Bed bugs (Cimex lectularius L.) exhibit limited ability to develop heat resistance
Bed bugs (Cimex lectularius L.) exhibit limited ability to develop heat resistance
Aaron R. Ashbrook 0 1
Michael E. Scharf 0 1
Gary W. Bennett 0 1
Ameya D. GondhalekarID 0 1
0 Editor: Pedro L. Oliveira , Universidade Federal do Rio de Janeiro , BRAZIL
1 Department of Entomology, Purdue University , West Lafayette, Indiana , United States of America
The global population growth of the bed bug, Cimex lectularius (L.), is attributed to their cryptic behavior, diverse insecticide resistance mechanisms, and lack of public awareness. Bed bug control can be challenging and typically requires chemical and non-chemical treatments. One common non-chemical method for bed bug management is thermal remediation. However, in certain instances, bed bugs are known to survive heat treatments. Bed bugs may be present after a heat treatment due to (i) abiotic factors associated with the inability to achieve lethal temperatures in harborage areas for a sufficient time period, (ii) re-infestation from insects that escaped to cooler areas during a heat treatment or (iii) development of physiological resistance that allows them to survive heat exposure. Previous research has investigated the optimal temperature and exposure time required for either achieving complete mortality or sublethally affecting their growth and development. However, no research has examined bed bug populations for their ability to develop resistance to heat exposure and variation in thermo-tolerance between different bed bug strains. The goals of this study were: i) to determine if bed bugs could be selected for heat resistance under a laboratory selection regime, and ii) to determine if bed bug populations with various heat exposure histories, insecticide resistance profiles, and geographic origins have differential temperature tolerances using two heat exposure techniques (step-function and rampfunction). Selection experiments found an initial increase in bed bug survivorship; however, survivorship did not increase past the fourth generation. Sublethal exposure to heat significantly reduced bed bug feeding and, in some cases, inhibited development. The stepfunction exposure technique revealed non-significant variation in heat tolerance between populations and the ramp-function exposure technique provided similar results. Based on these study outcomes, the ability of bed bugs to develop heat resistance appears to be limited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This research was funded by a grant
award from the Pest Management Foundation
(#208527) to ADG, MES and GWB. MES received a
grant award from US Department of Housing and
Development (INHHU0026-14); and ADG
supported the graduate assistantship for the first
author, ARA. Additionally, ARA was supported by
merit-based scholarships from Gerald Leep Family,
J. T. Eaton and Company, Pi Chi Omega, National
Of the ~100 species of blood feeding parasitic pests within the family Cimicidae, only the bed
bug, Cimex lectularius (L.), and the tropical bed bug, Cimex hemipterus (F.), are associated
with the recent global population resurgence [
1, 2, 3
]. Both, C. lectularius and C. hemipterus
Pest Management Foundation and R. O. William.
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
share hosts and their populations overlap in certain areas [
1, 4, 5
]. Yet, the ability of these
organisms to tolerate environmental conditions influences their geographic distribution
because they show differential temperature preference (lectularius 28?29?C, hemipterus 32?
1, 4, 6
]. This allows for widespread distribution of C. lectularius in temperate regions,
whereas C. hemipterus infestations are primarily in tropical/subtropical regions. However,
both species have been recently found outside of the previously mentioned areas [
1, 4, 7?9
likely because they are commonly found in stable indoor environments and are usually
sheltered from the outdoor temperature extremes [
Bed bugs are known to negatively influence humans as their bites can leave behind itchy
red welts [
]. Elimination of bed bugs can be costly as it entails application of chemical
insecticides and the use of non-chemical control techniques [
]. To avoid the challenges
associated with locating all insects in an infestation, pesticide label restrictions on where a product
can be applied within a residence and the potential for an insecticide resistant population to be
present, whole residence heating is used for bed bug elimination [
]. Entire home heating
is achieved by circulating heated air (55?65?C) indoors for six to eight hours with the ultimate
goal of heating bed bug containing objects to >50?C . Thermal remediation has many
advantages. Not only can it eliminate all bed bug life stages within a residence, but it can also
be used in areas or on objects where insecticides cannot be applied [
setup of a heat treatment requires less preparation by occupants and it provides more
immediate relief to them [
]. However, there are also some drawbacks to the use of heat for bed
bug disinfestation. For example, large scale heat treatments are time intensive, costly and do
not provide any residual protection against bed bugs [
]. Heat exposure may also damage
temperature-sensitive items [
]. Lastly, achieving the necessary lethal temperatures in
thermally insulated areas such as cracks and crevices of walls or furniture where bed bugs
prefer to reside is sometimes challenging.
