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)

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. (Heteroptera: 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 involvement 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 signiÞcant 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, GangloffKaufmann 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 turnovers 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 and Immunology, The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, e-mail: [email protected].

attributed to second-hand goods and furniture, increased 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 analogs, 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 developed 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). Pyrethrin resistance has been reported as a consequence of DDT cross-resistance (Busvine 1958). Pyrethrin and pyrethroid resistance in bed bugs is of considerable public health importance because a main incentive 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 nonchemical means for bed bug control need to be explored. Recent alternative ideas for control of arthropods of medical, veterinary, and agricultural importance include the targeting or manipulation of obligate endosymbiotic bacteria required by the arthropod (Beard et al. 1998). The idea is that by manipulating or eliminating symbionts required by the arthropod for bloodmeal digestion, reproduction, or development,

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

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SAKAMOTO AND RASGON: Wolbachia IN C. lectularius

arthropods can be sterilized or killed, resulting in reduction or elimination of populations. These strategies have the potential to be efÞcacious and costeffective. In principle, endosymbionts can be targeted speciÞcally, and thus these strategies should be safe and have minimal nontarget effects. Wolbachia is an ␣-proteobacterium that infects 20 Ð 70% of known arthropods and many Þlarial nematodes (Werren et al. 1995, Jeyaprakash and Hoy 2000). Wolbachia infections are associated with reproductive host effects including male killing, parthenogenesis, cytoplasmic incompatibility, and feminization (Werren 1997). Wolbachia have been previously described from the human bed bug and several other cimicids (Hypsa and Aksoy 1997, Rasgon and Scott 2004, Sakamoto et al. 2006). It is possible that Wolbachia might be useful in bed bug control strategies, either as a means of manipulating populations or as a novel target for control efforts. However, it is necessary to determine how prevalent Wolbachia infections are in wild bugs before developing such strategies. Previous studies investigating Wolbachia presence in C. lectularius relied on colony specimens or single wild specimens, but thus far the prevalence of infection of wild bed bug populations is unknown (Hypsa and Aksoy 1997, Rasgon and Scott 2004, Sakamoto et al. 2006). This study was undertaken to investigate the extent of Wolbachia infections in wild C. lectularius populations. We examined infections from Þve North American and one African geographic regions. Our data suggest that Wolbachia is prevalent in C. lectularius.

Table 1. coordinates

697

Date of specimen collection, locality, and GPS

Locality

Longitude

Latitude

Date collected

CA: Long Beach CA: Mixed sample CT: Stamford FL: Juno Beach FL: Miami FL: Largo NY: Deerpark NY: Geneva NY: Ithaca 2003 NY: Ithaca 2005 NY: Nesconset NY: E. Northport NY: Woodside NY: Mixed sample Toronto, Ontario: Shelter 1 Toronto, Ontario: Shelter 2 Zambia (B8) Zambia (C7) Zambia (Chid 2) Zambia (Lup) Zambia (WP12-A10) Zambia (WP44) Zambia (WP107) Zambia (WP108) Zambia (WP110) Zambia (WP111) Zambia (WP127) Zambia (WP129) Zambia (WP130) Zambia (WP131) Zambia (WP133)

33⬚ 46⬘ N Ñ 41⬚ 02⬘ N 26⬚ 49⬘ N 25⬚ 46⬘ N 27⬚ 54⬘ N 40⬚ 46⬘ N 42⬚ 51⬘ N 42⬚ 26⬘ N 42⬚ 26⬘ N 40⬚ 50⬘ N 40⬚ 50⬘ N 40⬚ 44⬘ N Ñ 43⬚ 39⬘ N 43⬚ 39⬘ N 16⬚ 29⬘ S 16⬚ 15⬘ S 16⬚ 15⬘ S 16⬚ 12⬘ S 16⬚ 19⬘ S 16⬚ 28⬘ S 16⬚ 21⬘ S 16⬚ 21⬘ S 16⬚ 21⬘ S 16⬚ 21⬘ S 16⬚ 22⬘ S 16⬚ 21⬘ S 16⬚ 21⬘ S 16⬚ 22⬘ S 16⬚ 22⬘ S

