Insecticide susceptibility status and resistance mechanism of Anopheles cracens Sallum and Peyton and Anopheles maculatus Theobald (Family:Culicidae) from knowlesi malaria endemic areas in Peninsular Malaysia

Ahmad Rohani, Abdul Rahman Aidil Azahary, Ke-Xin Yu, Mohamed Nor Zurainee,Wan Mohamad Ali Wan Najdah, Han Lim Lee

1Medical Entomology Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

2Department of Parasitology, University of Malaya, Kuala Lumpur, Malaysia

3Faculty of Health and Life Sciences, Management and Science University, 40100 Shah Alam, Selangor, Malaysia

Keywords:Insecticide susceptibility Vector control WHO adult bioassay Biochemical enzyme assay

ABSTRACT Objective: To assess the insecticide susceptibility status of Anopheles cracens (An. cracens)and Anopheles maculatus (An. maculatus) from knowlesi malaria endemic areas in Peninsular Malaysia towards DDT, malathion and deltamethrin and to determine the resistance mechanism involved. Methods: Adult and larval mosquitos were collected for surveillance. Susceptibility status of Anopheles was determined using the standard WHO adult bioassay, larval bioassay and biochemical enzyme assay. Results: WHO adult bioassay results indicated An. cracens collected from Kampung Sungai Ular, Pahang was resistant towards 4% DDT, while An.maculatus collected from Kampung Sokor, Kelantan and Kampung Sungai Lui, Selangor exhibited resistance towards 4% DDT. However, the enzyme activity profiles varied according to strains and species. The resistance ratio of larval bioassay, showed that all strains and species tested were susceptible to malathion and temephos. Conclusions: Since only a few anopheline strains exhibited low level of insecticide resistance towards malathion, DDT and temephos.These insecticides are still considered effective for vector control program towards An. cracens and An. maculatus.

1. Introduction

Anophelesmosquitoes are the main vectors that transmit human malaria which is a disease caused by the infections of the protozoan parasites such asPlasmodium falciparum,Plasmodium vivax,Plasmodium malariaeandPlasmodium ovale. Malaria is one of the most important tropical diseases in the world. In 2015, 214 million cases of malaria were reported, along with an estimated 438 000 of malaria deaths globally[1]. Being the prominent vectors of malaria, theAnophelesmosquitoes are widely distributed around the world, but only around 40 species are important vectors of malaria.Anopheles balabacensis,Anopheles campestris,Anopheles epiroticus,Anopheles flavirostris,Anopheles latens,Anopheles letiferandAnopheles maculatus(An. maculatus) are examples ofAnophelesspecies that are responsible for spreading human malaria in Malaysia[2].

Recently, human malaria cases caused by simianPlasmodium knowlesi(P. knowlesi) has been reported and found in most states of Peninsular Malaysia.P. knowlesiis a zoonotic malaria parasite of long tailed (Macaca fascicularis) and pig tailed (Macaca nemestrina) macaques. In South East Asia,P. knowlesiparasite was common and transmitted by mosquitoes of theAnopheles leucosphyrusgroup. In Peninsular Malaysia,Anopheles cracens(An. cracens),Anopheles hackeriandAnopheles introlatushave been shown to be the vector ofP. knowlesiinfection, whileAnopheles balabacensisandAnopheles latenswere identified as the vector in Sabah and Sarawak, respectively[3,4].

Vector control is an important component of the WHO Global Strategy for Malaria Control, for which its objective is to break the transmission of malaria parasite using indoor residual spraying or pyrethroid-impregnated materials. Malaria vector control activities in Malaysia focus mainly on the use of insecticide-treated bed nets and indoor residual spraying. Since 1998, deltamethrin wettable powder has replaced DDT as the main pyrethroid used in residual spraying, while permethrin was used to treat the bednets. Fogging with malathion technical grade is carried out as a supplementary measure in the event of outbreak to control malaria transmission.In addition, temephos (Abate®) is used as a larvacide for anti-larval activities[5,6].