If lethal temperatures are not achieved, bed bugs may detect and respond behaviorally to
sublethally heated areas by fleeing to cooler areas such as wall voids, deep within furniture, or
in neighboring unheated apartments [
]. Bed bugs stunned by sublethal heat exposure
could fall into protected areas and recover afterwards [
]. In one case, it was observed that
bed bugs escaped from a heat-treated apartment to an adjacent unheated unit to avoid heat
]. Loudon [
] reported that a single bed bug moved from the heated exterior to
the cooler interior of a luggage case in an attempt to escape lethal heat exposure. Furthermore,
when bed bugs are placed in an arena at room temperature (25?C), they can detect and orient
towards a heated copper coil (28?C to 48?C) that is 10?30 mm away [
], which indicates they
are good at responding to heated objects at short ranges. The abovementioned abiotic
challenges in achieving lethal temperatures in harborage areas combined with the ability of bed
bugs to behaviorally or physiologically respond to sublethal temperature exposure could
theoretically select them for increased heat resistance.
There are several examples of arthropods adapting to temperature extremes. Heat exposing
Drosophila melanogaster in the laboratory resulted in greater temperature resistance within a
few generations of selection [
]. Gray (2013) showed that plastic temperature tolerance
traits can be selected within Culex pipiens if they are reared at different temperatures .
Tetranychus cinnabarinus, a greenhouse pest, was selected for resistance to abamectin and also
showed some cross-resistance to heat exposure due to increased expression of heat shock
proteins (HSP) [
]. A springtail species, Orchesella cincta, was shown to significantly increase
expression of the HSP70 family proteins after exposure to non-lethal high temperatures (heat
hardening) prior to prolonged heat exposure . Although bed bugs do not display heat
], repeated sublethal heat exposure could potentially select them for heat
resistance, which would be problematic for the use of thermal remediation for their control.
2 / 17
Some of the previous temperature tolerance studies focusing on bed bugs have utilized two
different exposure techniques. The first technique, ?step-function?, is where the insects are
exposed to a rapid increase in temperature [
15, 30, 31
]. The second technique is
?ramp-function?, where the insects are exposed to a slow rate of rising temperatures [
another thermal biology study by Rukke et. al. [
], the effects of rearing bed bugs at
elevated temperatures (34 to 38?C) on survivorship, development and reproduction were
reported. However, none of the previous studies have investigated different bed bug
populations for variation in thermo-tolerance.
To address the knowledge gaps associated with the potential for bed bugs to develop heat
resistance as well as the absence of data on variation in thermo-tolerance of different bed
bug populations the goal of this research was two-fold. The first goal was to determine if a
laboratory strain of bed bugs could be selected for heat resistance through sublethal heat
exposure over multiple generations. The second goal was to utilize the step-function and
rampfunction heat exposure techniques to evaluate the temperature tolerance of different bed bug
Materials and methods
The insecticide-susceptible Harlan laboratory strain was used for heat selection experiments
and as a reference population for thermo-tolerance comparisons. Information on the ten field
populations used for heat tolerance screening are outlined in Table 1. Throughout this
manuscript, the terms ?strain? and ?population? are used interchangeably. Field populations of bed
bugs were collected from infested locations by pest management professionals (PMPs) and
university researchers after obtaining verbal authorization from anonymous private property
and business owners. No field studies were conducted for this research. All bed bug
populations were maintained at 25 ?1?C, 50 ?10% RH and a 12:12 h (L:D) cycle in a
temperaturecontrolled environmental chamber (Percival Scientific, Perry, IA). They were fed on
defibrinated rabbit blood purchased from Hemostat Laboratories (Dixon, CA] using the membrane
feeding method [
]. Heat selection experiments used large nymphs (4th?5th) that were starved
for seven days prior to heat exposure (step-function technique). Similarly, adult bed bugs (1:1
male to female ratio) used for step-function and ramp-function experiments were fed seven
days prior to their use. All field strains were laboratory-adapted and fed readily on defibrinated
Heat resistance selection study
Determination of lethal time estimates for late instar nymphs of the Harlan strain. In
order to select the Harlan strain for heat resistance, a LT75 (lethal time to kill 75% of the test
population at 45?C) was determined for 4th?5th instar nymphs by utilizing the step-function
heat exposure method [
15, 16, 29
]. For the LT75 determination, ten Harlan strain nymphs
were placed into a 15-mL glass test tube (Fisher Scientific, Pittsburg, PA) with a strip of
notecard paper (Roaring Spring Paper Products, Roaring Spring, PA) for harborage (Fig 1A). Test
tube openings were capped with Parafilm (Bemis NA, Neenah, WI). These tubes were then
placed in a 12x6 plastic rack which was then placed in a water bath (Isotemp 210, Fisher
Scientific, Dubuque, IA) heated to 45?C (Fig 1B). Rubber bands were used to secure the test tubes
and prevent them from floating in the water bath. The exposure periods for nymphs in the
45?C water bath were 10, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25 mins. After the exposure
period had elapsed, test tubes were removed from the water bath and bed bugs were placed in
a 35x10mm Petri dish (Fisher Scientific, Pittsburg, PA) with a Whatman No. 1 filter paper disc
(GE Healthcare, Pittsburg, PA) (Fig 1D). Petri dishes were held in an environmental chamber
with temperature, humidity, and light conditions identical to those used for rearing. Mortality
Fig 1. A. Bed bugs in a glass test tube with a strip of filter paper for harborage prior to heat exposure. B. An example of
how the bed bugs were heat exposed in the water bath. C. After heat exposure in the water bath, the bed bugs were
stunned and have fallen to the bottom of the test tube. D. Stunned bed bugs being placed in a Petri dish after heat
4 / 17
was scored 24 h after exposure by prodding the insects with a toothpick. Insects were scored as
dead if they could not move or right themselves after being prodded.
