caves and bed bugs (Cimex lectularius L.) were found buried in Egyptian tombs (Panagiotakopulu and Buckland 1999). The bed bug is known to feed on its host at night, while they sleep and locate their hosts through thermal and olfactory cues (Johnson 1941, Usinger 1966, Aboul-Nasr and Erakey 1967, Aboul-Nasr and Erakey 1968). A combination of vision, mechanoreception, and chemoreception may be used in bed bugs to locate hosts and seek harborages (Singh et al. 2015, Benoit et al. 2016). Not much is known about bed bugs leaving and returning to harborages with no empirical data on natural behavior, so most of what is known about leaving and returning to harborages comes from laboratory studies (Doggett 2018).

Thermoreceptors and hygroreceptors detect heat and moisture, respectively, in locating hosts and habitats. The most distal and thickest antennal bristles in adult cockroaches (Periplaneta Americana L.) are directly beneath a segment or ventral side of the antennae and are found on alternating segments that have hygroreceptive sensilla located on the distal half (Tichy and Kallina 2010). Cockroaches have ~20 hygroreceptive sensilla per antenna with rarely more than one per segment. Stick insects (Caurausius morosus Br.) only have one hygroreceptive sensillum present on the 12th antennal segment with a field of ~50 olfactory and mechanoreceptive sensilla (Tichy and Kallina 2010). Unique infrared thermoreceptors have positive thermotaxis and direct the buprestid, Melanophila acuminate DeGeer, toward a heat source located several kilimeters away (Evans 1964, Lazzari and Núñez 1989, Harrison et al. 2012, DeVries et al. 2016).

Many insect species use their antennae to perceive their surrounding environment. The antennae in Rhodnius prolixus Stål have olfactory and thermal sensors that serve to perceive air currents and contact (Wigglesworth and Gillett 1934). Under water deprivation conditions, upwind movements to warm and humid airstreams were observed in the mosquito, Aedes aegypti L. (Bar-Zeev et al. 1977). With water circulating through a coil at (38°C), bed bugs were observed to have a significant preference to heat at a distance of 10 mm but no preference at 30 mm (DeVries et al. 2016). Additionally, blood feeders were used to indicate the percentage of feeding at specific temperatures with 38–43°C being the optimum temperature and 48°C being too high discouraging feeding (DeVries et al. 2016). Heat is an important stimulus in probing and feeding in bed bugs (DeVries et al. 2016). Bed bugs respond to humidity difference after removing the F2 and F1 segment of the antennae (Fig. 1; Hafez and Erakey 1964). Bed bugs are indifferent to ranges of humidity after removing the entire antennae and when the F2, F1, and pedicel are amputated (Hafez and Erakey 1964). These results show the presence of thermoreceptors and hygroreceptors somewhere in the antennae segments but also represents the need for more experiments to help locate specific receptors (DeVries et al. 2016).

Fig. 1.

Intact antennae comparison with complete antennectomized antennae on female bed bug to observe response to heat after treatment.

Better Understanding Sensory Structure and Physiology

The objective of this study was to identify the minimum distance at which a bed bug displays orientation toward a heated target that is representative of human body temperature (36°C). Studies have used the ‘servosphere’ or ‘locomotory compensators’ as a way to observe insect reactions to environmental cues in measuring locomotion (Kramer 1976, Hawkins 1978, Bell and Kramer 1979, Visser and Taanman 1987, Noldus et al. 2002, Nagaya et al. 2017). A modified servosphere was built to quantify the orientation response a bed bug has to a heated source using a camera and Noldus EthoVision software. I hypothesize that heat will activate a positive taxis response in bed bugs. Further research presented here focuses on the attraction to heat based on antennae segments when a bed bug displays toward a heated target. Scanning electron images were taken of male and female antennae sections to further identify sensilla present per segment. The purpose of this study was to evaluate the effects of heat on complete antenectomized segments of the bed bug antennae to locate thermoreceptors with precision. Learning the specific function of antennae segments will help locate and understand thermoreceptors and possible hygro-receptors for bed bug host interaction.

