Susceptibility of Malaysian Tropical Bed Bug Cimex hemipterus F. (Hemiptera: Cimicidae) Populations to Deltamethrin and Propoxur Insecticides Authors:

Bed bugs were collected across Peninsular Malaysia to investigate their resistance to two types of insecticide classes, pyrethroid and carbamate. In this study, commercially-available deltamethrin and propoxur were used to test the resistance status *Corresponding author: abdhafiz@usm.my Zulaikha Zahran and Abdul hafiz Ab Majid 92 of tropical bed bug, Cimex hemipterus in Malaysia. Their resistance status was evaluated using insecticide-impregnated papers, a bioassay method recommended by the World Health Organization (WHO). Ten adult bed bugs in each population were placed inside the tubes with papers impregnated with 0.05% deltamethrin and 0.1% propoxur. At first, the bugs were exposed for 24 h, then, the exposure period was extended to 14 days. The bioassay was performed in triplicates. The highest resistance level was exhibited by the AR population with a lethal time value (LT50) of 466.119 h in the deltamethrin-containing tube. In contrast, propoxur was highly effective against the IP population due to its lethal time value (LT50 = 153.032 h) was the lowest. A significant difference (p < 0.005) was found between insecticide classes and bed bug populations. Mean mortality among AR, SW, HM, PY and PC populations showed a significant difference when exposed to deltamethrin. In conclusion, propoxur showed high efficacy in controlling bed bug populations. However, its efficacy was only at particular places. Meanwhile, a treatment using deltamethrin should be avoided by pest control professionals due to its low efficacy.


