VECfOR CONTROL, PEsT MANAGEMENT, RESISTANCE, REPELLENTS

Insecticide Resistance in Bedbugs in Thailand and LaboratoryEvaluation of Insecticides for the Control of Cimex hemipterus and

Cimex lectularius (Hemiptera: Cimicidae)

APIWAT TAWATSIN,t USAVADEE THAVARA,1JAKKRAWAHNCHOMPOOSHI,l YU1THANA PHUSUP/NISAHAT JONJANG,J CHAYADAKHUMSAWADS,J PAYU BHAKDEENUAt~/PATHOM SAWANPANYALERT,l PREECHA ASAVADACHANUKORN,2

Mm S. MULLA,3 PADET SIlUYASATIEN,4AND MUSTAPHA DEBBOUN5

J. Med. Entomol. 48(5): 1023-1030(2011);DOl: 1O.1603/ME11003ABSTRACT Bedbugs are found in many countries around the world, and in some regions they areresistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined theirresistance to insecticides. The surveys were carried out in six provinces that attract large numbers offoreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Hatchathani, Phuket, and Krabi. Bedbugs werecollected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimexhemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimexleciularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani.The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (di-chlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates(malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, A-cyhalothrin,etofenprox) in tests using World Health Organization insecticide-impregnated papers. The newinsecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phe-nylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), car-bamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofen-prox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four differentactive ingredients were effective against the bedbugs. The results obtained from this study suggestedthat both species of bedbugs in Thailand have developed marked resistance to various groups ofinsecticides, especially those in the pyrethroid group, which are the most common insecticides usedfor pest control. Therefore, an integrated pest management should be implemented for managingbedbugs in Thailand.

KEY WORDS bedbugs, insecticides, resistance, control, Thailand

Bedbugs are nocturnal, bloodsucking insects that feedon humans and are mainly important as biting nui-sance. People who are bitten by bedbugs may sufferfrom intense itching, inflammation, and seconclary in-fection after scratching, and sleepless episodes. Bed-bugs have been recognized in association with humans>3,000 yr and were found in Egyptian tombs (Pana-

Use of vertebrate animals was done under Bescarch Protocol Olll2:350 and approved by the Animal Care and Use Committee of theFaculty of Medicine, Chulalongkorn University, Thailand, Certifica-tion 002/2551.l National Institute of Health, Department of Medical Sciences,

Ministry of Public Health, Nonthaburi 11000, Thailand.2 Department of Statistics, Faculty of Commerce and Accountancy,

Chulalongkorn University, Bangkok 10330, Thailand.3 Department of Entomology, University of California, Riverside,

CA 92521.4 Corresponding author: Department of Parasitology, Faculty of

Medicine, Chulalongkorn University, Bangkok 10330, Thailand(e-mail: padet.s@chula.ac.th).

5 United States Army Medical Department Center and School, FortSam Houston, San Antonio, TX 78234.

giotakopulu and Buckland 1999). Recently, bedbugshave been implicated in the transmission of methicil-lin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus [aecium in some patients hos-pitalized in Vancouver, Canada (Lowe and Romney2011) .

Two species of bedbugs are considered as humanpests: the tropical bedbug, Cimex hemipterus (F.),which occurs mainly in tropical countries, and thecommon bedbug, Cimex lectularius L., which appearsin most parts of the world (Harlan et al. 2008). Inrecent years, bedbug infestations have been increas-ingly reported in the United Kingdom (Boase 2008)and other countries, such as Denmark, Norway, Swe-den, Switzerland, Australia, and the United States(Doggett et al. 2004, Kilpinen et al. 2008, Mueller et al.2008, Potter et al. 2008). The reason for this increaseis unknown; however, several factors, such as an in-crease in international travel, reduction in the use ofresidual insecticides indoors, and insecticide resis-

0022-2585/11/1023-10.30$04.00/0 © 2011 Entomological Society of America

1024 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 5

tance, may be contributing to the resurgence (Potter2005).

In Thailand, most people younger than SO yr oldhave never encountered bedbugs because they dis-appeared from the country for decades. Recently, thebedbug infestations have been found in various places,such as hotels and guesthouses providing accommo-dation to foreign travelers. Assorted insecticide prod-ucts for control of crawling pests are currently beingused for bedbug remediation by the pest control com-panies in Thailand. However, these products havenever been empirically tested to determine whetherthey are effective against the local populations of bed-bugs. The purpose of our studies was to determine thepresence or absence of insecticide resistance in bed-bug populations collected from wide geographic areasin Thailand. The potential activities of several insec-ticides commonly used for controlling crawling pestswere also examined for the control of bedbugs.

Materials and Methods

Bedbug Survey and Rearing. The bedbug surveyswere conducted in six provinces: Bangkok, Chonburi,Chiang Mai, Ubon Batchathani, Phuket, and Krabi,which attract large numbers of foreign tourists (Fig.1). Bedbugs were sampled from at least five hotels ineach province. All rooms of the surveyed hotels wereinspected for bedbugs. In each room, a thorough in-spection was carried out of the mattress, bed frame,areas around the bed, and all furniture in the room.The bedbugs found in each hotel were identified usingkeys Pratt and Stojanovich (1967) and then colonizedin the laboratory. A colony of tropical bedbugs, C.hemipterus, from each province was established fromindividuals collected from hotels or guesthouses inBangkok, Phuket, Krabi, or Chonburi. The colony ofcommon bedbugs, C. lectularius, was derived fromindividuals collected from a hotel in Chiang Mai prov-ince. The colonies of bedbugs were reared in thelaboratory and maintained at 26-28°C, 60-80% RH,and a photoperiod of 12:12 (L:D) h. The bedbugs werereared in plastic cups (.360-ml capacity, 12 cm high,8-cm-wide mouth, and 5-cm diameter at the bottom)covered with fine mesh chiffon cloth and fitted withrubber bands. Inside the cups, 10 pieces of cardboard(4 X 8 cm) were folded and stood on edge so that thebedbugs could crawl up the cardboard and insert theirmouthparts through the mesh top to feed. Once eachweek, a hamster host was anesthetized and placed ina tray. The rearing cup of the bedbugs was inverted soits mesh top made contact with the abdominal surfaceof the hamster and the bedbugs were allowed to feedfor 15 min. Adult bedbugs were fed to repletion 5 dbefore testing.