Methods and Materials

Bed Bug Rearing

A laboratory colony of bed bugs was used for all experiments. The strain Jersey City was collected in 2008 in Jersey City, New Jersey and maintained in the laboratory for 7 yr. Bed bug males and females of Cimex lectularius were collected and reared on a 14:10 (L:D) cycle at 24°C in 65% relative humidity (RH). All were fed defibrinated rabbit blood heated to 40°C with circulating water bath in an artificial feeding system once per week (Hemostat Laboratories, Dixon, CA). Additionally, all experiments were conducted during the first half of scotophase (under red-light) and at 25°C and 50% (±5%) relative humidity. All temperatures including experimental apparatuses were checked using a Surface thermometer (Thermocouple Thermometer, Professional Instrument Manufactures).

Modified Servosphere

Bedbugs and other insects are able to move 360° at a stationary point (Image 1), using a lightweight spherical ball suspended by industrial oxygenated air. Air was released through a polyethylene vacuum tube that is connected to a 1.5-ml microcentrifuge tube. This allows the bed bug to move freely in all directions. Initially, a temperature at 38°C was used to assess the direction/degree at which the insect reacts to changes in distance from stimulus source. To obtain the bed bugs’ initial reaction to heat, a 0.5-cm foam board was cut into smaller platform to create a target heat insulator blocker that prevents heat from reaching the insect. The insulator box was modified to have an enclosure with a 10.16 × 20.32-cm rectangular view to monitor insect reaction on spherical ball. The target heat insulator blocker is moved through the rectangular window and functions to prevent change to detection settings used by the EthoVision XT version 11.5 recording system (Noldus Information Technology Inc., Leesburg, VA; Noldus et al. 2002). HOBO (UX100-003) or temperature and humidity monitors (Onset Computer Corporation, Bourne, MA) were installed on a computer and are used to track variables in a dark room. Ambient room temperature was monitored to produce some heat in nonheated target for better control of stimuli. The heat source was placed precisely at different distances near the bed bug on the servosphere (Image 1). Distances were chosen to observe the response to heat at close proximity with a 1.27 cm or 0.5 in difference between values. Data on starved adult males and females with intact antennae at 2.54-cm distance was collected during the first scotophase, which is the first half of the feeding period between 8 and 12 pm. This was repeated with distances of 3.81 and 5.08 cm.

Image 1.

Bed bug resting on modified servosphere.

Antenectomization

The series of experiments include thermoreceptors in the antennae of C. lectularius. Data collection began by removing antennal segments with expected involvement in thermoreception (Figs. 1 and 2). Bed bugs were anesthetized using a CO₂ pad and antennae segments were removed using a surgical blade. Bed bugs recovered in the same rearing conditions and were subjected to experiments 24 h after surgery.

Fig. 2.

Segmented antenectomized antennae on female bed bug to observe response to heat after treatment.

Orientation in Response to Heat

To show a response to temperature (thermotaxis), the track-sphere device described above was used. A NIR camera with an IR filter (Infrared 850 light filter, Heliopan, North White Plains, NY) was used to record bed bug response under dark conditions (Image 4). The camera was positioned ~38 cm directly above the center of sphere. Light for the recordings were provided by two IR sources. EthoVision was used to capture video images and to track the bed bug during 5-min bioassays. All replicates were recorded using the Noldus EthoVision. Camera and orientation data are represented as 0° to 360° values. The 0° point is the heated target located in arena. NCSS 12 software was used to analyze values for circular statistics (NCSS 2018). Two variables were calculated from each arena: distance to target (cm) and angular direction relative to the heated target (deg/s). The same variables were calculated from the activity of bed bugs recorded in the control experiment at 25°C. In total, 20 different bed bug replicates were used along with 20 bed bugs in the control replicate for each distance (2.54, 3.81, and 5.08 cm). Trials were recorded individually; with control trials being the presence of a 25°C target versus a 25°C target representing (room temperature). Bed bugs were randomized in positions of four cardinal planes using a random number generator. Bed bugs were placed into container vials (7.5 ml) 5 min prior to the start of the experiment. Each bed bug was given 5 min to acclimate to placement on sphere. A 30-s window was used to remove barrier from heat source before tracking initial orientation response to heat source (Image 2). The bed bugs were observed and tracked using the EthoVision camera for 5 min.

Image 4.

Petri dish arena setup with light provided by two IR sources.

Image 2.

The modified servosphere with barrier from heat source prior to trials.