INTRODUCTION
Over the last two decades, the resurgence of bed bugs has become one of the primary problems in bed bug control strategy worldwide. Generally, the elimination of the bed bug infestations involves the use of chemical insecticides. However, a continual application of chemical insecticides has resulted in bed bug resistance (Dang et al. 2017) and have created widespread worries among pest control professionals. To make matter worse, a cryptic and nocturnal lifestyle of the bed bugs further complicate the elimination strategy (Reinhardt & Siva-Jothy 2007). Numerous studies have demonstrated that the evolution of insecticide resistance likely plays a major role in the resurgence of these blood-sucking insects (Reinhardt & Siva-Jothy 2007;Dang et al. 2017).
The main reason linked to this matter is that the evolution of their body systems, causing multiple resistance mechanisms to rapidly develop against pesticides applied (Adelman et al. 2011;Bai et al. 2011). After DDT was banned for its persistence and negative environmental impact (Carson 1963), other classes of insecticides with reduced susceptibilities such as carbamate, phenylpyrazole, neonicotinoid, organothiophosphate and pyrrole were tested for their toxicity in controlling Cimex lectularius colonies in poultry facilities (Steelman et al. 2008). In Australia, bed bug populations showed lower mortality rate for most insecticide classes tested, except imidacloprid and pirimiphos-methyl .
To date, a majority of pest control companies prefer to use synthetic pyrethroids as they are known to effectively kill insects such as mosquitoes, flies, head louse and bed bugs (Zhu et al. 2016). However, evolved resistance to the pyrethroids by insects including bed bugs has led to difficulties and posed more challenges in suppressing their infestation. A report by Zhu et al. (2010) proved that common bed bugs in the USA showed no response towards deltamethrin are associated with kdr-type resistance triggered by two major amino acids i.e. V419L 93 and L925I. Field-collected strains in infested homes in Denmark also reported the low efficacy of permethrin and deltamethrin, while the alternative insecticide, chlorpyrifos resulted in higher mortality percentage (Kilpinen et al. 2011).
Two knockdown resistance (kdr) mutations associated with resistance to pyrethroids have been recently detected in C. hemipterus (Dang et al. 2015). These kdr mutations affect the efficacy of pyrethroid, a class of insecticide that attack a voltage-gated sodium channel (VGSC) in the nervous system. An alteration of sequences of binding sites in the sodium channel counteracts the mode of action of pyrethroid and DDT, resulting in the insensitivity to both classes of insecticides (Soderlund & Knipple 2003). The kdr mutations have also been detected in several hematophagous insects such as mosquitoes, Anopheles gambiae, Culex pipiens, cat flea, Ctenocephalides felis and C. hemipterus (Bass et al. 2004;Dang et al. 2015).
In general, C. hemipterus is more susceptible than C. lectularius. However, some of them possess a higher tolerance level towards insecticides which can be passed on to their progeny (Busvine 1958). Pest control companies in Southeast Asia rely mostly on chemical and spray methods as treatments (Wang et al. 2011;Ab Majid & Zahran 2015a;2015b). However, due to resistant factors, the companies hardly reduce pest populations despite various insecticides are readily available in the market. Pyrethroid, a class of insecticide which has been widely used as a control agent was reported to demonstrate a little or no effect on bed bugs and other insect vectors (Wood et al. 2010;Miller 2014). Bed bugs may also develop their resistance to other classes of insecticides with similar active ingredients if no alternative control measure is immediately sought to avoid the circumstance (Moore & Miller 2006).
Numerous findings related to chemical and non-chemical methods that resulted in effective control of bed bugs have been reported by many. These include the application of diatomaceous earth and spraying of insecticides such as chlorfenapyr, propoxur, and other classes (Wang et al. 2009, Manuel 2010. Unlike termites, bed bugs lack grooming activity to affect other members in a population. Nevertheless, horizontal transfer through contact and ingestion of insecticides might occur among bed bugs (Akhtar & Isman 2013). A few studies found that simultaneous practices of two control methods such as a combination of heating and chemical treatment can effectively eliminate resistant populations compared to a single method; either by spraying or dust application (Wang et al. 2009, Akhtar & Isman 2013. Tropical bed bugs sampled in Bangkok, Phuket and Krabi of Thailand have developed resistance to several insecticide classes i.e. organochlorines, carbamates, and pyrethroids but noted a higher mortality when treated with imidacloprid and chlorfenapyr (Tawatsin et al. 2011).
Multiple resistance mechanisms have been identified in bed bugs including penetration resistance through cuticle thickening or remodeling, metabolic resistance by increasing the activities of detoxification enzymes (e.g. cytochrome P450 monooxygenases and esterases), and knockdown resistance by kdr mutations (Dang et al. 2017). Other potential resistance mechanisms such as behavioural and physiological changes (e.g. increasing esterase activities by point mutations, glutathione S-transferase, target site insensitivity including altered AChEs, GABA receptor insensitivity, and altered nAChRs), and symbiontmediated mechanisms are yet to be discovered. It is also worthwhile to mention that research addressing the mechanisms of insecticide resistance in the tropical bed bug, C. hemipterus is generally lacking although the mechanisms are likely similar to the temperate species, C. lectularius.
In Malaysia, the tropical bed bugs have been reported exhibiting a resistance to chlorinated hydrocarbons, particularly DDT, HCH, d-allethrin, and dieldrin group (Dang et al. 2017). However, the resistance status of the Malaysian bed bugs to other classes of insecticides such as pyrethroid and carbamate is currently unknown. Therefore, this study was conducted to evaluate the resistance status of C. hemipterus collected across Peninsular Malaysia to two major classes of insecticides i.e. pyrethroid (deltamethrin) and carbamate (propoxur). The efficacy of both insecticides on the bed bugs was also determined.

Bed Bugs Rearing and Population Selection
Bed bugs were acquired from the sample collection across Peninsular Malaysia. They were maintained, reared under room conditions (25 ± 2C, 50%-60% of relative humidity) and were fed weekly on expired human blood supplied by Penang General Hospital. Modifications of artificial feeding system were set up by stretching parafilm membrane on a glass jar filled with the expired blood based on the feeding maintenance of bed bugs by Montes et al. (2002) and Abd Rahim et al. (2015). Ten populations were selected based on the resistance status of recent bed bugs control reports and feedback from questionnaires given to the pest control companies in Malaysia (Ab Majid & Zahran 2015a). They were represented by each cluster obtained from the previous sampling. Bed bugs were separated and maintained according to field and laboratory strains. F1, F2 and F3 generations were labeled and kept in different containers.

Insecticide Preparation
Two insecticides i.e. deltamethrin and propoxur were used to evaluate the resistance of bed bugs using insecticide-impregnated papers from the World Health Organization (WHO) test kit (Busvine 1958). The papers (12 cm x 15 cm) were impregnated with 0.1% propoxur and 0.05% deltamethrin. Impregnated papers then were cut into smaller pieces (3 x 5 cm), rolled and inserted into glass test tubes of 12.5 cm x 1.5 cm. When the bugs were exposed to the treated papers, the tubes were covered with fine net cloth to prevent them from escaping. All tubes were placed in a container at room condition (temperature, 25 ± 2ºC, relative humidity, 50%-60%).