Insecticide Resistance Bioassay. The insecticide re-sistance of bedbug populations from the five provinceswas examined using the World Health Organization(WHO) test kit to make insecticide-impregnated pa-pers. Each paper (12 X 15 cm) was impregnated withdichlorodiphenyl trichloroethane (DDT; 4%), diel-drin (0.8%), malathion (.5%), or fenitrothion (1%).

Nt

Ubon Ratchathani

Bangkok

Fig. 1. Locations for bedbug surveys in Thailand.

Each insecticide-impregnated paper was cut into 3 X5-cm pieces, rolled, and placed inside a glass test tube(length X internal diameter: 12.5 X 1..5cm). A pieceof clean paper (3 X 5 cm) similar to the impregnatedpaper was rolled and placed inside another glass testtube as the control. Ten adult bedbugs of either sexwere carefully transferred into each tube and closedwith fine-mesh gauze. The tubes were set upright inthe holding rack and placed in a plastic box (length Xwide X high: 20 X 20 X 15 cm) linecl inside with dampcloths to obviate very low humidity. The box wasclosed for the appropriate exposure period. The di-agnostic exposure times for DDT (4%), dieldrin(0.8%), malathion (5%), and fenitrothion (1%) were5 and 2 d, 16 and 5 h, respectively. The concentrationsof insecticides and exposure times used are recom-mended by WHO (1981) and are based on the pre-vious established baseline data (our unpublisheddata). At the end of the exposure time, mortality ratesof the bedbugs exposed to DDT and dieldrin wererecorded. Bedbugs exposed to malathion and fenitro-thion were transferred to clean tubes, each containinga piece of clean paper, after the exposure period, andmortality rates were recorded after 24 h (WHO 1981) .Each bioassay was replicated five times against eachcolony of bedbugs.

September 2011 TAWATSIN ET AL.: BEDBUG INSECTICIDE REsISTANCE IN THAILAND 1025

The efficacy of insecticides against the colonizedbedbugs also was determined using the WHO test kitto create insecticide-impregnated papers. The insec-ticides impregnated on the papers (12 X 15 cm) wereDDT (4%), dieldrin (0.8%), bendiocarb (0.1%),propoxur (0.1%), malathion (5%), fenitrothion (1%),cyfluthrin (0.15%), deltamethrin (0.05%), permethrin(0.75%), A-cyhalothrin (0.0.5%), or etofenprox (0.5%).The tests were conducted similarly to the resistancebioassay, as described previously; however, the bed-bugs were continuously exposed to the insecticide-impregnated papers for 2 wk. Mortality in controlgroups exposed to untreated papers was determinedfor each insecticide group (organochlorine control,organophosphate/ carbamate control, and pyrethroidcontrol). The mortality rates of the bedbugs exposedto each insecticide and of the four control groups wererecorded daily for the 2-wk exposure period. Eachbioassay was replicated five times. The mortality ratesof the treated groups were corrected by Abbott's for-mula when the mortality rates of the control groupswere between 5 and 20% (Abbott 1925). Insecticidalefficacy of each insecticide was determined by calcu-lating the median lethal time (LT.5o) using Probit anal-ysis (Finney 1971, SAS Institute 2005). Significantdifferences among the treatments were determinedby the failure of the 95% confidence limits (CL) tooverlap.

Bioassay Against Insecticide Products. Ten insecti-cides were tested for insecticidal efficacy by directapplication bioassays against the five colonies of bed-bugs in this study. The insecticides are used commonlyfor controlling crawling pests, such as cockroaches,ants, bedbugs, and termites. The active ingredients arebifenthrin (10% wt.vol, emusifiable concentrate [EC],dilution 1:100), cypermethrin (10% wt.vol, EC, dilu-tion 1:100), esfenvalerate (5% wt.vol, suspension con-centrate [SC], dilution 1:100), etofenprox (10% wt.wt,water emulsion [E\;V] , dilution 1:25), fenobucarb(20% wt.vol, EC, dilution 1:40), propoxur (20% wt:vol,EC, dilution 1:800), diazinon (23% wt:wt, flowablemicro encapsulation, dilution 1:50), chlorfenapyr (24%wt.vol, SC, dilution 1:385), fipronil (2.5% wt:vol, EC,dilution 1:100), and imidacloprid (20% wt.vol, SC, di-lution 1:400). Each insecticide was suspended in tapwater at the labeled rate and transferred to a spray-nozzle plastic bottle (40-ml capacity). Ten adult bed-bugs were transferred from the rearing cups into apetri dish (diameter X height: 90 mm X 15 mm). Thebedbugs in each petri dish were sprayed with eightshots of each product (average amount used 0.78 - 0.92g, after allowing for loss during spraying) from a dis-tance of =10 cm. The control group was sprayed withwater only. After spraying, the bedbugs in each petridish were transferred into a new, clean petri dish todetermine mortality. Mortality rates of bedbugs ex-posed to each product and in the control were re-corded daily for 1 wk. Each bioassay was replicatedfour times. The mortality rates of the treated groupswere corrected by Abbott's formula when the mor-tality rate of the control group was between ,5and 20%(Abbott 192,5). Insecticidal efficacy of each product

was determined by calculating the median lethal timevalues (LT 50) (SAS Institute 2005) . Significant differ-ences among the treatments were determined by thefailure of the 95% CL to overlap.