Activation in Response to Heat (Antenectomized Copper Experiment)

To observe response to heat, four Petri dish arenas were created to observe the response to a heat source at 40°C (Image 3). The camera was positioned ~74 cm directly above the center of the experimental arena. Light for the recordings were provided by two IR illuminators (AT-8SB850mm, 130°, AXTON, North Salt Lake, UT). Bed bugs were separated with harborage in plastic Petri dish 10–15 d after feeding. Bed bugs were allowed to acclimate in plastic Petri dish container 1 wk prior to experimentation. Starvation was induced to replicate the scenario of host seeking to ensure activation to heat source was during insects’ need for a bloodmeal. During the first half of scotophase, a copper cap (0.635 cm in diameter) heated via power supply KORAD DC Power Supply (Songbai Industrial Park, Guangming New District, Shenzhen, P.R. China) was heated to 40°C. EthoVision XT software was used to capture video images and track bed bug movements. Seven variables were calculated from each arena: cumulative duration in heated zone (s), mean distance to heat (cm), number of visits to heat, mean velocity (cm/s), latency to first visit to heat (s), and angular velocity (deg/s). The same variables were calculated from the activity of bed bugs recorded in the control experiment. In total, 11 different replicates along with 11 different control replicates were conducted for intact bed bugs at 25°C (positive control) and antenectomized bed bug at 25°C (negative control). Trials were recorded in pairs, with control 25°C target versus a control 25°C target (room temperature). Bed bugs were randomized and prepared as in previous experiments. Bed bugs were placed into container vials (7.5ml) 5 min prior to start the experiment. Each bed bug was given 5 min to acclimate to placement in Petri dish. A 10-s window was used to release bed bug to arena before tracking initial response to heated coil.

Image 3.

Petri dish arenas with a copper heat source in the center.

Segmented Antenectomized

To observe response to heat, four Petri dish arenas were created to observe the response to a heat source at 40°C. The same camera setup was used to record bed bug response under dark conditions in the antenectomized experiment. Six variables were calculated from each arena: cumulative duration in heated zone, mean distance to heat, frequency of visits to heat, mean velocity to heat, latency to first visit to heated zone, and angular velocity to heated zone. The same variables were calculated from the activity of bed bugs recorded in the control experiment at 25°C. In total, 11 replicates were used along with 11 control replicates for each treatment.

Statistics

The following parameters were calculated for each video: distance-to-heat source (cm) and angular direction (deg/s) toward heated target All means are accompanied by their SEs and N. The significance criterion employed for all tests was P < 0.05. All statistical software used was written by NCSS, a statistics package produced and distributed by NCSS, LLC (NCSS 2018). Vector concentration and resultant length gathered using NCSS Statistical software (NCSS 2018). Watson–Williams test statistic used to show the mean of a set of angles in the data set representing a trial per replicate (NCSS 2018). Raleigh’s test statistic was used to look for uniformity distribution among angular data toward heat source (NCSS 2018). A pairwise regression was conducted to analyze significant difference between distances. Data with normal distributions were analyzed using one-way or two-way analysis of variance (ANOVA) followed by Tukey’s posthoc analysis. Data sets, with non-normally distributed data, were transformed using Johnson distributions and then analyzed with ANOVAs (Wang and Zhou 2016). Data sets with other distributions were analyzed using generalized linear models for their particular distribution (Sall et al. 2017).

SEM Images

The procedure was modified from ( Harraca et al. 2010, Fischer et al. 2012). Bed bugs were immersed in a buffered fixative solution (glutaraldehyde). To process samples, 2-ml Eppendorf tubes were used. Glutaraldehyde was washed out with imidazole buffer and then exchanged with 50% ethanol. The glutaraldehyde wash out with the imidazole buffer and the 50% ethanol exchange were repeated twice. The final step was conducted using absolute ethanol for ~2 h with a few minutes of gentle mixing between changes occurred. The procedure required the samples to be washed free of fixative solution and then dehydrated with graded ethanol solutions and finally washed in HMDS before air drying (Fischer et al. 2012).

