Accepted author version posted online: 23 November 2018

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1 Accepted uthor version posted online: 23 November 2018 Susceptibility of Mlysin Tropicl Bed Bug (Cimex hemipterus L.) Popultions to Deltmethrin nd Propoxur Insecticides Zulikh Zhrn nd Abdul Hfiz Ab Mjid* Household nd Structurl Urbn Entomology Lbortory, Vector Control Reserch Unit, School of Biologicl Sciences, Universiti Sins Mlysi, Pulu Pinng, Mlysi. *Corresponding uthor: Running title: Tropicl bed bug susceptibility on insecticides Abstrk: Pepijt telh dikumpul dri seluruh Semennjung Mlysi untuk meliht thp kerintngn terhdp du kels rcun serngg, piretroid dn krbmt. Dlm kjin ini, deltmethrin dn propoxur yng tersedi secr komersil digunkn untuk menguji sttus kerintngn pepijt tropik, Cimex hemipterus di Mlysi. Sttus kerintngn merek dinili dengn menggunkn kerts yng direspi rcun serngg, sutu kedh bioesei yng disyorkn oleh Pertubuhn Kesihtn Seduni (WHO). Sepuluh pepijt dews dlm setip populsi diletkkn di dlm setip tiub dengn kerts yng direspi 0.05% deltmethrin dn 0.1% propoxur. Pd mulny, pepijt terdedh selm 24 jm, kemudin tempoh pendedhn dilnjutkn hingg 14 hri. Bioesei dilkukn dlm triplikt. Thp kerintngn tertinggi dipmerkn oleh populsi AR dengn nili ms mut (LT 50 ) sebnyk j di dlm tiub yng mengndungi deltmetrin. Seblikny, propoxur sngt berkesn terhdp populsi IP kern nili ms mut yng pling rendh, (LT 50 = j). Perbezn yng signifikn (p <0.005) didpti ntr kels rcun serngg dn populsi pepijt. Purt kemtin di klngn populsi AR, SW, HM, PY, dn PC menunjukkn perbezn yng signifikn pbil terdedh kepd deltmetrin. Sebgi kesimpuln, propoxur menunjukkn keberkesnn yng tinggi dlm mengwl populsi pepijt. Wlu bgimnpun, keberkesnnny hny di tempt tertentu. Sementr itu, rwtn menggunkn deltmethrin hrus dielkkn oleh profesionl kwln perosk kern keberkesnnny yng rendh. Kt kunci: Cimex hemipterus, kerintngn rcun serngg, ms mut, kerentnn, Mlysi Abstrct: Bed bugs were collected cross Peninsulr Mlysi to investigte their resistnce to two types of insecticide clsses, pyrethroid nd crbmte. In this study, commercilly-vilble deltmethrin nd propoxur were used to test the resistnce sttus of tropicl bed bug, Cimex hemipterus in Mlysi. Their resistnce sttus ws evluted using insecticide-impregnted ppers, biossy method recommended by the World Helth Orgniztion (WHO). Ten dult bed bugs in ech popultion were plced inside the tubes with ppers impregnted with 0.05% deltmethrin nd 0.1% propoxur. At first, the bugs were exposed for 24 hours, then, the exposure period ws extended to 14 dys. The biossy ws performed in triplictes. The highest resistnce level ws exhibited by the AR popultion with lethl time vlue (LT 50 ) of h in the deltmethrin-contining tube. In contrst, propoxur ws highly effective ginst the IP popultion due to its lethl time vlue (LT 50 = h) ws the lowest. A significnt difference (p < 0.005) ws found between insecticide clsses nd bed bug popultions. Men mortlity mong AR, SW, HM, PY nd PC popultions showed significnt difference when exposed to deltmethrin. In 1

2 conclusion, propoxur showed high efficcy in controlling bed bug popultions. However, its efficcy ws only t prticulr plces. Menwhile, tretment using deltmethrin should be voided by pest control professionls due to its low efficcy. Keywords: Cimex hemipterus, insecticide resistnce, lethl time, susceptibility, Mlysi INTRODUCTION Over the lst two decdes, the resurgence of bed bugs hs become one of the primry problems in bed bug control strtegy worldwide. Generlly, the elimintion of the bed bug infesttions involves the use of chemicl insecticides. However, continul ppliction of chemicl insecticides hs resulted in bed bug resistnce (Dng et l. 2017) nd hve creted widespred worries mong pest control professionls. To mke mtter worse, cryptic nd nocturnl lifestyle of the bed bugs further complicte the elimintion strtegy (Reinhrdt nd Siv-Jothy 2007). Numerous studies hve demonstrted tht the evolution of insecticide resistnce likely plys mjor role in the resurgence of these blood-sucking insects (Reinhrdt nd Sive-Jothy 2007; Dng et l. 2017). The min reson linked to this mtter is tht the evolution of their body systems, cusing multiple resistnce mechnisms to rpidly develop ginst pesticides pplied (Adelmn et l. 2011; Bi et. l, 2011). After DDT ws bnned for its persistence nd negtive environmentl impct (Crson, 1963), other clsses of insecticides with reduced susceptibilities such s crbmte, phenylpyrzole, neonicotinoid, orgnothiophosphte, nd pyrrole were tested for their toxicity in controlling Cimex lectulrius colonies in poultry fcilities (Steelmn et l. 2008). In Austrli, bed bug popultions showed lower mortlity rte for most insecticide clsses tested, except imidcloprid nd pirimiphos-methyl (Lilly et l. 2015). To dte, mjority of pest control compnies preferbly use synthetic pyrethroids s they re known to effectively kill insects such s mosquitoes, flies, hed louse nd bed bugs (Zhu et l. 2016). However, evolved resistnce to the pyrethroids by insects including bed bugs hs led to difficulties nd posed more chllenges in suppressing their infesttion. A report by Zhu et l. (2010) proved tht common bed bugs in the USA showed no response towrds deltmethrin re ssocited with kdr-type resistnce triggered by two mjor mino cids i.e. V419L nd L925I. Field-collected strins in infested homes in Denmrk lso reported the low efficcy of permethrin nd deltmethrin, while the lterntive insecticide, chlorpyrifos resulted in higher mortlity percentge (Kilpinen et l. 2011). Two knockdown resistnce (kdr) muttions ssocited with resistnce to pyrethroids hve been recently detected in C. hemipterus (Dng et l. 2015). These kdr muttions ffect the efficcy of pyrethroid, clss of insecticide tht ttck voltge-gted sodium chnnel (VGSC) in the nervous system. An ltertion of sequences of binding sites in the sodium chnnel countercts the mode of ction of pyrethroid nd DDT, resulting in the insensitivity to both clsses of insecticides (Soderlund nd Knipple 2003). The kdr muttions hve lso been detected in severl hemtophgous insects such s mosquitoes, Anopheles gmbie, Culex pipiens, ct fle, Ctenocephlides felis nd C. hemipterus (Bss et l. 2004, Dng et l. 2015). In generl, C. hemipterus is more susceptible thn C. lectulrius. However, some of them possess higher tolernce level towrds insecticides which cn be pssed on to their progeny (Busvine 1958). Pest control compnies in Southest Asi rely mostly on chemicl nd spry methods s tretments (Wng et l. 2011, Ab Mjid nd Zhrn 2015b). However, due to resistnt fctors, the compnies hrdly reduce pest popultions despite vrious insecticides re redily vilble in the mrket. Pyrethroid, clss of insecticide which hs been widely used s control gent ws reported to demonstrte little or no 2

3 effect on bed bugs nd other insect vectors (Wood et l. 2010, Miller 2013). Bed bugs my lso develop their resistnce to other clsses of insecticides with similr ctive ingredients if no lterntive control mesure is immeditely sought to void the circumstnce (Moore nd Miller 2006). Numerous findings relted to chemicl nd non-chemicl methods tht resulted in effective control of bed bugs hve been reported by mny. These include the ppliction of ditomceous erth nd sprying of insecticides such s chlorfenpyr, propoxur, nd other clsses (Wng et l. 2009, Mnuel 2010). Unlike termites, bed bugs lck grooming ctivity to ffect other members in popultion. Nevertheless, horizontl trnsfer through contct nd ingestion of insecticides might occur mong bed bugs (Akhtr nd Ismn 2013). A few studies found tht simultneous prctices of two control methods such s combintion of heting nd chemicl tretment cn effectively eliminte resistnt popultions compred to single method; either by sprying or dust ppliction (Wng et l. 