Bioassay of Commercial Aerosol Products. Eightcommercial aerosol products also were tested for in-secticidal efficacy by direct application bioassaysagainst the five colonies of bedbugs. These productsare used for the control of mosquitoes, cockroaches,ants, and other household crawling insects. The testedproducts were designated as A-I (permethrin 0.2.5%wt.wt, d-allethrin 0.1% wt.wt, S-bioallethrin 0.1% wt:wt, d-tetramethrin 0.2.5% wt:wt), A-2 (cypermethrin0.1% wt:wt, prallethrin 0.03% wt:wt, imiprothrin 0.03%wt:wt), A-3 (imiprothrin 0.05% wt:wt, cyfluthrin0.025% wt:wt, cl-allethrin 0.2% wt.wt, permethrin0.1%), A-4 (cypermethrin 0.17% wt:wt, imiprothrin0.04% wt.wt}, A-5 (imiprothrin 0.02% wt.wt, perme-thrin 0.03% wt.wt, esbiothrin 0.1% wt.wt), A-6 (d-tetramethrin 0.2% wt.wt, prallethrin 0.14% wt:wt), A-7(tetramethrin 0.35% wt:wt, cypermethrin 0.1.5% wt:wt), and A-8 (etofenprox 0.6% wt:wt). The aerosolproducts were oil-based formulations, except A-8,which was a water-based formulation. Each can of testaerosol was shaken well 20 times (up and down di-rection), and the discharge rate of each can was pre-determined by weighing (0.7 ± 0.1 g). Ten adult bed-bugs were transferred from rearing cups into a petridish (diameter X height: 90 mm X 1.5mm) and weresprayed with one application of each product (averageamount used 0.65-0.82 g, after allowing for loss duringspraying) from a distance of 30 cm. The control groupwas not sprayed. Immediately after spraying, the bed-bugs in each petri dish were subsequently transferredinto a new, clean petri dish to determine mortality.The mortality rates of bedbugs exposed to each prod-uct and in the control were recorded at 48 h postex-posure. Each bioassay was replicated five times. Themortality rates of the treated groups were correctedby Abbott's formula when the mortality rates of thecontrol groups were between 5 and 20% (Abbott1925). Comparison of insecticidal activity among thetest aerosols against each colony of bedbugs was con-ducted using a one-way analysis of variance and Dun-can multiple range test (SAS Institute 2005), and alldifferences were considered significant at P < 0.05.

Results

A total of 12 of 50 hotels from five provinces wasinfested with bedbugs (Table 1). In addition to com-monly finding bedbugs on mattress seams, woodenbed frames, and inside the base of the bed ensembleand bed heads, they were also found on bedside tables,bed lamps, switch plates, underneath carpet edges,wall crevices, screw holes, picture frames, and cur-tains. The tropical bedbug, C. hemipterus, was found inhotels in Bangkok, Chonburi, Phuket, and Krabi prov-inces, whereas the common bedbug, G.Zectularius, wasonly found in hotels in Chiang Mai. Surprisingly, nobedbugs were found in the 24 hotels surveyed in UbonRatchathani Province.

1026 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 5

Table 1. Prevalence of bed hugs in hotels in six provinces ofThailand

Total No. hotels SpeciesProvinces hotels infested withsurveyed bedbugs found

Bangkok 5 3 C. hemipterusChonburi 5 1 C. hemipterusPhuket 5 3 C. lieinipterusKrabi 5 1 C. hemipterusChiang Mai 6 4 C. leetu lariusUbon Ratchathani 24 0 NoneTotal 50 ]2

Bedbugs colonized from the five provinces showedsignificant resistance against DDT, dieldrin, mala-thion, and fenitrothion, with average mortality rang-ing from 0 to 20% (Table 2). No mortality was ob-served in the control group. The susceptibility ofbedbugs to various insecticides using insecticide-im-pregnated papers of the WHO test kit is shown inTable 3. It is evident that both species of the bedbugswere susceptible to a high concentration of dieldrin(4%) with LT50 of <1 d (ranging from 0.3 to 0.9 d);however, they showed high LTso values from 9.8 to18.0 and 9.1-14.8 d when exposed to the low concen-tration of dieldrin (0.8%) and high concentration ofDDT (4%), respectively. Fenitrothion demonstratedrelatively low LTso values between 2.9 and 8.5 dagainst C. hemipierus, but high LT50 (17.1 d) for C.lectularius. The bedbugs from Phuket and Chonburiwere more susceptible to malathion (LTso 4.8 and7.8 d, respectively) than were those from Krabi,Chiang Mai, and Bangkok (LT5oranging from 10.9 to11.7 d). Bedbugs from Chonburi were more suscep-tible to etofenprox and A-cvhalothrin (LTso 6.8 and8.3 d, respectively) than were the bedbugs from Bang-kok, Phuket, Krabi, and Chiang Mai (LT5o 16.0-20.0and 10.9-15.2 d, respectively, for Table 3). The otherinsecticides (DDT, bendiocarb, propoxur, cy£luthrin,deltamethrin, and permethrin ) demonstrated rela-tively high LTso values ranging from 9.1 to 18.0 d forbedbugs from the five provinces. Moderate levels ofmortality (5-15%) were observed in the controlgroups from the five colonies of bedbugs in the in-secticidal efficacy tests.