Results

Orientation in Response to Heat

The objective of the experiment was to identify the minimum distance that a bed bug displays orientation response toward a heated target that represents human body temperature (36°C). Values are placed in the interface as block terms with values 1–4 symbolizing control (no heat), 2.54, 3.81, and 5.08 cm. The closer the resultant vector is to 1, the more focused the data set is around the mean direction (NCSS 2018). The system can discern the orientation response of bed bugs to a heated target at varying distances. Following Rayleigh test, all treatments except 2.54 cm had a uniform von Mises distribution (Fig. 3 and Table 1). Watson–Williams test shows that the mean of a set of angles itself is a vector in the data set representing a trial per replicate (F = 10.3196 P < 0.00001). Control versus 2.54 cm (Fig. 4; χ ² = 12.816, P < 0.0010, df = 19; U²-test; P < 0.005; n = 20); control versus 3.81 cm (Fig. 4; χ ² = 27.7982, P < 0.00001, df = 19; U²-test; P < 0.005; n = 20); control versus 5.08 cm (Fig. 4; χ ² = 3.9545, P < 0.0540, df = 19; U²-test; P < 0.4044; n = 20); 2.54 cm versus 3.81 cm (Fig. 4; χ ² = 1.5009, P = 0.2281, df = 19); 2.54 cm versus 5.08 cm (Fig. 4; χ ² = 11.1262, P < 0.0019); and 3.81 cm versus 5.08 cm (Fig. 4; χ ² = 12.4119, P < 0.0011). Bed bugs oriented to a heat source placed at 2.54 and 3.81 cm away (Fig. 4; χ ² = 12.816, P < 0.0010; Fig. 4; χ ² = 27.7982, P < 0.00001). No orientation responses were detected when the heat source was 5.08 cm away from the bed bug (Fig. 4; χ ² = 3.9545, P < 0.0540).

Fig. 3.

Circular plot of angles by groups in female Cimex lectularius. Twenty replicates per treatment are shown. The closer the resultant vector is to 1, the more focused the data set is around the mean direction. Minimum distance that a bed bug detects human body temperature (36°C) is at least 3.81 cm.

Table 1.

Analysis of data with circular statistics shows uniformly distributed known as Von mises distribution for all but 2.54 cm

Mean Group	Raleigh’s test statistic	Raleigh’s test statistic prob level	Mean direction (Theta)	Mean resultant length (R bar)	Circular dispersion (Delta)
Control (25°C)	1.7658	0.4136	102.5788	0.2116	6.4957
2.54 cm (1 in.)	18.07	0.0001*	354.4372	0.6469*	0.8297
3.81 cm (1.5 in.)	1.77	0.4124	317.9279	0.2119	9.6046
5.08 cm (2 in.)	0.44	0.78790	184.8764	0.1101	20.4486

Watson–Williams test shows the mean of a set of angles is itself a vector in the data set representing a trial per replicate. The closer the resultant vector is to 1, the more focused the data set is around the mean direction. The system can discern the orientation response of bed bugs to a heated target at varying distances. Bed bugs oriented to a heat source placed at 2.54 and 3.81 cm away. No orientation responses were detected when the heat source was 2 in. away from the bed bug. Minimum distance that a bed bug detects human body temperature (36°C) is at least 3.81 cm.

Fig. 4.

Circular plot of distance: 20 experimental replicates were completed for 2.54, 3.81, and 5.08 cm. All replicates were recorded using the Noldus EthoVision camera and data are represented as 0° to 360° values.

Activation in Response to Heat: Sensory Manipulation Complete Antenectomized Experiments

To observe bed bug response to a 40°C heat source, four Petri dish arenas were created. In total, 11 replicates were used along with common control groups for each treatment of intact bed bugs at 25°C (positive control) and antenectomized bed bugs at 25°C (negative control). Trials were recorded in pairs, with control 25°C target versus a control 25°C target (room temperature).