2009, Akhtr nd Ismn 2013). Tropicl bed bugs smpled in Bngkok, Phuket, nd Krbi of Thilnd hve developed resistnce to severl insecticide clsses i.e. orgnochlorines, crbmtes, nd pyrethroids but noted higher mortlity when treted with imidcloprid nd chlorfenpyr (Twtsin et l. 2011). Multiple resistnce mechnisms hve been identified in bed bugs including penetrtion resistnce through cuticle thickening or remodeling, metbolic resistnce by incresing the ctivities of detoxifiction enzymes (e.g. cytochrome P450 monooxygenses nd esterses), nd knockdown resistnce by kdr muttions (Dng et l. 2017). Other potentil resistnce mechnisms such s behviorl nd physiologicl chnges (e.g. incresing esterse ctivities by point muttions, glutthione S-trnsferse, trget site insensitivity including ltered AChEs, GABA receptor insensitivity, nd ltered nachrs), nd symbiont-medited mechnisms re yet to be discovered. It is lso worthwhile to mention tht reserch ddressing the mechnisms of insecticide resistnce in the tropicl bed bug, C. hemipterus is generlly lcking lthough the mechnisms re likely similr to the temperte species, C. lectulrius. In Mlysi, the tropicl bed bugs hve been reported exhibiting resistnce to chlorinted hydrocrbons, prticulrly DDT, HCH, d-llethrin, nd dieldrin group (Dng et l., 2017). However, the resistnce sttus of the Mlysin bed bugs to other clsses of insecticides such s pyrethroid nd crbmte is currently unknown. Therefore, this study ws conducted to evlute the resistnce sttus of C. hemipterus collected cross Peninsulr Mlysi to two mjor clsses of insecticides i.e. pyrethroid (deltmethrin) nd crbmte (propoxur). The efficcy of both insecticides on the bed bugs ws lso determined. 3

4 MATERIALS AND METHODS Bed Bugs Rering nd Popultion Selection Bed bugs were cquired from the smple collection cross Peninsulr Mlysi. They were mintined, rered under room conditions (25±2 C, 50-60% of reltive humidity) nd were fed weekly on expired humn blood supplied by Penng Generl Hospitl. Modifictions of rtificil feeding system were set up by stretching prfilm membrne on glss jr filled with the expired blood bsed on the feeding mintennce of bed bugs by Montes et l. (2002) nd Abd Rhim et l. (2016). Ten popultions were selected bsed on the resistnce sttus of recent bed bugs control reports nd feedbck from questionnires given to the pest control compnies in Mlysi (Ab Mjid nd Zhrn, 2015). They were represented by ech cluster obtined from the previous smpling. Bed bugs were seprted nd mintined ccording to field nd lbortory strins. F1, F2 nd F3 genertions were lbeled nd kept in different continers. Insecticide Preprtion Two insecticides i.e. deltmethrin nd propoxur were used to evlute the resistnce of bed bugs using insecticide-impregnted ppers from the World Helth Orgniztion (WHO) test kit (Busvine, 1958). The ppers (12cm x 15cm) were impregnted with 0.1% propoxur nd 0.05% deltmethrin. Impregnted ppers then were cut into smller pieces (3 x 5 cm), rolled nd inserted into glss test tubes of 12.5 cm x 1.5 cm. When the bugs were exposed to the treted ppers, the tubes were covered with fine net cloth to prevent them from escping. All tubes were plced in continer t room condition (temperture, 25±2ºC, reltive humidity, 50-60%). Resistnce Biossy The resistnce biossy ws conducted following methods by Busvine (1958) nd Twtsin et l. (2011). Only dult bed bugs from the F2 genertion were used in this biossy. Ech biossy required 40 bed bugs from ech popultion. The selected popultions re tbulted in Tble 1. Ten dults from ech popultion were tested by introducing them into the test tube contining 0.05% deltmethrin-impregnted ppers - or 0.1% propoxurimpregnted ppers. The concentrtions of insecticides nd exposure times were s recommended by World Helth Orgniztion (WHO) (Busvine nd Nsh (1953), Twtsin et l. (2011), DeVries et l. (2015) nd bsed on the previously estblished bseline dt (unpublished