Susceptibilities of the bedbug colonies to the com-mercial insecticides differed markedly (Table 4).Among the five colonies, the bedbugs from Bangkokexhibited the longest LTso against almost all insecti-cides tested, except imidacloprid, bifenthrin, and

propoxur. Imidacloprid was the most effective insec-ticide against both species of bedbugs with LTso be-tween 0.03 and 0.9 d. Chlorfenapyr and fipronil, how-ever, demonstrated lower activity than that ofimidacloprid with LT50 of =1..5-8.7 and 0.8-12.7 d,respectively. Propoxur, esfenvalerate, etofenprox, di-azinon, fenobucarb, and bifenthrin showed relativelylow insecticidal activity against C. hemipterus withLT50ranging from 10.4to 17.7d. It is interesting to notethat bifenthrin was not effective against C. hemipterus(colonized from Krabi) and C Iectularius in this studybecause no mortality was observed in all tests. Simi-larly, cypermethrin and propoxur were also ineffec-tive against C. hemipterus (colonized from Bangkok)and C. lectularius, respectively. No mortality was ob-served in the control groups of bedbugs tested.

The insecticidal activity of commercial aerosolproducts against bedbugs colonized from variousprovinces is shown in Table 5. The aerosols A-2 andA-3 (containing three and four active ingredients)demonstrated excellent efficacy against all bedbugcolonies with complete mortality (100%) within 48 h.The bedbugs from Chonburi were the most suscepti-ble to all of the aerosols tested. In comparison, theaerosols A-I, A-4, and A-6 provided higher levels ofinsecticidal activity (90-100%) than did A-5 and A-7(.5.5-85%) against the bedbugs colonized from Bang-kok, Phuket, Krabi, and Chiang Mai. The aerosol A-8containing etofenprox (0.6%) alone showed the leastactivity against bedbugs, with mortality rates rangingfrom 0 to .30%.No mortality was observed in bedbugsamong the untreated group.

Discussion

The tropical bedbug, C. hemipterus, disappearedfrom Thailand during the 1970s,probably as a result ofthe intensive application of DDT in malaria controlprograms. Beginning in 2007, Thailand experienced aresurgence of bedbug infestations of C.hemipterus andC. lectularius in hotels in many provinces where tour-ism is prevalent. We collected bedbugs from hotels inBangkok, Chiang Mai, Chonburi, Phuket, and Krabi,but not from hotels in Ubon Ratchathani. Bangkok, thecapital of Thailand, is visited by at least 12 millionforeign tourists each year. Chiang Mai, Chonburi,Phuket, and Krabi provinces are visited annually by2-5 million tourists in each province. In contrast, UbonRatchathani is visited by =60,000 - 80,000foreign tour-ists each year. Because bedbugs have been found only

Table 2. J.\IIortnlityof C. hemlpterus and C. lectularius to organochiorine and organophosphate Insecticides using the \VHO test method

Insecticide (%)Bangkok Chiang ~'Iaia

Average mortality (%) of bedbugs

Chonburi Phuket Krabi

DDT4%Dieldrin 0.8%Malathion 5%Fenitrothion 1%

oooo

W W 05 ~ 55 10 0o 0 0

1055o

No mortality was observed in the control group.a C. lectularius; all other locations were C. hemipterus.

September 2011 TAWATSIN ET AL.: BEDBUG INSECTICIDE REsISTANCE IN THAILAND 1027

Table 3. ~Iedian lethal time (LT50) of bedbugs colonized from five provinces in Thailand after exposure to insecticide-impregnatedpapers

Insecticide (%)Chiang Mai"

LTso (95% CL) in days of bedbugs colonized from five provinces

Bangkok Chonburi Phuket Krabi

DDT4%Dieldrin 4%Dieldrin 0.8%Bendiocarb 0.1%Propoxur 0.1%Malathion 5%Fenitrothion 1%Cyfluthrin 0.l5%Deltamethrin 0.05%Pennethrin 0.75%A-Cyhalothrin 0.05%Etofenprox 0.5%

12.0 (11.6-12.6)0.9 (0.1-1.9)

18.0 (14.6-36.5)12.5 (11.7-13.7)12.3 (11.5-13.4)11.7 (11.2-12.3)8.5 (8.1-8.9)

13.8 (13.1-15.0)18.0 (16.0-23.3)15.2 (13.9-17.4)15.0 (13.3-18.0)16.1 (14.3-19.2)

10.3 (95-11.3)0.3 (0.l-0.4)9.8 (8.4-11.6)

ILl (1O.6-1l.8)11.0 (10.4-11.8)7.8 (6.5-9.2)4.9 (4.6-5.2)

1l.5 (10.7-12.4)10.7 (9.6-122)10.0 (95-10.6)8.3 (7.7-9.0)6.8 (5.7-8.0)

9.1 (8.4-9.8)0.3 (0.1-0.9)

11.9 (10.5-14.0)11.9 (10.6-13.9)12.8 (1l.9-14.0)4.8 (3.3-S.9)2.9 (l.3-4.0)

14.6 (13.7-16.0)12.5 (1l.4-14.1)15.3 (139-17.6)10.9 (9.1-14.5)16.0 (13.1-2:3.6)

14.8 (14.2-15.7)0.4 (0.2-1.1)

16.8 (13.1-31.9)11.0 (10.1-12.1)11.9 (11.1-12.8)10.9 (10.1-11.8)7.5 (7.0-7.9)

14.9 (14.0-16.2)16.6 (15.2-19.3)13.7 (13.2-H.4)IS.2 (14.3-164)16.6 (14.8-19.7)

13.9 (132-15.0)0.3 (0.2-0.4)

12.7 (11.9-14.1)13.5 (12.6-14.9)10.4 (9.8-10.9)11.5 (11.1-12.0)17.1 (15.8-19.8)15.1 (14.1-16.6)13.5 (13.1-14.0)12.2 (1l.0-13.9)15.0 (13.5-17.8)20.0 (16.8-29.1)

Moderate mortality (5-1S%) was observed in the control groups, and they were subjected for mortality correction in the relevant treatedgroups. Significant differences among the treatments were determined by the failure of the 95% CL to overlap.

a C. lectularius, all other locations were C. hemipterus.

in places providing accommodation to foreign travel-ers, it is likely that the bedbugs were introduced intoThailand by foreign tourists traveling from the in-fested areas. The common bedbug, C. leciularius, wasprobably imported from foreign countries as theywere first recorded in Thailand in 2010 (Suwannayodet al. 2010), and the tropical bedbug, c. hemipterus,might be introduced into cities from foreign countriestoo.