There was a treatment effect of antenectomized bed bugs and the cumulative duration in heated zone (s) at 2 min (Fig. 5a; F_3,40 = 4.5243, P_treatment = 0.0080*). Intact bed bugs spent the longest cumulative time in the heated zone compared with other treatments in 2- and 5-min durations (Fig. 5a; F_3,40 = 4.5243, P_treatment = 0.0080*; F_3,40 = 11.2462, P_antennae = 0.0018*; F_3,40 = 0.4841, P_temp = 0.4906; and F₃,₄₀ =1.8426, P_{antennae*temp} = 0.1823) and 5 min (Fig. 5b; F_3,40 = 6.4338, P_treatment = 0.0012*; F_3,40 = 15.8283, P_antennae = 0.0003*; F_3,40 = 3.3168, P_temp = 0.0761; and F_3,40 = 0.1564, P_{antennae*temp} = 0.6946). There was a treatment effect in the mean distance to heat source. Heated intact bed bugs show shortest mean distance to heat source at all time points (Fig. 6a; F_3,39 = 4.8928, P_treatment = 0.0056*; F_3,39 = 12.7350, P_antennae = 0.0010*; F_3,39 = 1.9762, P_temp = 0.1677; and F₃,₃₉ = 0.1103, P_{antennae*temp} = 0.7415) and 5 min (Fig. 6b; F_3,40 = 3.6918, P_treatment = 0.0195*; F_3,40 = 8.8805, P_antennae = 0.0049*; F_3,40 = 2.1567, P_temp = 0.1498; and F_3,40 = 0.0383, P_{antennae*temp} = 0.8458). There was a treatment effect in the number of visits to heat source. Heated intact bed bugs visited the heated zone more in comparison with bed bugs without antennae at all time points (Fig. 7a; F_3,40 = 4.5092, P_treatment = 0.0081*; F_3,40 = 11.2384, P_antennae = 0.0018*; F_3,40 = 1.8183, P_temp = 0.1851; and F_3,40 = 0.4708, P_{antennae*temp} = 0.4966) and 5 min (Fig. 7b; F_3,40 = 3.6918, P_treatment = 0.0195*; F_3,40 = 8.8805, P_antennae = 0.0049*; F_3,40 = 2.1567, P_temp = 0.1498; and F_3,40 = 0.0383, P_{antennae*temp} = 0.8458).

Fig. 5.

(a and b) Bed bug duration in heated zone.

Fig. 6.

(a and b) Bed bug distance to heated zone.

Fig. 7.

(a and b) Number of bed bug visits to heated zone.

There was no significant difference found in mean velocity of bed bugs at 2 min (Fig. 8a; F_3,40 =1.0184, P_treatment = 0.3947). Heated intact bed bugs visited the heated zone more in comparison with bed bugs without antennae at all time points (Fig. 8a; F_3,40 =1.0184, P_treatment = 0.3947; Fig. 6a; F_3,40 = 0.9978, P_antennae = 0.3239; F_3,40 = 1.0013, P_temp = 0.3230; F_3,40 = 1.0561, P_{antennae*temp} = 0.3103) and 5 min (Fig. 8b; F_3,40 = 1.0775, P_treatment = 0.3695; F_3,40 = 0.7880, P_antennae = 0.3800; F_3,40 = 1.8151, P_temp = 0.1855; and F_3,40 = 0.6295, P_{antennae*temp} = 0.4322). There was a significant difference found in latency to first visit to heated zone in bed bugs at 2 min (Fig. 9a; F_3,40 = 8.7895, P_antennae = 0.0051). Heated intact bed bugs took less time to find the heated zone at all time point (Fig. 9a; F_3,40 = 3.8763, P_treatment = 0.0159*; F_3,40 = 8.7895, P_antennae = 0.0051; F_3,40 = 2.7358, P_temp = 0.1060; and F_3,40 = 0.1037, P_{antennae*temp} = 0.7492) and 5 min (Fig. 9b; F_3,40 = 4.2056, P_treatment = 0.0112*; F_3,40 = 7.9649, P_antennae = 0.0074*; F_3,40 =4.6513, P_temp = 0.0371*; and F_3,40 = 0.0006, P_{antennae*temp} = 0.9811).

Fig. 8.

(a and b) Speed of bed bug toward heated zone.

Fig. 9.

(a and b) Latency of bed bug visits to heated zone.

Bed bug angular velocity to heated zone was not different at 2 and 5 min (Fig. 10a; F_3,40 = 1.7965, P_treatment = 0.1634; F_3,40 = 0.1258, P_antennae = 0.7247; F_3,40 = 2.7597, P_temp = 0.1045; and F_3,40 = 2.5039, P_{antennae*temp} = 0.1214) or 5 min (Fig. 10b; F_3,40 = 0.2767, P_treatment = 0.8419; F_3,40 = 0.0058, P_antennae = 0.9396; F_3,40 = 0.7722, P_temp = 0.3848; and F_3,40 = 0.0520, P_{antennae*temp} = 0.8207).

Fig. 10.

(a and b) Bed bug angular velocity toward heated zone.