dt). Ech tretment ws done in triplictes with one control. Filter ppers were impregnted with stndrd oil solutions of insecticides incorported with the insecticide resistnce kit provided by the World Helth Orgniztion (WHO) (Busvine nd Nsh 1953, Twtsin 2011). Bed bugs exposed to ppers impregnted with silicone oil (deltmethrin tretment), nd risell oil (propoxur tretment) were used s controls. Bed bugs were fed on expired humn blood for 24 hours prior to experimentl procedures. The bed bugs were exposed for 24 hours for preliminry testing. Observtions on their mortlity were continued nd recorded for 14 dys. They were strved during the continuous biossy. Mortlity ws scored bsed on their posture nd movement in the tubes. Ded bugs were in n upsidedown position with no signs of movement fter 10 seconds of observtion. Survived bed bugs were kept in 70% lcohol for further observtion of resistnce in the bugs. Control groups exceeding 10% of mortlity were corrected using Abbott s formul (Abbott 1925). 4

5 Sttisticl Anlysis Percentge of mortlity in ech tretment ws clculted nd nlyzed dily t 24 hours intervl. Probit nlysis ws used in determining lethl times (hours) of the bugs t LT 50, LT 90, nd LT 99 with confidence limit of 10-90%. Evlutions of lethl times were mde by using softwre SPSS Version 22.0 (IBM Corp., Armonk, NY, USA). Men comprison ws performed using one-wy nd two-wy nlysis of vrince (ANOVA) to determine the significnt difference between the popultions nd the effect of insecticide on bed bug popultions. RESULTS Bsed on the results, tropicl bed bugs were less susceptible ginst deltmethrin compred to propoxur (Figure 1 nd Figure 2). From the first until the third dy of ppliction, ll popultions showed low mortlity within the rnge of 3-20% in the deltmethrin tretment. Among the tested popultions, the KT nd HM exhibited 20% higher in mortlity fter one week of exposure. The PC popultion recorded the highest mortlity (>70%) on dy 10 compred to the other popultions. On dy 14, the AR popultion exhibited the lowest mortlity (40%). The other popultions exhibited moderte to high mortlity; rnging from 60% to 80%. Two popultions, PC nd SW hd the highest mortlity rte (90%), further indicting tht both popultions were highly susceptible towrds deltmethrin. They were, however, still considered s resistnt to this insecticide. Survived bed bugs might hve developed resistnce in them due to the previous tretment of different insecticides by pest control opertors. Menwhile, propoxur showed high efficcy in killing bed bugs s it cused n overll mortlity of 90% throughout the tretment period (Figure 1). Only one popultion i.e. TI noted high mortlity on the first dy of exposure i.e. 20% nd incresed to 37% fter dy 3. Nine other popultions displyed low mortlity rte until the third dy of exposure, but the mortlity incresed to nerly 50% fter dy 7. On the 10 th dy, ll popultions reched 80-90% mortlity except for two popultions (KT nd HM), where the mortlity rtes were 60% nd 67%, respectively. Three popultions nmely IP, PY, nd PC noted 100% mortlity fter the 14 th dy of the tretment. The lowest mortlity ws observed in the KT popultion with 87%, while ~ 90% were recorded for the other seven popultions. Lethl time (in hours) ws performed to determine the efficcy of insecticides ginst bed bugs (Tble 2). The results showed tht the efficcy of ech insecticide clss ws different in ech popultion of bed bugs. A few popultions hd high LT 50 vlues which cn be linked to the resistnce fctor. For deltmethrin, the highest LT 50 vlue ws detected in the AR popultion with h. However, three popultions i.e. KG, HM, nd PY hd low LT 50 vlues with h, h, nd h, respectively. The PC popultion exhibited the lowest lethl vlue ( h), indicting it s the most susceptible popultion to deltmethrin. Trends of LT 50 (in hours) over time for ll popultions re presented in Tble 2 nd Tble 4. Most of the tested popultions showed high susceptibility to propoxur fter eight dys of exposure. Approximtely h ws required to kill 50% of the IP popultion, exhibiting the lowest resistnce compred to others. Another popultion exhibited low resistnce ws the TI with h. The remining eight popultions showed incresed lethl time vlues but were still considered to hve low resistnce since up to 30-53% of bed bugs were killed within 7-8 dys of exposure periods. The KT popultion, on the other hnd, hd the highest LT 50 with h, exhibiting the lowest percentge of mortlity. 5