Resistance Bioassay
The resistance bioassay was conducted following methods by Busvine (1958) and Tawatsin et al. (2011). Only adult bed bugs from the F2 generation were used in this bioassay. Each bioassay required 40 bed bugs from each population. The selected populations are tabulated in Table 1. Ten adults from each population were tested by introducing them into the test tube containing 0.05% deltamethrin-impregnated papers -or 0.1% propoxur-impregnated papers. The concentrations of insecticides and exposure times were as recommended by WHO (Busvine & Nash 1953;Tawatsin et al. 2011;DeVries et al. 2015) and based on the previously established baseline data (unpublished data). Each treatment was done in triplicates with one control. Filter papers were impregnated with standard oil solutions of insecticides incorporated with the insecticide resistance kit provided by the WHO (Busvine & Nash 1953;Tawatsin et al. 2011). Bed bugs exposed to papers impregnated with silicone oil (deltamethrin treatment), and risella oil (propoxur treatment) were used as controls. Bed bugs were fed on expired human blood for 24 h prior to experimental procedures. The bed bugs were exposed for 24 h for preliminary testing. Observations on their mortality were continued and recorded for 14 days. They were starved during the continuous bioassay. Mortality was scored based on their posture and movement in the tubes. Dead bugs were in an upside-down position with no signs of movement after 10 sec of observation. Survived bed bugs were kept in 70% alcohol for further observation of resistance in the bugs. Control groups exceeding 10% of mortality were corrected using Abbott's formula (Abbott 1925).

Statistical Analysis
Percentage of mortality in each treatment was calculated and analysed daily at 24 h interval. Probit analysis was used in determining lethal times (hours) of the bugs at LT 50 , LT 90 , and LT 99 with a confidence limit of 10%-90%. Evaluations of lethal times were made by using software SPSS Version 22.0 (IBM Corp., Armonk, NY, USA). Mean comparison was performed using one-way and two-way analysis of variance (ANOVA) to determine the significant difference between the populations and the effect of insecticide on bed bug populations.

RESULTS
Based on the results, tropical bed bugs were less susceptible against deltamethrin compared to propoxur ( Fig. 1 and Fig. 2). From the first until the third day of application, all populations showed low mortality within the range of 3%-20% in the deltamethrin treatment. Among the tested populations, the KT and HM exhibited 20% higher in mortality after one week of exposure. The PC population recorded the highest mortality (>70%) on day 10 compared to the other populations. On day 14, the AR population exhibited the lowest mortality (40%). The other populations exhibited a moderate to high mortality; ranging from 60% to 80%. Two populations, PC and SW had the highest mortality rate (90%), further indicating that both populations were highly susceptible towards deltamethrin. They were, however, still considered as resistant to this insecticide. Survived bed bugs might have developed resistance in them due to the previous treatment of different insecticides by pest control operators. Meanwhile, propoxur showed high efficacy in killing bed bugs as it caused an overall mortality of 90% throughout the treatment period (Fig. 1). Only one population i.e. TI noted high mortality on the first day of exposure i.e. 20% and increased to 37% after day 3. Nine other populations displayed a low mortality rate until the third day of exposure, but the mortality increased to nearly 50% after day 7. On the 10th day, all populations reached 80%-90% mortality except for two populations (KT and HM), where the mortality rates were 60% and 67%, respectively. Three populations namely IP, PY, and PC noted 100% mortality after the 14th day of the treatment. The lowest mortality was observed in the KT population with 87%, while ~ 90% were recorded for the other seven populations.
Lethal time (in hours) was performed to determine the efficacy of insecticides against bed bugs (    Table 2. Most of the tested populations showed high susceptibility to propoxur after eight days of exposure. Approximately 153.032 h was required to kill 50% of the IP population, exhibiting the lowest resistance compared to others. Another population exhibited low resistance was the TI with 158.754 h. The remaining eight populations showed increased lethal time values but were still considered to have low resistance since up to 30%-53% of bed bugs were killed within 7-8 days of exposure periods. The KT population, on the other hand, had the highest LT 50 with 203.334 h, exhibiting the lowest percentage of mortality. A two-way analysis of variance (ANOVA) was performed to determine the effect of insecticide groups on different populations of bed bugs. Based on the results, there was a significant interaction (F (9, 40) = 2.741 at p < 0.005) between insecticide groups and different populations of bed bugs. Comparison between two main active ingredients revealed that propoxur was highly effective (p < 0.0001) than deltamethrin in controlling bed bugs. Mean values of mortality in bed bugs for propoxur and deltamethrin were 9.5 and 7.6, respectively. Interactions between bed bug populations, however, had different results depending on which insecticides they were treated.