Activation in Response to Heat: Sensory Manipulation Segmented Antenectomized Experiments

Treated bed bugs show a significant difference in antennae and time spent in heated zone at 2 min. Bed bugs with scapus only spent less time in heated zone at 2 and 5 min (Fig. 11a; F_3,40 = 6.1124, P = 0.0016*) and 5 min (Fig. 11b; F_3,40 = 6.3367, P = 0.0013*). Combined data for 2 and 5 min show significance in treatment when observing the time bed bugs spent in zone (F_7,80 = 5.4276, P_treatment < 0.0001*; F_3,40 = 6.5814, P_antennae = 0.0005*; F_3,40 = 0.6976, P_time = 0.4061; and F_3,40 = 0.2664, P_{antennae*time} = 0.8494).

Fig. 11.

(a and b) Duration of bed bug in heated zone.

Bed bugs show no difference in mean distance to heat source across treatments at 2 and 5 min (Fig. 12a; F_3,40 = 2.2300, P_antennae = 0.0996) and 5 min (Fig. 12b; F_3,40 = 2.1442, P_antennae = 0.1098). Combined data for 2 and 5 min show no significance in in mean distance to heat source in antennae versus time (Fig. 12a and b; F_7,80 = 2.0109, P_treatment = 0.0638; F_3,40 =2.1453, P_antennae = 0.1010; F_3,40 = 0.9640, P_time = 0.3291; and F_3,40 = 0.0157, P_{antennae*time} = 0.9973). Treated bed bugs show a significant difference in frequency of visits to heated zone at 2 min. Bed bugs show a difference in frequency of visits to a heat source across treatments at 2 and 5 min. (Fig. 13a; F_3,40 = 6.1768, P_antennae = 0.0015*) and 5 min (Fig. 13b; F_3,40 = 6.3544, P_antennae 0.0013*). Combined data for 2 and 5 min show significance in treatment when observing the frequency of bed bugs visits to heat source in antennae versus time (Fig. 13a and b; F_7,80 = 5.4693, P_treatment < .0001*; F_7,80 = 6.6306, P_antennae = 0.005*; F_7,80 = 0.7306, P_time = 0.3952; and F_7,80 = 0.2750, P_{antennae*time} = 0.8433). Treated bed bugs show no significant difference in speed to heated zone at 2 min (Fig. 14a; χ ² = 1.053, P = 0.7882) and but a difference was present at 5 min (Fig. 14b; χ ² = 17.067, P = 0.0007). Combined data for 2 and 5 min show no significance in treatment when observing the speed of bed bugs to heat source in antennae versus time (Fig. 14a and b; χ ² = 1.603, P_antenna = 0.6585; χ ² = 2.459, P_time = 0.1191; and χ ² = 6.225, P_antenna*time = 0.1012).

Fig. 12.

(a and b) Bed bug distance to heated zone.

Fig. 13.

(a and b) Number of bed bug visits to heated zone.

Fig. 14.

(a and b) Speed of bed bug toward heated zone.

Bed bugs show significant difference in latency toward heat source across treatments at 2 and 5 min. (Fig. 15a; F_3,40 = 7.0501, P_antennae = 0.0006*) and at 5 min (Fig. 15b; F_3,40 = 6.4751, P_antennae = 0.0011*). Combined data for 2 and 5 min show significance in treatment when measuring the amount of time it took a bed bug to reach the heat source in antennae versus time (Fig. 15a and b; F_7,80 = 5.8465, P_treatment < .0001*; F_7,80 = 7.3396, P_antennae = 0.0002*; F_7,80 = 0.2790, P_time = 0.5988; and F_7,80 = 0.5112, P_{antennae*time} = 0.6757). Treated bed bugs show no significant difference in angular velocity to heated zone at 2 min (Fig. 16a; F_3,3 = 0.699, P = 0.5584) or 5 min (Fig. 16b; F_3,3 = 0.558, P = 0.6457). Combined data for 2 and 5 min show no significance in treatment when observing the angular velocity of bed bugs to heat source in antennae versus time (Fig. 16a and b; F_3,3 = 0.586, P_antennae = 0.6257; F_1,1 = 1.818, P_time = 0.1814; and F_3,3 = 0.371, P_{antennae*time} = 0.7738).

Fig. 15.

(a and b) Latency of bed bug toward heated zone.

Fig. 16.

(a and b) Bed bug angular velocity toward heated zone.

