POPULATION BIOLOGY/GENETICS

Geographic Distribution of Wolbachia Infections in Cimex lectularius

(Heteroptera: Cimicidae)
JOYCE M. SAKAMOTO1 AND JASON L. RASGON2

J. Med. Entomol. 43(4): 696Ð700 (2006)

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ABSTRACT Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory
colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information
exists regarding the extent of infection in natural populations. We assayed C. lectularius populations
from Þve North American regions (California, Connecticut, Florida, New York, and Toronto, Canada)
and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction
(PCR). Wolbachia infections were prevalent in all populations assayed (83Ð100%). There were no
signiÞcant differences in infection frequency between geographic regions, between sexes, or between
life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control
strategies is discussed.

KEY WORDS Wolbachia, Cimex lectularius, endosymbiont, pesticide resistance, control strategies

The human bed bug, Cimex lectularius L. (Heterop-
tera: Cimicidae) has been found in association with
humans since ancient times (Panagiotakopulu and
Buckland 1999). Human bed bugs are not known to
vector any pathogens in the wild, although there is
laboratory evidence to suggest their potential involve-
ment in mechanical transmission of hepatitis B virus
(Blow et al. 2001). Bed bug infestations have often
been considered a consequence of poor hygiene and
poverty, and indeed, infestations in ⬎50% of homes
have been common in developing countries (Temu et
al. 1999, Boase 2001). Bed bugs have not been a sig-
niÞcant household pest in developed countries since
the development and widespread use of DDT after the
beginning of World War II, and changes in sanitary
standards (Usinger 1966, Temu et al. 1999, Gangloff-
Kaufmann and Shultz 2003). Within the last 20 yr,
however, there have been an increasing number of
reports of human bed bug infestations in developed
countries (King et al. 1989, Krueger 2000, Boase 2001).
Areas afßicted are often places of high occupant turn-
overs such as short-term lodgings, apartments, college
dormitories, prisons, and homeless shelters (Jones
2004, Hwang et al. 2005). Although bed bugs have
traditionally been associated with poverty and poor
hygiene, bed bug infestations have recently become a
troublesome liability for hotels (Posner et al. 2003)
and other short-stay lodges (Ryan et al. 2004). The
increased incidence of bed bug outbreaks has been
1 Department of Microbiology and Immunology, University of
Maryland School of Medicine, Baltimore, MD 21201. Corresponding
author.
2 The W. Harry Feinstone Department of Molecular Microbiology
attributed to second-hand goods and furniture, in-
creased international trafÞc, and growing pesticide
resistance (King et al. 1989, Boase 2001, Jones 2004).
Current chemical methods used to control bed
bugs include pyrethroids, insect juvenile hormone an-
alogs, and organophosphates such as malathion (Boase
2001, Jacobs 2003). It has been noted that bed bug
populations quickly develop resistance to chemical
pesticides. Shortly after the start of World War II,
the use of DDT reduced bed bug populations in de-
veloped countries (Usinger 1966). DDT resistance
was Þrst reported in 1947 and since been observed in
both C. lectularius and the tropical bed bug Cimex
hemipterus (F.) (Busvine 1957, Usinger 1966). By
1971, bed bugs were described as being resistant to
organophosphates (e.g., malathion) (Feroz 1971). Py-
rethrin resistance has been reported as a consequence
of DDT cross-resistance (Busvine 1958). Pyrethrin
and pyrethroid resistance in bed bugs is of consider-
able public health importance because a main incen-
tive to use of pyrethroid-treated bed-nets for control
of malaria-transmitting mosquitoes is bed bug control
(WHO 1997, Temu et al. 1999). Pyrethroid resistance
in bed bugs has led to a reluctance of villagers to
continue treating bed-nets, resulting in increased
risk for contracting malaria (WHO 1997, Myamba et al.
2002). In light of these examples, alternative non-
chemical means for bed bug control need to be ex-
plored.
Recent alternative ideas for control of arthropods of
medical, veterinary, and agricultural importance in-
clude the targeting or manipulation of obligate endo-
symbiotic bacteria required by the arthropod (Beard

and Immunology, The Johns Hopkins Malaria Research Institute,

et al. 1998). The idea is that by manipulating or elim-
Bloomberg School of Public Health, Johns Hopkins University, Bal- inating symbionts required by the arthropod for
timore, MD 21205, e-mail: jrasgon@jhsph.edu. bloodmeal digestion, reproduction, or development,