6 A two-wy nlysis of vrince (ANOVA) ws performed to determine the effect of insecticide groups on different popultions of bed bugs. Bsed on the results, there ws significnt interction (F (9, 40) = t p < 0.005) between insecticide groups nd different popultions of bed bugs. Comprison between two min ctive ingredients reveled tht propoxur ws highly effective (p<0.0001) thn deltmethrin in controlling bed bugs. Men vlues of mortlity in bed bugs for propoxur nd deltmethrin were 9.5 nd 7.6, respectively. Interctions between bed bug popultions, however, hd different results depending on which insecticides they were treted. Therefore, one-wy ANOVA ws used to investigte the differences in bed bugs popultions treted with propoxur nd deltmethrin. As shown in Figure 1 nd Tble 2, exposure to propoxur exhibited no significnt difference mong the popultions (F (9, 20) = 1.468, p > 0.05) while, popultions treted with deltmethrin showed significnt difference mong the popultions (F (9, 20) = 3.453, p < 0.05). A significnt interction of men mortlity ws found between popultions of AR, SW, HM, PY, nd PC (Figure 2 nd Tble 2). DISCUSSION To dte, pyrethroid-bsed insecticides re widely used in Mlysi to control bed bug infesttions in vrious premises (Ab Mjid nd Zhrn 2015). Filure in chemicl control is the mjor fctor of resistnce development following repetitive exposure of similr insecticides to bed bugs. As such, biossy ws used to ssess the effect of two insecticides ginst popultions of bed bugs in Peninsulr Mlysi. The method is reltively simple to perform nd provides stndrdized dt in monitoring insecticide resistnce (Dng et l. 2017). Bsed on the results, deltmethrin ws less effective in controlling the popultions. Similr results were obtined in the tropicl bed bug popultions in Sri Lnk where high KT 50 vlues were observed, rnging from 0.5 to 24 hours nd from 2.5 to 47 hours fter being exposed to permethrin nd deltmethrin for 24 hours of (Krunrtne et l. 2007). Low doses of bifenthrin nd lph-cypermethrin were used to tret bed bugs in Thilnd hd cused high effective dose (ED 50 ) vlues compred to other urbn pests (Suwnnyod et l. 2010). Bed bugs might hve their systems triggered by these pyrethroidbsed insecticides, leding to developed resistnce in them (Krunrtne et l. 2007). While C. hemipterus showed resistnce towrds pyrethroid-bsed insecticides, C. lectulrius hve lredy developed theirs in mny Europen countries (Romero et l. 2007; Goddrd, 2013; Lilly et l. 2015). Rrely reports on tropicl bed bugs re published especilly on their susceptibility to chemicl pesticides. On the other hnd, topicl biossys on common bed bugs in Sydney, Austrli showed tht the pests were highly resistnt to permethrin ( times), deltmethrin ( times) nd bendiocrb (250 times) due to their high LD 50 vlues compred to other insecticide groups (Lilly et l. 2015). They seemed to thicken the cuticle s defense mechnism, which ws positively correlted with knockdown time of survived bugs fter 24 hours forced-exposure (Lilly et l. 2016). Furthermore, the effect of feeding nd strvtion sttus cn influence the insecticide tolernce in bed bugs. In this study, bed bugs were fed on expired humn blood for 24 hours prior to experimentl procedures to prevent mortlity due to strvtion. Bsed on the study by Busvine (1958), the tolernce level might be compromised due to strvtion which could led to bised results. Devries et l. (2015) lso suggested tht the blood mel hd little impct on tolernce of 9-dy nd 2-dy strved C. lectulrious to deltmethrin. Thus, we decided to feed the bed bugs prior to biossy to prevent mortlity due to strvtion which could give bised results. 6