Clearly, the control programme in Malaysia relies heavily on the chemical insecticides to curb the malaria situation. However,widespread resistance of vectors towards insecticides may limit the usage of insecticides and the effectiveness of insecticides in vector control programme. Due to these factors, resistance monitoring programme should be carried out constantly for early detection of the problem and rapid assimilation of information on the resistant mosquito population to strategies counter-measures. The aim of the current study was to assess the insecticide susceptibility status ofAn. cracensandAn. maculatusfrom knowlesi malaria endemic areas in Peninsular Malaysia towards DDT, malathion and deltamethrin and to determine the resistance mechanism involved.

2. Materials and methods

2.1. Study sites

This study was carried out in Kampung Mela Kuala Lipis Pahang,Kampung Sungai Ular Balok Pahang, Lata Cemerung Dungun Terengganu, Kampung Sokor Tanah Merah Kelantan, Mersing Reserved Forest Johor, Kampung Sahom Kampar Perak, Gerik Hulu Perak and Kampung Sungai Lui Hulu Langat Selangor. Both Kampung Mela and Kampung Sungai Ular Pahang are rubber estate, oil palm plantation and fruit orchard areas. Meanwhile,Lata Cemerung is a camping site, Kampung Sokor is a gold mining site, Mersing Reserved Forest is a logging track, Kampung Sahom is an aboriginal village, Gerik Hulu Perak is a forest near to the border of Thailand; and Kampung Sungai Lui is a village near to the border of Negeri Sembilan and Pahang. Figure 1 is the map of Peninsular Malaysia showing location of the 8 study sites selected. The area selection was based on knowlesi malaria cases reported for the year 2011 to 2014. In these study areas more than 80% of the villagers exploited their land for rubber and oil palm plantations and to a lesser extent for fruit orchards.

Figure 1. Location of 8 study sites in Peninsular Malaysia.

2.2. Adult surveillance

Adult mosquitoes were caught monthly from March 2014 to October 2015. The sampling was conducted by using human landing catches and CO2-baited Centers for Disease Control light traps (CDC-LT) simultaneously. Outdoor collection of mosquitoes was done separately from the 8 sites, for 3 consecutive nights at each site from 18: 00 to 6: 00 h. Alive adult mosquito were brought back to the lab and blood fed by using mice for colonization purpose.

Human landing catches were conducted by 3 teams of 2 catchers per team. They were randomly assigned into 3 teams and the teams were also randomly assigned for different location within the same study site. All mosquitoes that landed on the bare legs were caught by using 50 mm×19 mm glass vials which were subsequently plugged with cotton wool. CDC-LT baited with CO2were fixed randomly at the sampling sites. The CDC-LT was operated by four 1.5 V batteries which drive the suction fan and a 1.5 w bulb. The CO2was emitted from 0.5 kg dry ice placed in a 6 square inches ice box suspended adjacent and slightly above the light trap.Morphological identification of collectedAnophelesmosquitoes was done by using standard taxonomy keys[7,8] at the site the following morning.

2.3. Establishment of Anopheles species colonization

A colony ofAnopheleswas established from wild-caught fully engorged adult female. After the engorged adult female was maintained for 4-5 days and/or until gravid in the insectarium, it was then placed in a screen-topped plastic cup for oviposition,which contains filter paper lined inside the cup and 25 mL of reverse osmosis water. The eggs attached to the moist side of the filter paper and/or floating on the water surface were rinsed and transferred to white plastic tray (25 cm×36 cm×6 cm) containing 1 500 mL reverse osmosis water with wet filter paper lining the inside. During the embryonation period, the eggs were exposed to a 40-watt light for warming the eggs until hatching. Once hatched the 1st instar larvae were transferred daily from the oviposition cup to white plastic tray (25 cm×36 cm×6 cm) containing 1 500 mL reverse osmosis water. Each tray can accommodate 80 1st instar larvae. Ground fish food was used as larval feed. After pupation,approximately 100 pupae were placed in a plastic cup (14.5 cm in diameter and 6 cm in depth) containing 150 mL of distilled water and were kept in 30 cm×30 cm×30 cm cage in the presence of 10% sucrose solution for adult emergence. The first generation of the colony was used for susceptibility test. The insectarium was maintained at (27±2) ℃, 70%-80% relative humidity,and illumination from a fluorescent lighting was provided approximately for 12 h/day.