Since early 2000, bedbug infestations have beenreported in various countries across the world, such asin the United Kingdom (Boase 2001), Tanzania (My-amba et al. 2002), Australia (Doggett et al. 2004),Canada (Hwang et al. 2005), United States (Potter etal. 2008), Sri Lanka (Karunaratne et aL 2007), Den-mark, Norway, Sweden (Kilpinen et al. 2008), andSwitzerland (Mueller et al. 2008). Recently, Boase(2008) noted that the potential explanations for thebedbugs' resurgence may be attributed to social andhuman factors (increased human movement for busi-ness and leisure), environmental (more central heat-ing, double glazing, and global warming), and pestcontrol (inadequate training of pest control techni-

cians, changes in the pattern of insecticide use, effec-tive insecticides withdrawn from use, and insecticideresistance). In comparison with Canada and theUnited States, where the bedbug infestations haveoccurred in diverse settings in urban environments,including homes, multi-unit apartments, hotels, dor-mitories, health care facilities, schools, laundries,movie theaters, and public transportation vehicles(Hwang et al. 2005, Potter et al. 2008), the infestationsof bedbugs in Thailand are now limited only to somehotels or guesthouses. It is, therefore, an urgent needto develop programs to control bedbugs in the infestedhotels before they economically damage hotel busi-ness or spread out to dwellings and other sectorsacross the country.

The conventional bioassay based on WHO insecti-cide-impregnated papers showed resistance in bed-bugs to DOT, dieldrin, malathion, and fenitrothion.However, this method provides the tentative diagnos-tic dosages for only some insecticides (DDT, dieldrin,propoxur, fenchlorphos, malathion, fenitrothion, andtrichlorphon) against bedbugs (WHO 1976), of whichsome have already been banned for controlling in-

Table 4. Median lethal time (LT50) ofhedhugs colonized from five provinces in Thailand after exposure to var-ious insecticide pr-oductsby direct spraying

Insecticide Concentration LTso (95% CL) in days of bedhugs colonized from five provincesapplied" (mg/L) Bangkok Chonburi Phuket Krabi Chiang Maib

Diazinon 4,600 34.2 (16.8-5S.4) 8.0 (7.S-9.0) 8.4 (6.9-1l.9) 7.7 (7.3-8.5) 18.3 (12.2-60J)Fenobucarb 5,000 42.8 (18.6-72.8) 11.6 (9 ..3-17.3) 6.0 (4.3-19.l) 10.0 (7.6-19.2) 8.8 (7.7-10.5)Propoxur 250 72.4 (23.7-91.2) 7.1 (6 ..3-9.3) 9.4 (3.8-17.6) 30.3 (14.6-63.8) NAc

Esfenvalerate 500 .54.9 (32.6-66.8) 7.7 (6.6-10.0) 9.7 (71-50.1) 8.9 (7.4-14.3) 19.9 (12.9-839)Cyperrnethrin 1,000 NAc 6.4 (5.7-7.0) 7.1 (62-9.0) 9.7 (7.3-20.7) 7.7 (7.3-8.5)Bifentbrin 1,000 36.6 (13.4-64.7) 7.S (5.9-14.0) 23.2 (10.7-46.4) NAc NAc

Etofenprox 4,000 37.7 (24.:3-55.8) 8.0 (69-1Ll) 11.2 (7.9-52.7) 10.1 (7.9-40.9) 10.1 (8.6-13.0)Chlorfenapyr 624 8.7 (7.4-11.6) 3.7 (2.2-4.9) 3.3 (0.7-5.1) 1.5 (O.S-3.2) 3.8 (l.5-5.5)Fipronil 250 12.7 (8.7-7Ll) 1.3 (0.4-2.2) 6.2 (5.0-9.5) 0.8 (0.3-1.8) 9.5 (7.7-14.3)Irnidacloprid 500 0.4 (0.1-1.4) 0.8 (0.2-2.4) 0.03 (0.01-1.S) 0.4 (0.1-1.2) 0.9 (0.3-l.8)

No mortality was observed in the control group. Significant differences among the treatments were determined by the failure of the 9S%CL to overlap.

a Concentration applied at the labeled rate of each insecticide product.b C. lectularius; all other locations were C. heinipterus.C Lethal time was not applicable because no mortality was observed.

1028 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 5

Table 5. Insecticidal efficacy of conuncrcial aerosol products against bedbugs colonized from five provinces in Thailand

Aerosol Active ingredient (%)Mortality (mean:! SE) of bedbugs colonized from five provinces

Bangkok Chonburi Phuket Krabi Chiang Mai"

95:! 2.2b 100 ± O.Oa 100 ± O.Oa 95 ± 2.2b 100 ± O.Oa

100 ± O.Oa 100 :! O.Oa lOO:! O.Oa lOO::!:O.Oa lOO:! O.Oa

lOO:! O.Oa WO:! O.Oa lOO:! O.Oa lOO::!:O.Oa lOO::!:O.Oa

90::!: 3.2b lOO:! O.Oa lOO::!:O.Oa 90::!: 3.2b lOO::!:O.On

55::!: 5.5d 100 ± O.Oa 60::!: 4.5c1 65:!: 2.7d 70::!: 3.5c

90::!: 3.5b lOO::!:O.Oa 90 ::!:3.5b lOO::!:O.Oa WO::!:O.Oa

75 :! 5.0c WO:!: O.On 80 ± 3.2c 85 ::!:1.6c 80 ± 2.2b

10 ± 1.6e 30 ± 1.6b o ± O.Oe 15 ± 3.5e o ± O.OdF = 90.04; F = 1,960; F = 228.24; F = 182.48; F = 554.29;

df = 7,32; cif = 7,32; cif = 7,32; cif = 7,32; df = 7,32.;P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001