0022-2585/06/0696Ð0700$04.00/0 䉷 2006 Entomological Society of America

July 2006 SAKAMOTO AND RASGON: Wolbachia IN C. lectularius 697

arthropods can be sterilized or killed, resulting in Table 1. Date of specimen collection, locality, and GPS
reduction or elimination of populations. These strat- coordinates
egies have the potential to be efÞcacious and cost-
Date
effective. In principle, endosymbionts can be targeted Locality Longitude Latitude
collected
speciÞcally, and thus these strategies should be safe
CA: Long Beach 33⬚ 46⬘ N 118⬚ 11⬘ W Nov. 2004
and have minimal nontarget effects. CA: Mixed sample Ñ Ñ Aug. 2004
Wolbachia is an ␣-proteobacterium that infects 20 Ð CT: Stamford 41⬚ 02⬘ N 73⬚ 32⬘ W 2004Ð2005
70% of known arthropods and many Þlarial nematodes FL: Juno Beach 26⬚ 49⬘ N 80⬚ 03⬘ W Jun. 2005
(Werren et al. 1995, Jeyaprakash and Hoy 2000). Wol- FL: Miami 25⬚ 46⬘ N 80⬚ 11⬘ W Apr. 2005
FL: Largo 27⬚ 54⬘ N 82⬚ 47⬘ W Aug. 2005
bachia infections are associated with reproductive NY: Deerpark 40⬚ 46⬘ N 73⬚ 18⬘ W Aug. 2005
host effects including male killing, parthenogenesis, NY: Geneva 42⬚ 51⬘ N 76⬚ 59⬘ W Aug. 2005
cytoplasmic incompatibility, and feminization (Wer- NY: Ithaca 2003 42⬚ 26⬘ N 76⬚ 29⬘ W May 2003
NY: Ithaca 2005 42⬚ 26⬘ N 76⬚ 29⬘ W Jun. 2005

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ren 1997). Wolbachia have been previously described
NY: Nesconset 40⬚ 50⬘ N 73⬚ 08⬘ W Sept. 2005
from the human bed bug and several other cimicids NY: E. Northport 40⬚ 50⬘ N 73⬚ 10⬘ W Sept. 2005
(Hypsa and Aksoy 1997, Rasgon and Scott 2004, Saka- NY: Woodside 40⬚ 44⬘ N 73⬚ 53⬘ W Sept. 2005
moto et al. 2006). It is possible that Wolbachia might NY: Mixed sample Ñ Ñ Aug. 2005
be useful in bed bug control strategies, either as a Toronto, Ontario: Shelter 1 43⬚ 39⬘ N 79⬚ 22⬘ W Sept. 2005
Toronto, Ontario: Shelter 2 43⬚ 39⬘ N 79⬚ 22⬘ W Sept. 2005
means of manipulating populations or as a novel target Zambia (B8) 16⬚ 29⬘ S 26⬚ 51⬘ E Feb. 2005
for control efforts. However, it is necessary to deter- Zambia (C7) 16⬚ 15⬘ S 26⬚ 51⬘ E Feb. 2005
mine how prevalent Wolbachia infections are in wild Zambia (Chid 2) 16⬚ 15⬘ S 26⬚ 28⬘ E Feb. 2005
bugs before developing such strategies. Previous stud- Zambia (Lup) 16⬚ 12⬘ S 26⬚ 27⬘ E Jan. 2005
Zambia (WP12-A10) 16⬚ 19⬘ S 26⬚ 34⬘ E Jan. 2005
ies investigating Wolbachia presence in C. lectularius Zambia (WP44) 16⬚ 28⬘ S 26⬚ 56⬘ E Jan. 2005
relied on colony specimens or single wild specimens, Zambia (WP107) 16⬚ 21⬘ S 26⬚ 46⬘ E May 2004
but thus far the prevalence of infection of wild bed bug Zambia (WP108) 16⬚ 21⬘ S 26⬚ 46⬘ E May 2004
populations is unknown (Hypsa and Aksoy 1997, Ras- Zambia (WP110) 16⬚ 21⬘ S 26⬚ 46⬘ E May 2004
Zambia (WP111) 16⬚ 21⬘ S 26⬚ 45⬘ E May 2004
gon and Scott 2004, Sakamoto et al. 2006). This study Zambia (WP127) 16⬚ 22⬘ S 26⬚ 45⬘ E May 2004
was undertaken to investigate the extent of Wolbachia Zambia (WP129) 16⬚ 21⬘ S 26⬚ 45⬘ E May 2004
infections in wild C. lectularius populations. We ex- Zambia (WP130) 16⬚ 21⬘ S 26⬚ 45⬘ E May 2004
amined infections from Þve North American and one Zambia (WP131) 16⬚ 22⬘ S 26⬚ 45⬘ E May 2004
Zambia (WP133) 16⬚ 22⬘ S 26⬚ 44⬘ E May 2004
African geographic regions. Our data suggest that
Wolbachia is prevalent in C. lectularius. GPS, global positioning system.
Seconds are omitted from coordinates to preserve anonymity of
infested dwellings.