2.4. Susceptibility test of Anopheles mosquitoes

Susceptibility status ofAnophelesmosquitoes was examined using three different bioassays, namely WHO adult bioassay, larval bioassay and biochemical enzyme assay.

2.4.1. Adult bioassay

WHO insecticide monitoring test kit comprised WHO test tubes (12.5 cm in length, 4.4 cm in diameter) and WHO impregnated papers (12 cm×15 cm), were purchased from Vector Control Research Unit, School of Biological Sciences,Universiti Sains Malaysia, Penang, which was designated by WHO for the production of WHO insecticide test kits. The test tubes were used for exposing the mosquitoes to the insecticides at WHO recommended diagnostic concentration. In this study,the mosquitoes were subjected to 4.00% dichloro-diphenyltrichloroethane (DDT), 5.00% malathion and 0.25% deltamethrin for 1 h. F1 generation of field-collectedAnophelesspecies from all localities were used in the adult bioassay.

Adult bioassay was conducted according to WHO standard procedure[9]. A total of 20 sugar-fed adult female mosquitoes aged 2-5 days old were transferred to the tube with green dot(holding tube) and kept isolated/aside for 1 h to acclimatize the mosquitoes. Mosquitoes were then transferred to the tube with red dot (exposure tube) and exposed to insecticide impregnated paper. Each insecticide testing involved three replicates and 3 controls. During the exposure, knockdown time was recorded every five minutes for 1 h. After 1 h, mosquitoes were transferred to a fresh paper cups, fed with 10% sugar solution and mortality was recorded at 24 h post-exposure. According to WHO criteria for determining resistance, a mortality of 98% to 100% indicates susceptibility, 90% to 97% of mortality indicates possibility of resistance that require further confirmation and less than 90% of mortality indicates resistance[9].

Data obtained during 1 h exposure time were subjected to Probit analysis (SPSS ver. 11.5) to compute the lethal time (LT50) values.Resistance ratio (RR) was calculated by dividing the LT50value of the field strain to the susceptible strain, which was maintained at the Medical Entomology Unit, Institute for Medical Research. RR was determined as follow:

2.4.2. Larval bioassay

Larval susceptibility test was conducted according to WHO larval susceptibility bioassay[10]. WHO supplied insecticides namely malathion (781.25 mg/L stock solution) and temephos (156.25 mg/L stock solution) were purchased from Vector Control Research Unit,School of Biological Sciences, Universiti Sains Malaysia, Penang.Malathion (3.125 mg/L) and temephos (0.250 mg/L) were tested at diagnostic dosages for larval mosquitoes. Various concentrations of malathion and temephos were prepared and used to determine the lethal concentration (LC50). All the insecticide solutions were prepared in disposable paper cups of 300 mL capacity with triplicates. The cups were left undisturbed for 30 min after pipetting of the required insecticide into them. A total of 25 3rd instar larvae were then introduced into each cup. Larval mortality was recorded after 24 h post exposure. If the mortality of control larvae is between 5% and 20%, the mortalities of treated groups should be corrected according to Abbott’s formula as shown below:

LC50value was calculated by using Probit analysis software(Probit SOFTWARE Ltd) to determine the knockdown time of the mosquito. RR value was calculated by dividing the LT50value of the field strain to the susceptible strain. When RR value is less than 5, the field population is considered susceptible, when RR value is between 5 and 10 the field mosquitoes are considered to have moderate resistance, and when RR is more than 10, the field mosquitoes are highly resistant.