A-I Permethrin 0.25% wt:wt d-Allethrin0.10% wt.wt S-Bioallethrin 0.10%wt.wt d-Tetramethrin 0.25% wt.wt

A-2 Cypermethrin 0.1% wt:wt Prallethrin0.03% wt.wt Imiprothrin 0.03%wt:wt

A-3 Imiprothrin 0.050% wt.wt Cyfluthrin0.025% wt:wt d-Allethrin 0.200%wt.wt Permethrin 0.100% wt.wt

A-4 Cypermethrin 0.17% wt.wtImiprothrin 0.04% wt:wt

A-,5 Imiprothrin 0.02% wt.wt Permethrin0.03% wt:wt Esbiothrin 0.10%wt.wt

A-6 d-Tetramethrin 0.20% wt:wtPrallethrin 0.14% wt:wt

A-7 Tetramethrin 0.35% wt:wtCypermethrin 0.15% wt.wt

A-8 Etofenprox 0.60% wt:wtANOVA

The average mortality in the same column (same province), followecl by the same letter, is not significantly different (P> 0.05, Duncan'smultiple range test). No mortality was observed in the control group. ANOVA, analysis of variance.

a C. lectularius; all other locations were C. hemipterus.

sects. It has a limitation to detect resistance to otherinsecticides by bedbugs and also requires a large num-ber oflive bedbugs for testing each time. Therefore, itis important to develop additional, practical, and spe-cific tools for long-term insecticide-resistance moni-toring and management of bedbugs. Recently, Seonget al. (2010) succeeded to develop a molecular resis-tance-monitoring tool based on the frequency of twopoint mutations (V419L and L925I) in the voltage-sensitive sodium channel o-subunit gene in conjunc-tion with the filter contact vial bioassay method todetermine the resistance of bedbugs to deltamethrinand A-cyhalothrin. It could be an effective and prac-tical tool for insecticide-resistance monitoring andmanagement of bedbugs in the future.

According to our results from WHO insecticide-impregnated papers, bedbugs from the five provincesshowed some level of resistance to insecticides, suchas organochlorines (DDT, dieldrin), carbamates(bendiocarb, propoxur), organophosphates (mala-thion, fenitrothion), and pyrethroids (cyfluthrin, del-tamethrin, permethrin, A-cyhalothrin, etofenprox).The bedbug colonies were confirmed for pyrethroiclresistance by polymerase chain reaction assay with aset of specific primers for voltage-sensitive sodiumchannel o-subunit gene and gene mutations related topyrethroid resistance (Malainual et al. 2010). In Thai-land, C. hemipterus was found to be resistant to DDTsince 1970s (WHO 1976). C. lectularius showed theability to develop resistance to bifenthrin and o-cyper-methrin (Suwannayod et al. 2010). Until recently,resistance to insecticides, especially in the pyrethroidgroup, was found in bedbugs worldwide. C.hemipierusin Tanzania was found to be resistant to permethrinand alphacypermethrin (Myamba et al. 2002),

whereas those in Sri Lanka were resistant to DDT,malathion, propoxur, permethrin, and deltamethrin(Karunaratne et at 2007). C. lectularius in the UnitedStates were found to be resistant to deltamethrin andA-cyhalothrin (Moore and Miller 2006, Romero et al.2007, Yoon et al. 2008, Zhu et al. 2010, Seong et al.2010), and those in Denmark to permethrin and del-tamethrin (Kilpinen et al. 2008).

Insecticides in the pyrethroid group, such as bifen-thrin, cypermethrin, esfenvalerate, and etofenprox,have been commonly used for controlling insect pestsin hotels by pest control operators (PCOs) in Thai-land, whereas those in organophosphate (diazinon)and carbamate group (fenobucarb, propoxur) havebeen used infrequently. The results obtained from thisstudy indicated clearly that these insecticides wererelatively ineffective against the bedbugs, and thiscould be a reason why PCOs cannot manage infesta-tions of bedbugs in hotels. This study also investigatedsome insecticides from other groups, such as irnida-cloprid (neonicotinoid group), chlorfenapyr (pyrrolegroup), and fipronil (phenylpyrazole group), andshowed that they were relatively effective against thebedbugs, possibly because of their different modes ofaction.lmidacloprid is a neonicotinoid insecticide thatacts on the nicotinic acetylcholine receptor, wherethe nervous system is then excessively stimulated anddeath eventually occurs. According to its specificityfor this type of receptor, which is more frequentlyfound in insect nervous system than that of otheranimals, imidacloprid has considerably low toxicity tomost animals. Chlorfenapyr, however, is a proinsec-ticide known as halogenated pyrrole that must bemetabolized into an active insecticide after enteringthe host, and it then disrupts the production of aden-

September 2011 TAWATSIN ET AL.: BEDBUG INSECI1CIDE REsISTANCE IN THAILAND 1029

osine triphosphate at the mitochondria, causing cel-lular death and, subsequently, host mortality. Becausehumans are unable to generate the active form of thisinsecticide, chorfenapyr is considered to have rela-tively low toxicity to humans. However, chorfenapyrmay not control bedbug populations in a timely man-ner because it was so slow acting when used as contactinsecticide (Moore and Miller 2006). Unlike those twoinsecticides, fipronil is an insecticide of the phe-nylpyrazole group that binds to the y-aminobutyricacid-gated chloride channel, resulting in overstimula-tion of the nervous system and leading to poisoningand death. However, bedbugs could be potentiallyresistant to fipronil because it has similar mode ofaction as the organochorine group, such as dieldrin,and to which bedbugs were found to be resistant.Thus, imidacloprid, chlofenapyr, and fipronil have to-tally different modes of action against insects as com-pared with insecticides in pyrethroid group, which acton sodium channel of neurons, or those in carbamateand organophosphate group, which inhibit cholines-terase in the nervous system. Imidacloprid, chlofe-napyr, and fipronil are classified as moderately haz-ardous pesticides (WHO class II) with acute oral LD50(rat) of =450,441, and 92 mg/kg, respectively (WHO2010).