Selection regimen. The abovementioned step-function heat exposure method and the
probit analysis-determined LT75 value (in mins) was used to select the Harlan 4th?5th instar
nymphs for heat resistance. An equal subset of nymphs not exposed to heat was maintained as
a control colony. Each glass test tube that was used to confine bed bugs during heat exposure
contained ten nymphs. Several test tubes were used for heat exposure experiments every
generation depending upon the availability of nymphs. After heat exposure at the 45?C LT75 time,
all nymphs from each individual test tube were transferred to a Petri dish with filter paper and
mortality was scored after 24 h. Surviving nymphs from individual Petri dishes were then
pooled in a single rearing container with mesh (Uline, Pleasant Prairie, WI), where they
developed into adults and reproduced. Both control and heat-selected colony nymphs were fed one
to two times weekly. The selection regime was continued from F0 to F7 generation (except F1)
and initially began by selecting 300 nymphs (distributed in 30 test tubes) at F0 generation. As
the selection process continued (F4 generation and beyond) less insects were used due to
lower colony numbers. Therefore, depending on the availability of insects in each generation,
between 50 and 300 nymphs were utilized for selection experiments.
Assessment of blood-feeding and molting ability of heat exposed bed bugs
During the resistance selection procedure, heat-selected bed bugs were also qualitatively
observed for sublethal effects such as the inability to feed to repletion and to successfully molt.
Qualitative observations of the sublethal heat impacts on bed bugs led to conducting
comparative experiments where the ability of heat exposed insects to feed was assessed. In order to
quantitatively evaluate how heat affected blood feeding, 4th?5th instar Harlan nymphs were
exposed to LT75 time at 45?C and mortality was scored 24 h later. Survivors of heat exposure
were then placed in jars and their ability to feed to repletion on defibrinated rabbit blood was
observed on days five, eight, ten and fourteen after heat exposure. Identical numbers of control
nymphs were placed in jars and also observed for their ability to feed to repletion at the same
time points mentioned above. The number of insects utilized for each replicate was
determined by the survival of the bed bugs in response to heat exposure at the LT75 time. Overall,
six replicates were performed with an average of 40 bed bugs per replicate.
Thermo-tolerance comparisons among bed bug strains
The procedures used for step-function thermo-tolerance comparison experiments were similar
to those used for determining LT75 estimates for the Harlan nymphs. For each population, ten
mixed sex adult insects (1:1 ratio) were placed into a 15-mL glass test tube with a strip of filter
paper as harborage. Test tubes were sealed with Parafilm, placed in a 12x6 plastic holding rack
and then transferred to a water bath heated to 45?C. Insects were exposed at 45?C for 10, 12,
13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 mins to generate exposure
timemortality data. Three to four replicates (10 adults per replicate) were performed for each time
point. Additional time points that provided 75?100% mortality were included in step-function
heat exposure experiments to increase the precision of LT99 estimations [
]. Test tubes were
removed from the water bath after the exposure period had elapsed and bed bugs were placed
in a 35x10mm Petri dish with a Whatman No. 1 filter paper disc. Petri dishes were held in an
environmental chamber with temperature, humidity, and light conditions identical to those
used for rearing. Mortality was scored 24 h after exposure using the parameters described
under determination of lethal time estimates for late instar nymphs. Control insects were held
in test tubes at room temperature and then transferred to Petri dishes.