7 A study on the resistnce levels in severl popultions of bed bugs in the United Sttes hs been recently reported by Romero nd Anderson (2016). They detected low lethl dose (LD 50 ) in four types of nicotinoids (imidcloprid, cetmiprid, dinotefurn, nd thimethoxm). Esterse ctivities hve been ssocited with the incresed resistnt popultions, which probbly cquired from the previous exposure to other insecticide groups (Mmidl et l. 2011). Although mny formultions re mrketed extensively, newer products, however, do not entirely solve the resistnt issues mong bed bug popultions cross the world. A mixture of imidcloprid nd bet-cyfluthrin in form of concentrted suspension, Temprid SC lso exhibited less efficcy ginst severl selected strins due to the presence of pyrethroid components (Wng et l. 2015; Wng et l. 2016; Gordon et l. 2014). In study by Ab Mjid nd Zhrn (2015b), dult bed bugs were not 100% killed, lthough they pplied Temprid t high concentrtion (630 ppm). In nother study, the exposure of the product on bed bugs exhibited high resistnce rtio in the first nymphl stge with vlue of 9.4 for Epic Center strin nd 10.0 for Hrln strin (Cmpbell nd Miller, 2015). Ashbrook et l. (2017) ssessed susceptibility levels of 10 bed bugs popultions from six different sttes in the USA within seven dys of exposure to two types of insecticides i.e. chlorfenpyr nd bifenthrin. Among these, they detected significntly reduced susceptibility in three nd five popultions to chlorfenpyr nd bifenthrin. They found tht popultions with more thn 25% survivors were deemed to hve reduced susceptibility to the insecticides. They lso found n unexpected correltion between chlorfenpyr nd bifenthrin susceptibility of the different bed bug popultions lthough the modes of ction of both insecticides re different (Ashbrook et l. 2017). Bsed on the results, high percentge of mortlity were observed when the popultions were exposed to propoxur. There ws significnt interction between mortlity percentge nd lethl time. It ws shown tht propoxur ws lso susceptible to resistnt popultions. Minimum dys required for 50% of the popultion to die ws 6.4 dys, wheres 58.8 dys ws the mximum dys required for 99% of the popultion to die (LT 50 = h, LT 99 = h). In the USA, residul biossy on bed bugs collected in poultry house exhibited 100% mortlity when 1% propoxur ws used (Goddrd, 2013). The bugs did not rpidly die with less thn 80% of mortlity ws noted when exposed to other nonpropoxur products within 24 hours. In generl, propoxur cts s neurotoxin tht inhibited the binding process of cetylcholine to the receptor which is lso similr to the role of orgnophosphte insecticide in the nervous systems of insects. However, the ppliction of propoxur is risky s it remins intct in the tmosphere in the infested plces for severl weeks (Berg, 2010). Aprt from the pungent smell of the ctive ingredient, propoxur is hrmful to children nd pregnnt mothers (Doggett et l. 2012). Reports from Thilnd nd Sri Lnk climed tht there were resistnce issues in the field popultions tested with propoxur (Twtsin et l. 2011; Krunrtne et l. 2007). Observtion on 24 hours exposure t concentrtion of 0.8% displyed tht 45.5% of bed bugs survived regrdless of their growth stges. This could be relted to the previous enzyme detoxifiction on other insecticide groups which resulted in multiple cross-resistnce in bed bugs. Thus, the United Sttes Environmentl Protection Agency (EPA, 2016) hs prohibited the use of propoxur, prticulrly round the children. The insecticide is, however, llowed to be used in urbn res including commercil nd industril buildings like office properties, wrehouses, hotels, mlls nd medicl centers (EPA, 2016). Ditomceous erth dust is nother option in bed bug control mesures for it hs similr chrcteristics to insecticides (Doggett, 2012; Berg, 2010). The effectiveness of this non-chemicl tretment in eliminting the infesttion is higher since it is ble to kill bed bugs t ll stges whether or not they hve been treted with insecticides (Berg, 2010). A study by Wng et l. (2012), suggested tht the non-chemicl tretment ws ble to eliminte bed bugs in the prtment building rther thn sprying residul insecticides which cn 7