It is noteworthy that the oil-based aerosols contain-ing multiple pyrethroid insecticides (2-4 differentactive ingredients at low concentrations) were effec-tive against the bedbugs, whereas the water-basedaerosol containing a single active ingredient at highconcentration (etofenprox 0.6% wt:wt) was ineffec-tive. This phenomenon could be the result of at leasttwo factors: formulation and synergistic effects amongactive ingredients. First, the oil-based formulation islikely to be more efficacious than the water-basedformulation because the former formulation consistsof oleophilic solvent such as kerosene, which hasstrong affinity to bind to the oleophilic surface ofinsect exoskeleton. The treated insects, as a result,could absorb the active ingredients in the oil-basedformulations more than that in the water-based for-mulation. Second, a combination of various active in-gredients could increase insecticidal activity of theaerosol; however, the degree of enhancement de-pends on properties of each active ingredient and theproportion of each active ingredient in the formula-tion. Also, the total concentration of active ingredientsof these aerosols was relatively high (0.15-0.70% wt:wt) as compared with the products containing singlepyrethroid insecticides used by PCOs. Although thebedbugs are resistant to pyrethroid insecticides, acombination of multiple active ingredients of the py-rethroid group in oil-based formulations as found inaerosols can manage these bedbugs. Recently, Barileet al. (2008) demonstrated the effects of a tank mixtureof deltamethrin se (0.06%) with formulated piperonylbutoxide (0.1%) synergized natural pyrethrum (1.0%)in the field. They found that the initial mortality of thebedbug population increased, and elimination of theinfestation was achieved within a shorter period (2-4

wk) with fewer reservices as compared with an ap-plication of deltamethrin se (0.06%) alone.

In conclusion, the results obtained from this studysuggested that the bedbugs in Thailand have devel-oped resistance to various groups of insecticides, es-pecially those in the pyrethroid group, which are themost common insecticides used for pest control. Bothspecies of the bedbugs were differently susceptible tothe insecticides tested. Based on insecticide efficacyand low toxicity to humans, imidacloprid is recom-mended for control of the bedbugs. This active ingre-dient could effectively control the insecticide-resis-tant bedbugs and may play an important role in themanagement of resistance to insecticides of bedbugsin Thailand and elsewhere. As mentioned by Harlan etal. (2008), research should be encouraged and carriedout to discover and make available new insecticideactive ingredients, products, devices, and techniquesthat will be effective in controlling bedbugs. There-fore, an integrated bedbug management plan shouldbe implemented for controlling bedbugs in associationwith insecticide resistance management. The planshould include education and training of involvedpersons, thorough inspection, application of effectiveinsecticides, insecticide resistance monitoring andmanagement, reduction of available harborages, andregular monitoring of bedbug infestations.

Acknowledgments

We are grateful to YaowalukChanbang and Suttida Su-wannayot,Faculty ofAgriculture,Chiang Mal University,forprovidinga colonyof the C. lectularius used in this study.Wethank the staffof the Biologyand EcologySection,NationalInstitute of Health, Thailand, for their assistancein the bed-bug survey,laboratory culture of bedbugs,and efficacytests.This work was supported by the National Research Councilof Thailand (Grant 2.5.52-68). This manuscript was approvedto be published by permission of the National ResearchCouncil of Thailand (NRCT 000.5/1147).

References Cited

Abbott, "v. S. 1925. A method of computing the effective-ness of an insecticide. J. Econ. Entomol. 18: 265-267.

Barile, J., R. Nauen, G. Nentwig,R. Pospischil,and B. Reid.2008. Laboratory and field evaluation of deltamethrinand bendiocarb to control Cimex lectularius (Heterop-tera: Cimicidae), pp. 105-111. In W. H. Robinson and D.Bajomi (eds.), Proceedings, Sixth International Confer-ence on Urban Pests, 13-16 July2008, Budapest, Hurigary.OOK-PressKft, Veszprem,Hungary.

Boase,C. 2001. Bedbugs:back from the brink. Pestic. Out-look 12: 159-162.

Boase,C. 2008. Bed bugs (Hemiptera: Cimicidae): an evi-dence-based analysisof the current situation,pp. 7-14. InW. H. Robinsonand D. Bajomi (eds.), Proceedings,SixthInternational Conference on Urban Pests, 13-16 July2008, Budapest, Hungary. OOK-Press Kft. Veszprem,Hungary.

Doggett, S, 1,., M. J. Ceary and R. C. Russell. 2004. Theresurgence of bed bugs in Australia:with notes on theirecology and control. Environ. Health 4: 30-38.

Finney, D. J. 1971. Probit Analysis.Cambridge UniversityPress, Cambridge,United Kingdom.

1030 JOURNAL OF MEDICAL ENTOlv!OLOGY Vol. 48, no. 5

Harlan, H. J., M. K. Faulde, and G. J. Baumann. 2008. Bed-bugs, pp. 131-153. In X. Bonnefoy, H. Kampen, and K.Sweeney (eds.), Public Health Significance of UrbanPests. World Health Organization, Geneva, Switzerland.