5 / 17
Procedures for the ramp-function heat exposure bioassay that utilizes a gradual or
incremental increase in temperature were somewhat similar to those used for the step-function
bioassays explained above. Briefly, 15-mL glass test tubes with 10 mixed sex adult bed bugs (1:1
ratio) per tube were placed in a 12x6 plastic holding rack that was transferred to a water bath
at room temperature. The water bath was then turned on and the bed bugs were exposed to
gradually rising temperatures at the rate of 0.57 ?C/min until the water temperature reached
45?C. Once the water bath reached 45?C (~37-min heating time), insects were held in the
water bath for a time that corresponded with the LT99 time for the Harlan strain. After the
ramp-up heat exposure period was completed, insects were placed into Petri dishes with filter
paper and mortality was scored 24 hours later using previously described criterion under
determination of the lethal time estimates for late instar nymphs? section. Three replicates of
ten mixed sex bed bugs per replicate were performed for each population, including the Harlan
strain, which was used as a positive control for all bioassay tests. Test tubes containing control
insects were held at room temperature during the ramp-up heat exposure experiments.
Time-mortality data for 4th?5th late instar nymphs were utilized for PROC probit analysis in
SAS 9.4 (SAS 2012, Cary, NC) to determine LT75 exposure time. The survivorship data for
nymphs from the F0 to F7 generations were analyzed using ANOVA followed by all pairs
Tukey?s test in SAS 9.4. Comparisons for feeding experiments were made in JMP 13.2 (SAS
institute 2016, Cary, NC) using a repeated measures MANOVA with an interaction effect
between nymphs and day. Nonparametric Wilcoxon tests were then conducted to determine if
feeding response on any particular day was statistically different between heat-exposed and
control insects. Exposure time-mortality data from the step-function experiments with adults
was analyzed by PROC probit in SAS 9.4 to determine lethal time (LT50 and LT99) estimates
and associated parameters for each population. The probit output values (intercept, slope, and
covariance) were further used to statistically compare heat-tolerance profiles between different
field populations as well as with the Harlan strain [
]. Mortality of field populations from the
ramp-function heat experiments were analyzed by ANOVA using the PROC GLM function
and means were separated using a Tukey?s test (P<0.05). Linear regression analysis was
performed in JMP 13.2 to determine if there was any correlation between the LT50 or LT99 values
and the latitude for the town/city where bed bug collections were made.
Response of Harlan strain nymphs to heat selection
Probit analysis conducted on time-mortality data for 4th?5th instar Harlan nymphs indicated a
time of 18.15 min at 45?C would kill 75% of test insects (LT75). However, for conducting
selection experiments, the lower fiducial limit of the LT75 estimate (i.e., 17.45 mins) was used after
performing empirical mortality validation tests, which showed that 17.45 min exposure caused
~75% mortality (Figure A in S1 File). The first round of selection (F0 generation) resulted in
an average of 26.3% ? 5.5% of nymphs surviving (Fig 2). Survivorship significantly increased
in the F2 and F3 generations to 50.5% ? 5.6%. and 55.5% ? 3.6%, respectively (ANOVA results:
df = 35, 102, F = 1.63, P <0.001). However, survivorship in the F4 generation reduced to
31.4% ? 16% survivorship, which was statistically similar to the F0 generation. Exposure of F5
to F7 generation nymphs to the LT75 resulted in similar survivorship with an average of 26% ?
9.5%, 20% ? 4.4%, 27.8% ? 9.1% surviving the exposure. Although some of the F7 selected
nymphs initially survived heat exposure, the attempt to establish the F8 generation was not
6 / 17
Fig 2. Bars depicting average survivorship of late-instar Harlan nymphs (4th?5th instar) after each generation (F0 to F7) of
selection or heat exposure at 45?C for 17.45 mins (LT75 time). Bars not connected by the same letter show statistically different
survivorship rate (P<0.05; Tukey?s test).
successful because bed bugs died out completely likely due to the adverse effects such as
reduced feeding and molting issues caused by the selection regime.
Impacts of heat exposure on feeding and molting success
As mentioned above, some sublethal effects of heat exposure were observed in surviving
insects. Initial qualitative observations suggested that fewer heat-exposed nymphs fed to
repletion when offered a blood meal, but all control insects readily fed. Similarly, after feeding,
some of the heat-exposed nymphs failed to escape from their exuvia and died during the
molting process (Fig 3). The heat-exposed insects that died during molting showed dark
pigmentation instead of the opaque and translucent appearance of normal teneral bed bugs. Molting
defects were not observed in nymphs of the control strain. To verify the qualitative
observations of reduced blood feeding by heat-exposed nymphs, a separate experiment was conducted
where the feeding response of controls and nymphs that had survived heat exposure was
quantitatively compared. Five, 8, 11, and 14 days after heat exposure, a significantly lower
proportion of heat selected nymphs fed to repletion in comparison to the control strain nymphs (Fig
4, Repeated measures MANOVA results: df = 3, 8, F = 14.85, P <0.0012, Wilcoxon test results;
day 5, Z = -2.80, P = 0.005; day 8, Z = -2.80, P = 0.005; day 11, Z = -2.74, P = 0.006 and day 14,
Z = -2.77, P = 0.0055.).