8 reintroduce the colony within short period of time. Furthermore, the suppression of bed bug popultions will be more effective if the implementtion of integrted control mngement is included in the tretment method. Heting nd steming in between C long with spreding the powdered ditomceous erth to tiny gps could definitely reduce the number of bugs in the infested house (Puckett et l. 2013; Akhtr nd Ismn, It is criticl to continue periodic monitoring nd to implement preventive control mesure fter successful elimintion of bed bugs hs been chieved to minimize the reinfesttion rte nd cost ssocited with bed bug controls (Bennet et l. 2016). It lso requires community-wide nd proctive, bed bug mngement progrms (Romero et l. 2017), in order to provide more effective, sustinble, economiclly vible, nd ffordble solutions to the housing residents nd premise owners. CONCLUSION It cn be concluded tht the tropicl bed bug, C. hemipterus ws highly susceptible to propoxur. Low lethl vlues indicted tht the insecticide ws effective in killing this bug. The pyrethroid-bsed ctive ingredient, deltmethrin, however, ws expected for its low efficcy towrds resistnt bugs. The only limittion of this study ws no susceptible popultions of C. hemipterus cn be used for comprison. A little informtion is vilble regrding tropicl bed bug, C. hemipterus nd its resistnce towrds insecticides (Dng et l. 2017). However, this study provided preliminry reference for locl pest control professionls to void the use of ny potentil resistnt products to control tropicl bed bug infesttions in Mlysi. ACKNOWLEDGEMENTS The uthors would like to thnk Universiti Sins Mlysi for funding the reserch (Short Term Grnt: 304/ PBIOLOGI/ ). We would lso like to thnk these individuls for ssisting us while smpling the bugs cross Peninsulr Mlysi: Wn Ahmd Syhir, Noor Hzwni, Nurdilhusn nd lstly to ll residents involved. 8

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11 Miller, D. M. (2014). Bed bug tretment using insecticides. Virgini: Virgini Deprtment of Agriculture nd Consumer Service. Montes, C., Cudrillero, C., & Vilell, D. (2002). Mintennce of lbortory colony of Cimex lectulrius (Hemipter: Cimicide) using n rtificil feeding technique. Journl of Medicl Entomology 39(4): Moore, D. J., & Miller, D. M. (2006). Lbortory evlutions of insecticide product efficcy for control of Cimex lectulrius. Journl of Entomologicl Science 99(6): Puckett, R., McDonl, D. L., & Gold, R. E. (2013). Comprison of multiple stem tretment durtions for control of bed bugs (Cimex lectulrius L.). Pest Mngement Science 69(9): Romero, A., & Anderson, T. D. (2016). High levels of resistnce in the common bed bug, Cimex lectulrius (Hemipter: Cimicide), to neonicotinoid insecticides. Journl of Medicl Entomology 53(3): Romero, A., Potter, M. F., Potter, D. A., & Hynes, K. F. (2007). Insecticide resistnce in the bed bug: fctor in the pest's sudden resurgence? Journl of Medicl Entomology 44(2): Romero, A., Sutherlnd, A. M., Gouge, D. H., Spfford, H., Nir, S., Lewis, V., Choe, D.