118⬚ 11⬘ W Ñ 73⬚ 32⬘ W 80⬚ 03⬘ W 80⬚ 11⬘ W 82⬚ 47⬘ W 73⬚ 18⬘ W 76⬚ 59⬘ W 76⬚ 29⬘ W 76⬚ 29⬘ W 73⬚ 08⬘ W 73⬚ 10⬘ W 73⬚ 53⬘ W Ñ 79⬚ 22⬘ W 79⬚ 22⬘ W 26⬚ 51⬘ E 26⬚ 51⬘ E 26⬚ 28⬘ E 26⬚ 27⬘ E 26⬚ 34⬘ E 26⬚ 56⬘ E 26⬚ 46⬘ E 26⬚ 46⬘ E 26⬚ 46⬘ E 26⬚ 45⬘ E 26⬚ 45⬘ E 26⬚ 45⬘ E 26⬚ 45⬘ E 26⬚ 45⬘ E 26⬚ 44⬘ E

Nov. 2004 Aug. 2004 2004Ð2005 Jun. 2005 Apr. 2005 Aug. 2005 Aug. 2005 Aug. 2005 May 2003 Jun. 2005 Sept. 2005 Sept. 2005 Sept. 2005 Aug. 2005 Sept. 2005 Sept. 2005 Feb. 2005 Feb. 2005 Feb. 2005 Jan. 2005 Jan. 2005 Jan. 2005 May 2004 May 2004 May 2004 May 2004 May 2004 May 2004 May 2004 May 2004 May 2004

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 further processing. DNA Extraction. Nonvoucher specimens were ground with sterilized plastic pestles, digested and extracted using DNEasy Mini Spin columns (QIAGEN, Valencia, CA) according to the manufacturers suggested protocol. For specimens kept as vouchers, we used a minimally destructive method for DNA extraction (Sakamoto et al. 2006) to preserve external morphology 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 fragment from Wolbachia 16S rDNA. Because the size of the amplicon is small, these primers consistently amplify Wolbachia DNA even from degraded infected specimens (Sakamoto et al. 2006). Each 25-␮l reaction consisted of 1 ␮l of template DNA, 0.4 ␮M each primer (INTF2, 5⬘-AGTCATCATGGCCTTTATGGA-3⬘; INTR2, 5⬘-TCATGTACTCGAGTTGCAGAGT-3⬘), 0.4 mM dNTPs, and 2.5 U of Taq Polymerase. 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 consistently failed to amplify Wolbachia-speciÞc frag-

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ments, we tested DNA template quality by attempting to amplify an ⬇400-bp fragment of the insect mitochondrial 12S rDNA (Simon et al. 1991). Primer sequences are as follows: 12SA1, 5⬘AAACTAGGATTAGATACCCTATTAT-3⬘ and 12SB1, 5⬘-AAGAGCGACGGGCGATGTGT-3⬘. PCR products were separated and visualized as described previously. Specimens that failed to amplify both Wolbachia and insect mitochondrial DNA were excluded from the analysis. Statistical Analysis. Differences in infection frequency between geographic regions were statistically compared by G test. Differences in infection frequency between sexes and between life stages were statistically compared by Fisher exact test. Ninety-Þve percent exact conÞdence intervals were calculated from the binomial distribution (Sokal and Rohlf 2003). Results Bed bug specimens from four states in the United States (California, New York, Florida, and Connecticut); from Toronto, Ontario, Canada; and from Macha, Zambia, were surveyed for incidence of Wolbachia infections. In total, 201 specimens were surveyed, and 191 specimens were positive for Wolbachia. Infection frequencies among geographic regions (California, Connecticut, Florida, New York, Toronto, and Zambia) ranged from 0.833 to 1.0, with a total frequency for all collections of 0.95 (Table 2). Infection frequencies among geographic regions did not differ statistically (G ⫽ 8.71, df ⫽ 5, P ⫽ 0.12). There were no statistically signiÞcant differences in infection frequency between sexes (female: 0.935, n ⫽ 75; male: 0.942, n ⫽ 52; Fisher exact test, two-tail P ⫽ 1.0), or between life stages (adults: 0.938, n ⫽ 119; nymphs: 0.946, n ⫽ 74; Fisher exact test, two-tail P ⫽ 1.0).