Heat tolerance comparison for different bed bug strains: Step-function
The baseline LT50 and LT99 (and 95% fiducial limits) estimates for the Harlan susceptible
strain adults at 45?C were 14.3 (13.7?14.8) and 23.21 (21.7?25.48) mins, respectively (Table 2).
Empirical data showed that 100% mortality of the Harlan adults as well as 4th ? 5th instar
7 / 17
Fig 3. A. A large nymph that survived heat exposure, but was unable to complete the molting process. B. A magnified
view of a heat exposed bed bug shown in the left image. This insect was attempting to molt, but failed to escape its
exoskeleton. The epicranial suture is circled in white appears to have opened, but the bed bug failed to escape through
it. C. Depicted in the image from left to right are three heat exposed nymphs that failed to successfully molt to next
instar after heat exposure. On the right is an exuvia from a nymph that did successfully molt. Photo credit: John
nymphs could be achieved with a 22-min exposure (Figures A and B in S1 File). Some
differences were observed in the responses of different populations to heat exposure at the LT50
level, wherein the KVS strain showed significantly higher heat tolerance or resistance ratios
in comparison to the Harlan, Raleigh, Hackensack, Richmond and Poultry House strains
(P<0.05; Table 2 and Table A in S1 File). However, the LT99 values of the KVS strain were
not significantly different from the Harlan and all field strains (P>0.05; Table 2 and Table B in
In spite of the lack of statistical support for differences in LT99 values for different strains, it
was observed that strains with previous heat exposure histories Raleigh and McCall had lower
LT99 estimates (22.3?26.3 mins) in comparison to some other populations such as Bradenton,
Knoxville, KVS and Poultry House (LT99 of 27.6 to 29.2 mins). These populations with the
highest LT99 values also tended to have the highest predicted LT50 values, except the Poultry
House strain, which had an LT50 value close to that of the Harlan strain. No correlation
was observed between the latitude of collection location and the LT50 or LT99 estimates for
8 / 17
Fig 4. Bars representing percentage of Harlan nymphs from heat exposed (dark grey bars) and control (white bars) treatments that fed to repletion. Bed bugs
that survived heat exposure at 45?C were offered blood meals at five, eight, eleven, and fourteen days after treatment (n = 40 per replicate). An equal number of
control bed bugs that were not exposed to heat were offered a blood meal at the same time intervals. Statistically significant differences were found between the two
treatment types and are denoted with an asterisks ( ). Nonparametric Wilcoxon tests showed that at all feeding intervals feeding responses of heat-exposed and
control nymphs were significantly different (P<0.05). Error bars indicate ? standard error (SE) values.
different field strains (LT50: R2 = 0.19, P > 0.21, LT99: R2 = 0.23, P > 0.16). Similarly, LT50 and
LT99 estimates of the strains with documented history of insecticide resistance (Richmond,
Knoxville and Poultry house) were not significantly different than that of the susceptible
Harlan strain (P>0.05; Table 2, Tables A and B in S1 File). Lastly, no control mortality occurred in
any of the bioassay experiments.
i Lethal time (LT50 and LT99) values with 95% fiducial limits (FL).
All values are expressed in mins. LT values within each column or category (i.e., LT50 or LT99) that are not connected by the same letter are significantly different as
their confidence intervals do not overlap with the number ?1? [
9 / 17
Heat tolerance comparisons for different bed bug strains: Ramp-function
No variability was found in temperature tolerance of bed bug populations in the
ramp-function heat exposure bioassays conducted at temperatures between 25 to 45?C (data not shown).
Complete (100%) mortality was achieved for all strains (ANOVA results, df = 9, 20, P > 0.99)
including the Harlan population. No mortality was observed in untreated controls.
Factors affecting heat resistance development in bed bugs
When inside a human dwelling, bed bugs face a variety of challenges, such as starvation,
desiccation, damage by traumatic insemination and local extinction through the implementation of
pest management strategies. In comparison to other control strategies such as the use of
insecticides, how bed bug populations respond to thermal challenges has been less studied. Late
instar (4th?5th) nymphs were utilized to determine if a C. lectularius laboratory population
could develop heat resistance. This life stage was chosen because the 4th?5th nymphs are close
in size to adults, but are still sexually immature. Therefore, these individuals were capable of
reproduction only if they survived heat selection and successfully molted to the adult life stage.
Additionally, no significant differences in temperature tolerance were observed between late
instar nymphs and adults (Figures A and B in S1 File).