Scanning Electron Microscopy

The sensilla found on C. lectularius are the same described in the literature (Levinson et al. 1974, Steinbrecht and Müller 1976; Fig. 17). Bed bug images were individually counted for sensilla type. Seven distinct types of sensilla were evident in the olfactory region (Figs. 17 and 18). Four small grooved pegs (type C) and six long smooth pegs (type D) were found in the first olfactory region (F2) (Fig. 19e and f). The position of the sensilla was constant, confirming previous observations (Levinson et al. 1974, Steinbrecht and Müller 1976; Fig. 19e and f).

Fig. 17.

Head of female Cimex lectularius with left antenna, anterior view using TEM. (SC) Scapus, (PE) Pedicellus, (F1) first Segment Flagellum, (F2) second Segment Flagellum, (O1 and O2), and Olfactory Region 1 and 2.

Fig. 18.

Cimex lectularius, male, F2 Olfactory region, 11.0 mm × 600 50 μm. Outer side of F2 house the C and E olfactory sensilla, 12 A1 /E type hairs in blue triangle, 4 D type in red rectangle, 5 C type in yellow star, and 2 F type in orange arrow.

Fig. 19.

(a) Cimex lectularius, male, F2 Olfactory region. SEM, 11.1 mm × 600 50 μm, there are 11 A type hairs located in this image in blue triangle. (b) Cimex lectularius, male, pedicellus. SEM, 11.0 mm × 600 50 μm, there are seven A/E type hairs found on the F2 in blue triangle and 1 F type in orange arrow.(c) Cimex lectularius, male, pedicellus. SEM, 11.1 mm × 600 50 m, 13 E type hairs found in pedicellus region of antennae in blue triangle. (d) Cimex lectularius, male, pedicellus, and partial scapus. SEM, 10.9 mm × 600 50 μm, 18 E type hairs in pedicellus region of antennae in blue triangle. (e) Cimex lectularius, male, pedicellus. SEM, 11 mm × 600 50 μm, 30 E type hairs in pedicellus region of antennae in blue triangle and 2 C type sensilla in yellow star. (f) Cimex lectularius, male, scapus. SEM, 10.8 mm × 600 50 μm, 17 E type hairs in blue triangle and 3 D type sensilla in red rectangle.

Discussion

Bed bugs oriented to a heat source at 2.54 cm supporting the idea that bed bugs use heat when in close proximity of a food source (Fig. 4). Based on the location, convectional air currents move away from the heat source, giving bed bugs cues along with olfactory function to orient toward host. Intact bed bugs were attracted to heat source and had the shortest latency to movement (Fig. 9) and traveled less (shortest distance) when moving toward the heated zone (Fig. 6). Intact bed bugs spent the longest time (Fig. 5), had the most visits (Fig. 7), and displayed the highest speed (Fig. 8) and angular velocity (Fig. 10), toward the heated zone compared to antenectomized bed bugs. These data reinforce knowledge about how hematophagous insects use their antennae to detect blood meals and validates the method used in this experiment prior to the implementation of antenectomization.

Intact, scapus and pedicel, and missing F2 antennae bed bugs had the highest speeds in the presence of a heat source at 2 and 5 min (Fig. 14a and b). Bed bugs with only scapus segment were slowest in speed toward heat source than other treatments (Fig. 14a and b). The speed at which bed bugs travel toward the heat source is related to how activated they are (DeVries et al. 2016). Data show activation and attraction to heat source in bed bugs of all treatments at 2 and 5 min (Fig. 14a and b). Attraction to heat source without antennae shows the possibility that thermosensilla are present along the body and may play a role in heat seeking behavior. Latency experiments show that thermosensilla are likely found in the first three segments of the antennae that include the F2, F1, and pedicel, and the heat seeking function is delayed in bed bugs with scapus only. Bed bugs appear to lose orientation and sensory function toward heat source after treatment. The higher the degree per second, the faster the orientation toward heat source. The angular velocity assay shows that bed bugs did not change direction toward heat source any different in any of the treatments. In this study, the orientation or movement of an organism in response to a stimulus was positive in terms of orientation and detection of stimuli in bed bugs. Thus, bed bugs have been identified to be activated and attracted to heat with use of their antennae and lack sensory capacity to find heat with the loss of antennae.