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Table 2. Wolbachia infection rates in C. lectularius as determined by PCR Region California

Pop.

n

Long Beach 1 Mixed sample 5 CA subtotal 6 Connecticut Stamford 66 Florida Juno Beach 2 Miami 20 Largo 3 FL subtotal 25 New York Deerpark 6 Geneva 1 Ithaca 2003 7 Ithaca 2005 6 Nesconset 2 E. Northport 1 Woodside 17 Mixed sample 8 NY subtotal 48 Toronto, Ontario Shelter 1 1 Shelter 2 9 Toronto subtotal 10 Zambia B8 6 C7 1 Chid 2 1 Lup 2 WP12-A10 1 WP44 1 WP107 7 WP108 1 WP110 1 WP111 6 WP127 3 WP129 1 WP130 11 WP131 2 WP133 2 Zambia subtotal 46 Total 201

Infection Frequency

95% CI

1.0 0.8 0.833 0.894 1.0 0.9 1.0 0.92 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.833 1.0 1.0 1.0 0.0 1.0 1.0 1.0 1.0 0.833 1.0 1.0 1.0 1.0 1.0 0.956 0.95

0.050, 1.000 0.284, 0.995 0.359, 0.996 0.767, 0.950 0.224, 1.000 0.683, 0.988 0.368, 1.000 0.740, 0.990 0.607, 1.000 0.050, 1.000 0.652, 1.000 0.607, 1.000 0.224, 1.000 0.050, 1.000 0.838, 1.000 0.688, 1.000 0.940, 1.000 0.050, 1.000 0.707, 1.000 0.741, 1.000 0.359, 0.996 0.050, 1.000 0.050, 1.000 0.224, 1.000 0.000, 0.950 0.050, 1.000 0.652, 1.000 0.050, 1.000 0.050, 1.000 0.359, 0.996 0.368, 1.000 0.050, 1.000 0.762, 1.000 0.050, 1.000 0.050, 1.000 0.852, 0.995 0.910, 0.976

The 95% exact conÞdence intervals were calculated from the binomial distribution.

Discussion Evolution of pesticide resistance has led to interest in novel control methods for medically important arthropods. One avenue of research includes manipulation of endosymbiotic bacteria carried by target arthropods. For control strategies exploiting endosymbionts to be developed, the endosymbiotic microbiota of target arthropods needs to be characterized. Targeting an inßuential bacterium for control strategies 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). Bu¨ chner described transovarial transmission of bacteriome endosymbionts via the nurse cells and nutritive cords to the developing ova (cited by Usinger 1966). Speculations have arisen as to the role that individual endosymbionts 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 containing 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. lectularius and the closely related species Cimex columbarius (Jenyns), (sometimes considered a subspecies), 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 reciprocal cross was fertile (Ueshima 1964). Similar reductions 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 oviposition (Usinger 1966). If an unmated female develops eggs, they generally do not develop and are resorbed (Usinger 1966). Cytoplasmic incompatibility

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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 symbionts 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 University, Ithaca, NY), William H. Kern (University of Florida, Fort Lauderdale, FL), Peggy Nusser (Lady Bug Pest Control, Fort Lauderdale, FL), Lynn Frank (Suburban Services, Smithtown, 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 Florida, 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 discussions regarding bed bug biology. Funding was provided by the Johns Hopkins Malaria Research Institute to J.L.R.

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Werren, J. H. 1997. Biology of Wolbachia. Annu. Rev. Entomol. 42: 587Ð 609. [WHO] World Health Organization. 1997. Chapter 4. Bedbugs, ßeas, lice, ticks and mites: ectoparasites that live on the body, in clothing and in beds, pp. 237Ð243. In Vector control series. World Health Organization, Geneva, Switzerland. Received 29 November 2005; accepted 12 March 2006.