The Harlan population was selected for heat resistance by exposing them to a
pre-determined LT75 over the F0 to F7 generations (Fig 2). During the selection regime, increased
survivorship was initially seen for the F2 and F3 generations. However, when F4 nymphs were heat
selected, their survivorship decreased relative to previous generations. In subsequent
generations (F5 to F7), survivorship declined further. Although some insects initially survived the
heat selection in the F7 generation, none survived long enough to establish the F8 generation
and eventually selection could not proceed further. Previous heat selection experiments with
other insect species have used a variety of techniques to determine if selection for heat
resistance is possible. Laboratory experiments that used ramp-function heat to select D.
melanogaster found a significant increase survivorship up to the F4 generation; however, survivorship
was not reported after this generation [
]. When two D. melanogaster populations were
reared at different temperatures for 4 years, the population reared at higher temperature was
better at tolerating step-function heat exposure [
]. However, rearing bed bugs at
temperatures greater than 30?C in order to select them for temperature tolerance would likely not
select them for heat resistance since research has shown that rearing bed bugs at these
temperatures causes mortality, sterility, and developmental issues [
33, 34, 38
The initial increase in survivorship followed by a decline in survivorship indicates that bed
bugs may have a limited ability to develop greater temperature resistance in a laboratory
setting. This could be due to many factors. One of the factors affecting the ability of bed bugs to
develop heat resistance, could be the lack of genetic diversity in a laboratory colony (Harlan
strain). Adapting insects to laboratory conditions can reduce the genetic diversity of a
population compared to wild type populations, which has previously occurred with the sandfly,
Lutzomyia longipalpis [
]. Genetic diversity of laboratory colonies such as the Harlan strain can
also be reduced when in culture for a long duration. Kim et al. [
] found that the Western
corn rootworm had decreased genetic diversity when in culture for ~190 generations.
Similarly, older laboratory colonies of D. melanogaster experience reduced genetic diversity in
comparison to recently established colonies [
]. Additionally, genetic studies on bed bugs
have found that field populations have low genetic diversity within populations [
] and the
10 / 17
Harlan population is likely no different. Given the low genetic diversity within different bed
bug populations in general, the findings of the laboratory selection study likely also hold true
for field populations, i.e., the ability of bed bugs to develop stable and significant levels of heat
resistance in a field setting could be very limited.
In addition to the low genetic diversity within bed bug populations, the sublethal effects of
heat exposure observed in this study, which were consistent with other studies [
15, 33, 34, 38
may further constrain the ability of bed bugs to develop heat resistance. Similar to previous
research  when bed bugs were exposed to sublethal heat they were initially stunned (Fig
1C) and could not walk, but some recovered and were capable of movement after 24 h. Bed
bugs require blood meals in order to molt and reproduce successfully [
heatexposed bed bugs showed a significantly reduced feeding preference relative to control
nymphs for up to 14 days post exposure (Fig 4). Similarly, in another study, reduction in bed
bug feeding was observed after exposure to sublethal levels of steam [
]. Bed bugs that did not
feed after heat exposure could have been avoiding further stress associated with consuming a
hot blood meal. Blood feeding has been shown to increase the body temperature and elicit
HSP expression in mosquitoes [
]. It has also been reported that bed bugs that feed on
overheated blood (39?C) will die, likely due to heat stress [
]. Although not investigated in this
study, the heat selection regime may also have impacted bed bug reproduction by eliminating
Wolbachia symbionts [
]. It has been previously shown that rearing bed bugs at 36?C can
significantly reduce Wolbachia cell counts from their mycetomes, which consequently reduces
egg viability for up to 10 weeks after exposure [
]. However, if bed bugs are briefly exposed
to steam, their reproduction is not impacted [
]. This indicates that bed bugs must be
heatexposed for a longer duration to eliminate their Wolbachia symbionts. In the future,
quantitative PCR experiments could be conducted to determine the heat exposure duration required
to eliminate the microbial symbionts of bed bugs using the step-function or ramp-function
In some instances, we found that nymphs that survived the step-function heat exposure
failed to escape from their exuvia during molting (Fig 3). Experiments with the flesh fly,
Sarcophaga crassipalpis, found that some adults were unable to successfully eclose from the
puparium after sublethal heat exposure [
]. Similar to findings mentioned above, Rukke et al. [
reported that C. lectularius nymphs reared at temperatures between 34 ?; 38?C for two to three
weeks failed to molt properly. Studies with other arthropods have shown that physiological
adjustments required for overcoming heat stress also have deleterious effects on reproduction
and development [
The deleterious effects of heat exposure on bed bugs, such as reduced blood feeding and
molting abnormalities, likely became an important factor regarding survivorship and
developmental ability of the heat-selected strain beyond the F4 generation (Fig 2). Eventually, the
heat-selected colony died out completely after the F7 heat exposure experiment. If the
heatassociated sublethal effects of this study are extrapolated to the field, the heat-exposed bed
bugs that survive may be less successful in passing their genes to the next generation, which
would further reduce the probability of heat resistance evolution.