Trends of LT 50 (in hours) over time for all populations are presented in
Therefore, one-way ANOVA was used to investigate the differences in bed bugs populations treated with propoxur and deltamethrin. As shown in Fig.  1 and Table 2, exposure to propoxur exhibited no significant difference among the populations (F (9, 20) = 1.468, p > 0.05) while, populations treated with deltamethrin showed a significant difference among the populations (F (9, 20) = 3.453, p < 0.05). A significant interaction of mean mortality was found between populations of AR, SW, HM, PY, and PC ( Fig. 2 and Table 2).

DISCUSSION
To date, pyrethroid-based insecticides are widely used in Malaysia to control bed bug infestations in various premises (Ab Majid & Zahran 2015a). Failure in chemical control is the major factor of resistance development following repetitive exposure of similar insecticides to bed bugs. As such, a bioassay was used to assess the effect of two insecticides against populations of bed bugs in Peninsular Malaysia. The method is relatively simple to perform and provides standardized data in monitoring insecticide resistance (Dang et al. 2017). Based on the results, deltamethrin was less effective in controlling the populations. Similar results were obtained in the tropical bed bug populations in Sri Lanka where high KT 50 values were observed, ranging from 0.5 h to 24 h and from 2.5 h to 47 h after being exposed to permethrin and deltamethrin for 24 h (Karunaratne et al. 2007). Low doses of bifenthrin and alpha-cypermethrin were used to treat bed bugs in Thailand had caused high effective dose (ED 50 ) values compared to other urban pests (Suwannayod et al. 2010). Bed bugs might have their systems triggered by these pyrethroid-based insecticides, leading to developed resistance in them (Karunaratne et al. 2007).
While C. hemipterus showed resistance towards pyrethroid-based insecticides, C. lectularius have already developed theirs in many European countries (Romero et al. 2007;Goddard 2013;Lilly et al. 2015). Rarely reports on tropical bed bugs are published especially on their susceptibility to chemical pesticides. On the other hand, topical bioassays on common bed bugs in Sydney, Australia showed that the pests were highly resistant to permethrin (1,235,000 times), deltamethrin (370,000 times) and bendiocarb (250 times) due to their high LD 50 values compared to other insecticide groups . They seemed to thicken the cuticle as a defense mechanism, which was positively correlated with a knockdown time of survived bugs after 24 h forced-exposure (Lilly et al. 2016). Furthermore, the effect of feeding and starvation status can influence the insecticide tolerance in bed bugs. In this study, bed bugs were fed on expired human blood for 24 h prior to experimental procedures to prevent mortality due to starvation. Based on the study by Busvine (1958), the tolerance level might be compromised due to starvation which could lead to biased results. DeVries et al. (2015) also suggested that the blood meal had a little impact on tolerance of 9-day and 2-day starved C. lectularious to deltamethrin. Thus, we decided to feed the bed bugs prior to bioassay to prevent mortality due to starvation which could give biased results.
A study on the resistance levels in several populations of bed bugs in the United States has been recently reported by Romero and Anderson (2016). They detected a low lethal dose (LD 50 ) in four types of nicotinoids (imidacloprid, acetamiprid, dinotefuran, and thiamethoxam). Esterase activities have been associated with the increased resistant populations, which probably acquired from the previous exposure to other insecticide groups . Although many formulations are marketed extensively, newer products, however, do not entirely solve the resistant issues among bed bug populations across the world. A mixture of imidacloprid and beta-cyfluthrin in a form of concentrated suspension, Temprid SC also exhibited less efficacy against several selected strains due to the presence of pyrethroid components (Wang et al. 2015;Wang et al. 2016;Gordon et al. 2014). In a study by Ab Majid and Zahran (2015b), adult bed bugs were not 100% killed, although they applied Temprid at a high concentration (630 ppm). In another study, the exposure of the product on bed bugs exhibited high resistance ratio in the first nymphal stage with a value of 9.4 for Epic Center strain and 10.0 for Harlan strain (Campbell & Miller 2015). Ashbrook et al. (2017) assessed susceptibility levels of 10 bed bugs populations from six different states in the USA within seven days of exposure to two types of insecticides i.e. chlorfenapyr and bifenthrin. Among these, they detected a significantly reduced susceptibility in three and five populations to chlorfenapyr and bifenthrin. They found that populations with more than 25% survivors were deemed to have reduced susceptibility to the insecticides. They also found an unexpected correlation between chlorfenapyr and bifenthrin susceptibility of the different bed bug populations although the modes of action of both insecticides are different (Ashbrook et al. 2017).