When specific segments were removed, bed bugs with only a scapus spent less time in heated zone while intact and scapus and pedicel bed bugs spent the most time in heated zone (Fig. 11a and b). Bed bugs missing the F2 segment did not behave differently than any other treatment (Fig. 11a and b). The time spent in a heated zone is a variable that shows the exploratory behavior of bed bugs. This is indicative that heat is stimulating bed bugs to remain in heated zone when the first three segments of antennae are intact. The size of the arena may play a role in the amount of space allowed for the bed bug to be away from stimuli. Minimum distance that a bed bug detects human body temperature (36^°C) is at least 2.54 cm. Bed bugs missing scapus were the least attracted to the heat source (Fig. 13a and b). Intact bed bugs made more visits to the heat source at 2 min (Fig. 13a). The trend shows bed bugs are still able to sense heat with the first three segments removed. Higher frequency of visits to the heat source occurred in intact, missing F2, and scapus and pedicel segmented bed bugs, revealing attraction to heat. Data from the assay confirm that bed bugs with scapus segment only sense heat less than other treatments. This confirms research conducted by (DeVries et al. 2016), indicating activation from temperature and the use of antennae for thermoreception.

The data presented here highlight the need for more bioassays regarding bed bug and host interactions and bloodmeal seeking behavior. The variables in the assay were chosen to observe the response and activation of bed bugs to heated stimuli. The variables show that when bed bugs are searching for a host; time, distance, the number of visits, orientation, and speed toward heat source are informative metrics. Bed bugs that were missing the first three segments of their antennae were not able to find the heat source as readily as other treatments because thermosensilla may be in use in the first three segments of antennae. The experiments show that the most distal segment plays a smaller role in heat sensing. An animal with damaged antennae may be unable to locate a host and secure a bloodmeal. It is imperative that bed bugs find a bloodmeal in order to advance insect life stages and have enough nutrients to mate and lay eggs.

Bed bugs must also remain cryptic from predators as they are in search of a host; making fast host detection important and indicating that more time spent searching may be detrimental. Bed bugs have strong exoskeletons as adults (Reinhardt and Siva-Jothy 2007). Antennae are pivotal in responses to external stimuli; thus future experiments must investigate the effects of these cues based on human hosts (Harraca et al. 2010, Olson et al. 2009). Like kissing bugs (Triatoma rubida L.;Ferreira et al. 2007), assays suggest that bed bugs use temperature irregularities on the surface of skin in order to locate blood vessels. Heat from the vertebrate guides the direction of penetration of the bed bug’s mouthparts in the form of a flexible fascicule made of a pair of mandibles and maxillae, which probes in various directions until a suitable size vessel is found (Dickerson and Lavoipierre 1959, Lazzari and Núñez 1989, Araujo et al. 2009). The evolution of a bloodmeal occurred several times within arthropods, leading to a great diversity of salivary composition among arthropods (Francischetti et al., 2010). These coincidences can be ascribed as products of convergent evolution since C. lectularius L. does not share a common blood feeding ancestor with any of the hematophagous insects (Francischetti et al. 2010). The process of taking a bloodmeal and finding the blood vessels quickly without disturbing the host is a key feature for the adaption of hematophagy in insects (Ferreira et al. 2007). Previous research showed that removal of terminal segments elicits no behavioral response to stimuli usually driven by olfactory cues (Harraca et al. 2010, Liu and Liu 2015). Female kissing bug antennae have sensilla between E type hairs with increased surface area of antennae structure. Previous work shows that a lack of antennae has a clear impact on the way triatomes use thermal information (Flores and Lazzari 1996, Barrozo et al. 2003, Lorenzo Figueiras et al. 2013). Many studies have described different host cues that help Rhodnius prolixus Stål and other blood-sucking insects find a bloodmeal using modified mouthparts that allow them to pierce blood vessels under the skin (Ferreira et al. 2007). Experimental result strongly supports that the thermal receptors located on the antennae are responsible for blood vessel detection in R. prolixus (Ferreira et al. 2007). Their experiment revealed that a thermo-tropotaxis mechanism is involved in guiding proboscis extension (Ferreira et al. 2007).

To follow up with the current assays, a modified arena that allows for monitoring of bedbugs not only on direction but also velocity and intensity of attraction to heat, CO₂, and moisture would be a strong future direction. Questions remain about how chemical cues and heated stimuli activate bed bug host selection behavior (Liu and Liu 2015). Behavioral responses are hard to study without proper visual equipment in place to observe nightly activities of bed bugs.