Minimal variation in thermo-tolerance of bed bug strains
The final goal of this study, was to test the ability of field strains to tolerate heat using both the
step-function and ramp-function heat exposure techniques (Tables 1 and 2, Tables A and B in
S1 File). Another objective of these experiments was to determine the influences that
geographic origin, insecticide resistance status and previous heat exposure history have on
temperature tolerance of bed bug field strains. Adult bed bugs (1:1 ratio of males and females)
11 / 17
were used for the thermo-tolerance bioassays because they are one of the most temperature
tolerant among the mobile life stages [
]. The temperature tolerance of early instar nymphs
was not determined. However, C. hempiterus first instar nymphs have lower temperature
tolerance in comparison to adult C. hempiterus [
] and C. lectularius may be similar in this regard.
Additionally, bed bugs starved for 7 d prior to heat exposure that were used in this study were
likely close to an optimal thermo-tolerant state [
]. Previous research has shown that bed
bugs that were fed 1 d and 21 d prior to heat exposure are less thermo-tolerant than insects fed
9 d fed prior to heat exposure [
]. Devries et al. [
] suggest that there is a metabolic state
around this optimal feeding status that maximizes bed bug thermo-tolerance, but what causes
this relationship between thermo-tolerance and metabolism is unclear.
Using the step function technique, some variability was observed in LT50 times (Table 2
and Table A in S1 File), however, none of the LT99 estimates were significantly different
(Table 2 and Table B in S1 File). No clear patterns emerged with respect to the LT estimates
and previous history of heat exposure, geographic origin or insecticide resistance status. In
comparison to other strains, the bed bug populations that had a history of heat exposure did
not show significantly higher LT99 values (e.g., Raleigh, NC, LT99 22.3 min, McCall, FL, LT99
26.3 min). This could have been due to the variety of demonstrated impacts of heat exposure
found in this study as well the fitness costs documented in other insect species [
15, 33, 34, 38,
]. Secondly, the geographic origin (latitude of collection location) of a bed bug
population also did not influence their temperature tolerance, likely since indoor environments are
relatively stable and based on the preference of the tenant. Bed bugs thus are probably not
exposed to sufficiently variable temperatures over many generations to change their
thermotolerance. In Japan, a study with 30 different Drosophila species found that the temperature
tolerance did not vary by the geographic latitude of a population, but rather the habitat type
(e.g., tree canopy versus open field conditions) . Lastly, pyrethroid resistant strains (e.g.,
Knoxville, Lafayette and Richmond) [
], did not show significantly different
thermo-tolerance based on the LT50 and LT99 values in comparison to the Harlan strain (Table 2, Tables
A and B in S1 File) indicating lack of correlation between insecticide resistance status and heat
tolerance. However, because of the unknown insecticide resistance status of the KVS strain
that shows significant thermo-tolerance at the LT50 level, we could not confirm if heat
tolerance of this strain is associated with pesticide resistance. Previously, abamectin (an avermectin
class insecticide) resistant mites were also shown to have cross-resistance to heat [
since this insecticide is not used for bed bug control, it is likely that they would not develop
cross resistance to heat in this way.
The absence of any significant differences in the thermo-tolerance among bed bug
populations were further verified using the ramp-function exposure technique. With the
rampfunction technique, the temperature is gradually increased, which is similar to how heat is
deployed in the field [
]. This method also allows bugs more time to physiologically
respond to thermal stress. However, complete mortality was achieved in all bed bug
populations that were tested using the ramp-function technique. It is possible that during the process
of establishing colonies of wild type bed bugs in a laboratory setting, the insects may have gone
through a significant bottleneck effect that could have further reduced or eliminated any
substantial differences in thermo-tolerance that were originally present. Additionally, how
arthropods express heat shock proteins, other stress-induced genes, and metabolites such as sugars
and amino acids in a field setting in response to thermal challenges is not well understood.
Instead of increasing expression of HSPs and stress-induced genes to survive heat exposure, a
more optimal response could be to flee to cooler areas to avoid heat stress, and this appears to
be the case when bed bugs are exposed to heat . Bed bugs express heat shock proteins
when heat exposed and it has been shown that they have 13 HSP genes [
]. However, HSP
12 / 17
gene expression profiles for the bed bug populations used in this study in response to heat
exposure are yet to be determined.