- H., Li, S., & Young, D. (2017). Pest mngement strtegies for bed bugs (Hemipter: Cimicide) in multiunit housing: literture review on field studies. Journl of Integrted Pest Mngement 8(1):1 10. Steelmn, C. D., Szlnski, A. L., Trout, R., McKern, J. A., Solorzno, C., & Austin, J. W. (2008). Susceptibility of the bed bug Cimex lectulrius L. (Heteropter: Cimicide) collected in poultry production fcilities to selected insecticides. Journl of Agriculturl nd Urbn Entomology 25(1): Suwnnyod, S., Chnbng, Y., & Burnpnichpn, S. (2010). The life cycle nd effectiveness of insecticides ginst the bed bugs of Thilnd. Southest Asin Journl of Tropicl Medicine nd Public Helth 41(3): Twtsin, A., Thvr, U., Chompoosri, J., Phusup, Y., Jonjng, N., Khumswds, C., Bhkdeenun, P., Swnpnylert, P., Asvdchnukorn, P., Mull, M. S., & Siriystien, P. (2011). Insecticide resistnce in bedbugs in Thilnd nd lbortory evlution of insecticides for the control of Cimex hemipterus nd Cimex lectulrius (Hemipter: Cimicide). Journl of Medicl Entomology 48(5): Wng, C., Gibb, T., & Bennett, G. (2009). Evlution of two lest toxic integrted pest mngement progrms for mnging bed bugs (Heteropter: Cimicide) with discussion of bed bug intercepting device. Journl of Medicl Entomology 46(3): Wng, C., Sltzmnn, K., Bennett, G., & Gibb, T. (2012). Comprison of three bed bug mngement strtegies in low-income prtment building. Insects 3(2):

12 Wng, C., Singh, N., Zh, C., & Cooper, R. (2016). Efficcy of selected insecticide sprys nd erosols ginst the common bed bug, Cimex lectulrius (Hemipter: Cimicide). Insects 7(1): 5. Wng, C., Tsi, W., Cooper, R., & White, J. (2011). Effectiveness of bed bug monitors for detecting nd trpping bed bugs in prtments. Journl of Economic Entomology 104(1): Wng, L., Ci, X., & Xu, Y. (2015). Sttus of urbn bed bug infesttions in Southern Chin: n nlysis of pest control service records in Shenzhen in 2012 nd Donggun in Journl of Medicl Entomology 52(1): Wood, O. R., Hnrhn, S., Coetzee, M., Koekmoer, L. L., & Brooke, B. D. (2010). Cuticle thickening ssocited with pyrethroid resistnce in the mjor mlri vector Anopheles funestus. Prsites nd Vectors 3(1): 67. Zhu, F., Lvine, L., O Nel, S., Lvine, M., Foss, C., & Wlsh, D. (2016). Insecticide resistnce nd mngement strtegies in urbn ecosystems. Insects 7(1): 2. Zhu, F., Wigginton, J., Romero, A., Moore, A., Ferguson, K., Plli, R., Potter, M. F., Hynes, K. F., & Plli, S. R. (2010). Widespred distribution of knockdown resistnce muttions in the bed bug, Cimex lectulrius (Hemipter: Cimicide), popultions in the United Sttes. Archives of Insect Biochemistry nd Physiology 73(4):

13 Men mortlity 10 AR TI SW KG IP PY PC KT PD HM Strin Figure 1. Effect of propoxur on strins of bed bugs. Brs of sme letters hd no significnt difference. b 10 b Men mortlity 8 b b b b b b b KT PD AR TI SW KG IP PY PC HM Strin Figure 2. Effect of deltmethrin on strins of bed bugs. Brs of sme letters hd no significnt difference. 13

14 Tble 1. Strins of bed bugs in the resistnce biossy. Loction nd strin/popultion Cluster Dte collected Totl bed bugs in both tretments Resistnce sttus Aru, Perlis (AR) 1 24 Sept Unknown Ipoh, Perk (IP) 1 17 Oct Highly resistnt Teluk Intn, Perk (TI) 1 18 Oct Resistnt Hutn Melintng, Perk 1 18 Oct Resistnt (HM) Sg. Petni, Kedh (PY) 1 26 Sept Resistnt Lngkwi, Kedh (PC) 1 27 Oct Resistnt Kul Terenggnu, 2 20 Aug Unknown Terenggnu (KT) Klng, Selngor (KG) 3 25 Nov Resistnt Port Dickson, Negeri 4 29 Nov Unknown Sembiln (PD) Senwng, Negeri Sembiln (SW) 4 29 Nov Unknown *Sttus on field strin/popultion were in ccordnce vi questionnires by professionls nd locl residents. 14

15 Tble 2. Comprison of lethl hours of two insecticides on ten popultions of bed bugs. Insecticide Strin Slope ± SE LT 50 (hours) LT 90 (hours) LT 99 (hours) group Deltmethrin (Pyrethroid) TI ± ( ) ( ) ( ) IP ± ( ) ( ) ( ) SW ± ( ) ( ) ( ) AR ± * * * PD ± ( ) ( ) ( ) PY ± ( ) ( ) ( ) KG ± ( ) ( ) ( ) KT ± ( ) ( ) ( ) HM ± ( ) ( ) ( ) PC ± ( ) ( ) ( ) Propoxur (Crbmte) TI ± ( ) ( ) ( ) IP ± ( ) ( ) ( ) SW ± ( ) ( ) ( ) AR ± ( ) ( ) ( ) PD ± ( ) ( ) ( ) PY ± ( ) ( ) ( ) KG ± ( ) ( ) ( ) KT ± ( ) ( ) ( ) HM ± ( ) ( ) ( ) PC ± ( ) ( ) ( ) *No time intervl shown 15