Materials and Methods

Insect Samples. Wild specimens were collected
from infested homes and homeless shelters, and from
human hosts inhabiting these dwellings in 2004 Ð2005.
Collection localities are listed in Table 1. Specimens
were placed into either 100% ethanol or dried with
silica desiccant and transported to the Johns Hopkins
Bloomberg School of Public Health (JHSPH) for fur-
ther processing.
DNA Extraction. Nonvoucher specimens were
ground with sterilized plastic pestles, digested and ex-
tracted using DNEasy Mini Spin columns (QIAGEN,
Valencia, CA) according to the manufacturers sug-
gested protocol. For specimens kept as vouchers, we
used a minimally destructive method for DNA extrac-
tion (Sakamoto et al. 2006) to preserve external mor-
phology of processed insects, and thus their value as
museum specimens. Insect abdomens were cut with a
sterile microscalpel (or pricked with a microdissecting
needle for nymphs), and specimens were digested
overnight (⬇18 h) in 180 ␮l of 1⫻ phosphate-buffered
saline, 20 ␮l of Proteinase K, and 200 ␮l of AL buffer
solution (QIAGEN). Digestate was vortexed with 200 ml
of 100% cold ethanol, applied onto DNEasy columns and
DNA bound, and washed and eluted according to the
manufacturerÕs suggested protocol. Exoskeletons were
mounted on slides using Euparal permanent mounting
medium (Bioquip Products, Rancho Dominguez, CA).
Wolbachia-Specific Polymerase Chain Reaction
(PCR). All PCR was conducted using known infected
colony C. lectularius specimens as a positive control
and a reaction containing all PCR ingredients except
template DNA as a negative control. Because DNA
template quality was unknown, we used a newly
described primer set (INTF2, INTR2) (Sakamoto
et al. 2006) that speciÞcally ampliÞes a 136-bp frag-
ment from Wolbachia 16S rDNA. Because the size of
the amplicon is small, these primers consistently
amplify Wolbachia DNA even from degraded in-
fected specimens (Sakamoto et al. 2006). Each 25-␮l
reaction consisted of 1 ␮l of template DNA, 0.4 ␮M
each primer (INTF2, 5⬘-AGTCATCATGGCCTTT-
ATGGA-3⬘; INTR2, 5⬘-TCATGTACTCGAGTTGCA-
GAGT-3⬘), 0.4 mM dNTPs, and 2.5 U of Taq Polymer-
ase. Fragments were ampliÞed on a PTC thermocycler
(Bio-Rad, Hercules, CA) using a program of 95⬚C for
5 min, followed by 40 cycles of 1 min each 95, 55, 72⬚C,
and a Þnal extension of 72⬚C for 5 min. Fragments were
separated by 1% agarose gel electrophoresis, stained
with ethidium bromide, and visualized by UV light.
SpeciÞc PCR ampliÞcation was conÞrmed by directly
sequencing puriÞed PCR product (Sakamoto et al.
2006).
Insect Mitochondrial DNA. For specimens that con-
sistently failed to amplify Wolbachia-speciÞc frag-
698 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 43, no. 4