Hwang, S. W., T.J. Svoboda, 1.J. De Jong, K. J. Kabasele, andE. Gogosis. 2005. Bed bug infestations in an urban en-vironment. Emerg. Infect. Dis. 11: 5.3.3-5.38.

Karunaratne, S.H.P. P., B. T. Damayanthi, M.H.J. Fareena, V.Imbuldeniya, and J. Hemingway. 2007. Insecticide re-sistance in the tropical bedbug Cimex hemipterus. Pestic.Biochem. Physio!. 88: 102-107.

Kilpinen, 0., K. V. Jensen, and M. Kristensen. 2008. Bed bugproblems in Denmark, with a European perspective, pp'395-399. In W. H. Robinson and D. Bajomi (eds.), Pro-ceedings, Sixth International Conference on Urban Pests,13-16 July 2008, Budapest, Hungary. OOK-Press KEt,Veszprcm, Hungary.

Lowe, C.. F., and M. G. Romney. 2011. Bedbugs as vectorsfor drug-resistant bacteria. Emerg. Infect. Dis. 17: 11.32-1134.

Malainual, N., T. Thet-em, A. Tawatsin, U. Thavara, and S.Tiewcharoen. 2010. Voltage-sensitive sodium channelgene in pyrethroids resistant bedbugs (Ci.mex hemi-pterus) in Thailand, pp. 135. In K. Rydzanicz and E. Lone(eds.), Proceedings, 17th European Society for VectorEcology Conference, 13-17 September 2010, Wroclaw,Poland. University of Wroclaw, Wroclaw, Poland.

Moore, D. J., and D. M. Miller. 2006. Laboratory evaluationsof insecticide product efficacy for control of Cirnex lectu-larius. J. Econ. Entomo!. 99: 2080-2086.

Mueller, G., I. L. Luescher, and M. Schmidt. 2008. Tempo-ral changes in the incidence of household arthropod pestsin Zurich, Switzerland, pp. 15-21. In W. H. Robinson andD. Bajomi (eds.), Proceedings, Sixth International Con-ference on Urban Pests, 13-16 July 2008, Budapest, Hun-gary. OOK-Press Kft, Veszprern, Hungary.

Myamba, J., C. A. Maxwell, A. Asidi, and e. F. Curtis. 2002.Pyrethroid resistance in tropical bedbugs, Cimexhemipterus, associated with use of treated bednets. Med.Vet. Entomo!. 16: 448-451.

Panagiotakopulu, E., and P. e. Buckland. 1999. Cimex lectu-larius L.S·om Pharaonic Egypt. Antiquity 73: 908-911.

Potter, M. F. 2005. A bed bug state of mind: emerging issuesin bed bug management. Pest Control Technol. 33: 82-97.

Potter, M. F., A. Romero, and K. F. Haynes, 2008. Battlingbed bugs in the USA, pp. 401-406. In W. H. Robinson and

D. Bajomi (eds.), Proceedings, Sixth International Con-ference on Urban Pests, 13-16 July 2008, Budapest, Hun-gary. OOK-Press Kft, Veszprem, Hungary.

Pratt, H. D., and C. J. Stojanovich. 1967. Bugs: pictorial keyto some species that may bite man, pp. 94. In PictorialKeys to Arthropods, Reptiles, Birds and Mammals of Pub-lic Health Significance. U.S. Department of Health, Ed-ucation, and Welfare, Atlanta, GA.

Romero, A., M. F. Potter, D. A. Potter, and K. F. Haynes.2007. Insecticide resistance in the bed bug: a factor in thepest's sudden resurgence? J. Med. Entomo!. 44: 175-178.

SAS Institute. 2005. SAS/STAT User's Guide, version 9.1.SAS Institute, Cary, Ne.

Seong, K. M., D.-Y. Lee, K. S. Yoon, D. H. Kwon, H. C. Kim,T. A. Klein, J. M. Clark, and S. H. Lee. 2010. Establish-ment of quantitative sequencing and filter contact vialbioassay for monitoring pyrethroid resistance in the com-mon bed bug, Cimex lectuiarius. J. Med. Entomol. 47:592-599.

Suwannayod, S., Y. Chanbang, and S. Buranapanichpan.2010. The life cycle and effectiveness of insecticidesagainst the bed bugs of Thailand. Southeast Asian J. Trop,Med. Public Health 41: 548-554.

[WHO] World Health Organization. 1976. Resistance ofvectors and reservoirs of disease to pesticides. Tech. Rep.Ser. 585: 1-88.

[WHO] World Health Organization. 1981. Instructions fordetermining the susceptibility or resistance of adult bed-bugs to insecticides. (WHO/VBC/81.809).

[WHO] \\lorld Health Organization. 2010. The WHO rec-ommended classification of pesticides by hazard andguidelines to classification 2009. (http://www.who.int/ipcs / publications / pesticides_hazard _2009.pdf).

Yoon, K. S., D. H. Kwon, J. P. Strycharz, e. S. Hollingsworth,S. H. Lee, and J. M. Clark. 2008. Biochemical and mo-lecular analysis of cleltamethrin resistance in the commonbed bug (Hemiptera: Cimicidae). J. Med. Entomo!. 45:1092-1101.

Zhu, F., J. Wigginton, A. Romero, A. Moore, K. Ferguson, R.Palli, M. F. Potter, K. F. Haynes, and S. R. Palli. 2010.Widespread distribution of knockdown resistance muta-tions in the bed bug, Cimex leciularius (Hemiptera: Cimi-cidae), populations in the United States. Arch. InsectBiochem. Physiol. 73: 245-2.57.

Received 7 January 2011; accepted 19 June 2011.