Based on the results, a high percentage of mortality were observed when the populations were exposed to propoxur. There was a significant interaction between mortality percentage and lethal time. It was shown that propoxur was also susceptible to resistant populations. Minimum days required for 50% of the population to die was 6.4 days, whereas 58.8 days was the maximum days required for 99% of the population to die (LT 50 = 153.302 h, LT 99 = 1413.259 h). In the USA, a residual bioassay on bed bugs collected in a poultry house exhibited 100% mortality when 1% propoxur was used (Goddard 2013). The bugs did not rapidly die with less than 80% of mortality was noted when exposed to other non-propoxur products within 24 hours. In general, propoxur acts as a neurotoxin that inhibited the binding process of acetylcholine to the receptor which is also similar to the role of organophosphate insecticide in the nervous systems of insects.
However, the application of propoxur is risky as it remains intact in the atmosphere in the infested places for several weeks (Berg 2010). Apart from the pungent smell of the active ingredient, propoxur is harmful to children and pregnant mothers (Doggett et al. 2012). Reports from Thailand and Sri Lanka claimed that there were resistance issues in the field populations tested with propoxur (Tawatsin et al. 2011;Karunaratne et al. 2007). Observation on 24 h exposure at a concentration of 0.8% displayed that 45.5% of bed bugs survived regardless of their growth stages. This could be related to the previous enzyme detoxification on other insecticide groups which resulted in multiple cross-resistance in bed bugs. Thus, the United States Environmental Protection Agency (EPA 2016) has prohibited the use of propoxur, particularly around the children. The insecticide is, however, allowed to be used in urban areas including commercial and industrial buildings like office properties, warehouses, hotels, malls and medical centers (EPA 2016).
Diatomaceous earth dust is another option in bed bug control measures for it has similar characteristics to insecticides (Doggett et al. 2012;Berg 2010). The effectiveness of this non-chemical treatment in eliminating the infestation is higher since it is able to kill bed bugs at all stages whether or not they have been treated with insecticides (Berg 2010). A study by Wang et al. (2012), suggested that the non-chemical treatment was able to eliminate bed bugs in the apartment building rather than spraying residual insecticides which can reintroduce the colony within a short period of time. Furthermore, the suppression of bed bug populations will be more effective if the implementation of integrated control management is included in the treatment method. Heating and steaming in between 50°C-60°C along with spreading the powdered diatomaceous earth to tiny gaps could definitely reduce the number of bugs in the infested house (Puckett et al. 2013;Akhtar & Isman 2013). It is critical to continue a periodic monitoring and to implement a preventive control measure after successful elimination of bed bugs has been achieved to minimise the re-infestation rate and cost associated with bed bug controls (Bennet et al. 2016). It also requires community-wide and proactive, bed bug management programs (Romero et al. 2017), in order to provide a more effective, sustainable, economically viable, and affordable solutions to the housing residents and premise owners.

CONCLUSION
It can be concluded that the tropical bed bug, C. hemipterus was highly susceptible to propoxur. Low lethal values indicated that the insecticide was effective in killing this bug. The pyrethroid-based active ingredient, deltamethrin, however, was expected for its low efficacy towards resistant bugs. The only limitation of this study was no susceptible populations of C. hemipterus can be used for comparison.
A little information is available regarding tropical bed bug, C. hemipterus and its resistance towards insecticides (Dang et al. 2017). However, this study provided a preliminary reference for local pest control professionals to avoid the use of any potential resistant products to control tropical bed bug infestations in Malaysia.