ments, we tested DNA template quality by attempting Table 2. Wolbachia infection rates in C. lectularius as deter-
to amplify an ⬇400-bp fragment of the insect mitochon- mined by PCR
drial 12S rDNA (Simon et al. 1991). Primer sequences
Infection
are as follows: 12SA1, 5⬘AAACTAGGATTAGATAC- Region Pop. n 95% CI
Frequency
CCTATTAT-3⬘ and 12SB1, 5⬘-AAGAGCGACGGGC-
California Long Beach 1 1.0 0.050, 1.000
GATGTGT-3⬘. PCR products were separated and visu- Mixed sample 5 0.8 0.284, 0.995
alized as described previously. Specimens that failed to CA subtotal 6 0.833 0.359, 0.996
amplify both Wolbachia and insect mitochondrial DNA Connecticut Stamford 66 0.894 0.767, 0.950
were excluded from the analysis. Florida Juno Beach 2 1.0 0.224, 1.000
Miami 20 0.9 0.683, 0.988
Statistical Analysis. Differences in infection fre- Largo 3 1.0 0.368, 1.000
quency between geographic regions were statistically FL subtotal 25 0.92 0.740, 0.990
compared by G test. Differences in infection fre- New York Deerpark 6 1.0 0.607, 1.000
Geneva 1 1.0 0.050, 1.000

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quency between sexes and between life stages were
Ithaca 2003 7 1.0 0.652, 1.000
statistically compared by Fisher exact test. Ninety-Þve Ithaca 2005 6 1.0 0.607, 1.000
percent exact conÞdence intervals were calculated Nesconset 2 1.0 0.224, 1.000
from the binomial distribution (Sokal and Rohlf 2003). E. Northport 1 1.0 0.050, 1.000
Woodside 17 1.0 0.838, 1.000
Mixed sample 8 1.0 0.688, 1.000
Results NY subtotal 48 1.0 0.940, 1.000
Toronto, Ontario Shelter 1 1 1.0 0.050, 1.000
Bed bug specimens from four states in the United Shelter 2 9 1.0 0.707, 1.000
States (California, New York, Florida, and Connecti- Toronto subtotal 10 1.0 0.741, 1.000
Zambia B8 6 0.833 0.359, 0.996
cut); from Toronto, Ontario, Canada; and from Macha, C7 1 1.0 0.050, 1.000
Zambia, were surveyed for incidence of Wolbachia Chid 2 1 1.0 0.050, 1.000
infections. In total, 201 specimens were surveyed, and Lup 2 1.0 0.224, 1.000
191 specimens were positive for Wolbachia. Infection WP12-A10 1 0.0 0.000, 0.950
WP44 1 1.0 0.050, 1.000
frequencies among geographic regions (California, WP107 7 1.0 0.652, 1.000
Connecticut, Florida, New York, Toronto, and Zam- WP108 1 1.0 0.050, 1.000
bia) ranged from 0.833 to 1.0, with a total frequency for WP110 1 1.0 0.050, 1.000
all collections of 0.95 (Table 2). Infection frequencies WP111 6 0.833 0.359, 0.996
WP127 3 1.0 0.368, 1.000
among geographic regions did not differ statistically WP129 1 1.0 0.050, 1.000
(G ⫽ 8.71, df ⫽ 5, P ⫽ 0.12). There were no statistically WP130 11 1.0 0.762, 1.000
signiÞcant differences in infection frequency between WP131 2 1.0 0.050, 1.000
sexes (female: 0.935, n ⫽ 75; male: 0.942, n ⫽ 52; Fisher WP133 2 1.0 0.050, 1.000
Zambia subtotal 46 0.956 0.852, 0.995
exact test, two-tail P ⫽ 1.0), or between life stages Total 201 0.95 0.910, 0.976
(adults: 0.938, n ⫽ 119; nymphs: 0.946, n ⫽ 74; Fisher
exact test, two-tail P ⫽ 1.0). The 95% exact conÞdence intervals were calculated from the bi-
nomial distribution.