With respect to the role of metabolites in thermal tolerance, Belgica antartica is known to
increase internal concentrations of trehalose to become more tolerant to both heat and cold
]. Arthropods can also increase the proportion of saturated lipids and cuticular
hydrocarbons (e.g., n-alkanes) in their cell membrane and cuticles, respectively, to help reduce water
loss and aid in temperature tolerance [
]. In response to rising environmental
temperatures, Orchesella cincta can increase the proportion of saturated lipids in their cellular
membranes . Similarly, when Pogonomyrex barbatus were exposed to higher temperatures and
lower humidity for 20 days, they increased the proportion of saturated cuticular hydrocarbons
in their exoskeleton . Bed bugs are similar to desert-adapted arthropods in their ability to
withstand desiccation [
] and have also shown the ability to evolve modified cuticles to resist
]. However, the roles of metabolites (trehalose) and changes in cuticular
hydrocarbon profiles in bed bug heat tolerance are not known and should be further investigated.
It is possible that small differences in LT50 and LT99 durations of different populations
(Table 2), although mostly non-significant, could allow some populations such as KVS, Poultry
house, and Bradenton to escape insufficiently heated areas in the field more effectively than
other bed bug populations. Research indicates that if bed bugs are exposed to sublethal
temperatures or if the heat in an area is uneven, they would move to an area with more suitable
]. Currently, the bed bug strains tested in this study are being examined for
differences in their heat repellency behavior by exposing them to rising environmental
temperatures in harborages that are gradually heated (ramp-function method.
Implications for bed bug control
The range of sublethal impacts caused by heat exposure as well as the upper physiological
limits of C. lectularius heat tolerance has implications for using lethal heat as a control measure
for bed bug elimination. First, if bed bugs remain after a heat treatment or are present in a
follow-up inspection, the chances that these insects have developed any substantial heat
resistance are low. The initial increase followed by a decrease in bed bug survivorship during heat
selection experiments in addition to the plethora of sublethal heat impacts, suggest that
individuals that are more heat resistant are quickly selected against (negative selection in a few
generations). An alternative explanation for insects remaining after a heat treatment is that they
were exposed to sublethal temperatures, escaped from high temperature zones, or were
reintroduced to the domicile [
]. If the resident complains of being bitten by bed bugs shortly
after a heat treatment, the latter explanation is likely; given that heat-exposed bed bugs will
feed at reduced rates for up to two weeks.
In order to ensure that all insects are eliminated within an infestation, temperatures 50?C
as well as a sufficient exposure period are required [
], especially if bed bugs are suspected to
be harboring deep within objects. Monitoring temperatures throughout heated areas in order
to identify heat sinks and/or insulated areas is critical for complete bed bug elimination [
Since bed bugs have been shown to travel long distances within an infestation [
] and can
detect heated objects [
], they will likely flee to cooler spots or adjacent housing units if
sublethal temperatures are used during thermal remediation [
]. Therefore, interception
measures should be utilized to trap bed bugs within areas that are heated to 50?C or higher.
Placing traps, sealing wall cracks or electrical outlets, and applying silicate dusts or insecticides
to create a barrier would prevent bed bugs from escaping. Additionally, if there are areas that
are not reaching temperatures 50?C, then insecticides can later be applied as spot treatments
to those areas and other control strategies can be deployed . This is a well-known practice
13 / 17
that is already utilized by some pest management companies. It is important to note that heat
is one of many tools available for bed bug elimination and should be deployed with other IPM
strategies and insecticides to maximize control.
S1 File. Supporting Figures and Tables.
S2 File. Raw data from all experiments.
heat-exposed bed bugs.
We thank the many university researchers and pest management professionals who helped
collect bed bug populations used in this study, and John Obermeyer for photographing the
Conceptualization: Aaron R. Ashbrook, Michael E. Scharf, Gary W. Bennett, Ameya D.
Data curation: Aaron R. Ashbrook, Ameya D. Gondhalekar.
Formal analysis: Aaron R. Ashbrook, Ameya D. Gondhalekar.
Funding acquisition: Michael E. Scharf, Gary W. Bennett, Ameya D. Gondhalekar.
Investigation: Aaron R. Ashbrook, Ameya D. Gondhalekar.
Methodology: Aaron R. Ashbrook, Michael E. Scharf, Ameya D. Gondhalekar.
Project administration: Ameya D. Gondhalekar.
Resources: Ameya D. Gondhalekar.
Supervision: Michael E. Scharf, Ameya D. Gondhalekar.
Validation: Aaron R. Ashbrook, Ameya D. Gondhalekar.
Visualization: Ameya D. Gondhalekar.
Writing ? original draft: Aaron R. Ashbrook, Ameya D. Gondhalekar.
Writing ? review & editing: Aaron R. Ashbrook, Michael E. Scharf, Gary W. Bennett, Ameya
14 / 17
15 / 17
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