Discussion
Evolution of pesticide resistance has led to interest
in novel control methods for medically important
arthropods. One avenue of research includes manip-
ulation of endosymbiotic bacteria carried by target
arthropods. For control strategies exploiting endo-
symbionts to be developed, the endosymbiotic micro-
biota of target arthropods needs to be characterized.
Targeting an inßuential bacterium for control strate-
gies will not be useful if the endosymbiont does not
occur at high prevalence. We have demonstrated that
Wolbachia infections are prevalent in natural bed bug
populations and as such, may be useful as a potential
target for control efforts.
Multiple endosymbionts have been observed
through light and electron microscopic studies of bed
bug tissues and organs (Arkwright et al. 1921, Chang
and Musgrave 1973, Usinger 1966). Büchner described
transovarial transmission of bacteriome endosymbi-
onts via the nurse cells and nutritive cords to the
developing ova (cited by Usinger 1966). Speculations
have arisen as to the role that individual endosymbi-
onts may play, if any, in bed bug biology. It has been
suggested by De Meillon and Golberg (1947) that bed
bugs might obtain B vitamins from at least one of their
endosymbionts. There is also evidence that blood con-
taining antibiotics may affect the ability of adult bed
bugs to lay viable eggs (Takano-Lee et al. 2003).
The speciÞc effect of Wolbachia on bed bug biology
is unknown. In experimental crosses between C. lectu-
larius and the closely related species Cimex colum-
barius (Jenyns), (sometimes considered a subspe-
cies), CI-like crossing patterns were observed.
SpeciÞcally, there was a signiÞcant decrease in the
number of eggs oviposited when C. lectularius males
mated with C. columbarius females, whereas the re-
ciprocal cross was fertile (Ueshima 1964). Similar re-
ductions in oviposition rate have been noted with
antibiotic treatment (Takano-Lee et al. 2003) and by
curing bed bugs of symbionts by heat (Chang 1974).
Cytoplasmic incompatibility is typically detected by a
reduction in egg hatch rate (Hoffmann and Turelli
1997). Bed bug eggs are fertilized and develop about
one-third to maturity within the ovaries before ovi-
position (Usinger 1966). If an unmated female devel-
ops eggs, they generally do not develop and are
resorbed (Usinger 1966). Cytoplasmic incompatibility
July 2006 SAKAMOTO AND RASGON: Wolbachia IN C. lectularius
expression would result in functionally unfertilized
eggs, which we speculate would be resorbed, resulting
in lower oviposition rates in an incompatible cross
rather than reduced hatch rate. Because multiple sym-
bionts are present in C. lectularius, speciÞc symbiont
curing must be conducted to prove or disprove this
hypothesis.
Acknowledgments
We thank the following people for providing specimens
used in this study: Douglas Norris and Rebekah Kent (Johns
Hopkins University, Baltimore, MD), Neeta Pardanani and
Joseph E. Kuntz (Stamford Health Department, Stamford,
CT), Fred Rozo (Western Exterminator Company, Anaheim,
CA), Laura Harrington and Becky Poulson (Cornell Univer-
sity, Ithaca, NY), William H. Kern (University of Florida, Fort
Lauderdale, FL), Peggy Nusser (Lady Bug Pest Control, Fort
Lauderdale, FL), Lynn Frank (Suburban Services, Smith-
town, NY), Stephen W. Hwang (Centre for Research on
Inner City Health, Toronto, Ontario, Canada), John Mangold
(Terminex International, McDonough, GA), and Susan
Hacker. Special thanks to Brian Cabrera (University of Flor-
ida, Fort Lauderdale, FL) and Harold Harlan (National Pest
Management Association, Fairfax, VA) for insights into new
outbreaks and live specimens and to Robin Todd (Insect
Control and Research, Inc., Baltimore, MD) for helpful dis-
cussions regarding bed bug biology. Funding was provided
by the Johns Hopkins Malaria Research Institute to J.L.R.
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zerland.
Received 29 November 2005; accepted 12 March 2006.
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