2.4.3. Biochemical enzyme assay

Biochemical assay of the resistance enzymes was conducted by using selected female adult field mosquitoes. Monooxygenases(MO) enzyme assay was performed according to Nazniet al.[11].The colour intensities were measured at wavelength 630 nm by using an immunoassay reader (Dynatech, Model MR 5000).Non-specific esterase (NSE) enzyme microassay was conducted according to Lee[12] and the reaction was measured at wavelength 450 nm. Glutathione-S-transferase (GST) enzyme was conducted as described by Lee and Chong[13]. The enzyme reaction was read at 450 nm, while the acetylcholinesterase (AChE) microassay was conducted according to Brogdonet al.[14] and the enzyme activity was assessed at 410 nm.

Protein assay of mosquito was conducted according to the method of Bradford[15]. A standard curve was prepared by using bovine serum albumin as the protein standard (Bio-rad Protein Assay Kit)and the protein concentration of mosquito was determined based on this standard curve.

2.5. Statistical analysis

LT50and LC50values were determined by using Probit analysis.One-way ANOVA followed by Tukey test was used to determine the significant differences between groups.

3. Results

3.1. Entomological surveillance of An. cracens and An.maculatus

During the survey performed from March 2014 until October 2015, a total of 492Anophelesfemale adults, which consisted ofAn. cracens(46.3%) andAn. maculatus(53.7%) were collected. As for mosquito larvae, a total of 110 were collected and the majority of them were those ofAn. cracens(Table 1).

Table 1 Total number of adult and larva of Anopheles mosquitoes collected according to species and sites.

3.2. Susceptibility test of Anopheles mosquitoes

3.2.1. WHO adult bioassay

An. cracenscollected from Kampung Sg. Ular and Kampung Sahom showed possible resistance to 5% malathion. Strains of Kampung Mela, Kampung Sokor and Kampung Sahom showed a possible resistance to 4% DDT, while strain from Kampung Sg.Ular showed confirmed resistant towards 4% DDT.An. cracensof all 6 strains tested remained susceptible to 0.05% deltamethrin(Table 2).

In the case ofAn. maculatus, Kampung Sg. Lui strain was the only strain that showed a possible resistance to 5% malathion.There was also one strain which was the Kampung Sokor strain that showed suggested resistance towards 0.05% deltamethrin.All other strains tested remained susceptible to 5% malathion and 0.05% deltamethrin. Strain of Gerik showed a suggested resistance to 4% DDT, while Kampung Sokor and Kampung Sg.Lui indicated confirmed resistant to 4% DDT (Table 2).

With regards to RR and LT50, the highest RR towards 5%malathion amongst theAn. cracenswas exhibited by Kampung Sahom strain (1.42) with LT50of 64.95 min which was found significantly higher when compared to other strains, except forKampung Sg. Ular.An. cracensof Kampung Sg. Ular strain showed the highest RR towards 4% DDT (1.36) with LT50of 67.92 min which was found significantly higher than Kampung Sokor,Lata Cemerung and Mersing. This strain also showed highest RR towards 0.05% deltamethrin (1.25) with LT50of 77.40 min which was significantly higher than all other strains tested. As forAn.maculatusit was the Kampung Sg. Lui strain that demonstrated the highest RR (0.92) towards 5% malathion with LT50of 33.27 min which was significantly higher only to that of Mersing strain.An. maculatusof Kampung Sg. Lui strain also was the strain that exhibited the highest RR towards 4% DDT (1.24; LT50of 60.33 min) and deltamethrin (1.45; LT50of 94.51 min) which was significantly higher than all others strain except Kampung Sahom strain (Table 3).

Table 2 Insecticide susceptibility of adult Anopheles mosquitoes from selected knowlesi malaria endemic areas in Peninsular Malaysia (1 h insecticide exposure).

Table 3 LT50 values (minutes) of insecticide against adult Anopheles species from selected knowlesi malaria endemic areas in Peninsular Malaysia.

3.2.2. WHO larval bioassay

The larval bioassay study showed that forAn. cracens, all strains remained susceptible to temephos. However,An. cracensof Lata Cemerung and Kampung Sahom strains demonstrated possible resistance towards malathion. MostAn. maculatusstrains were also found susceptible to temephos but two strains, those of Kampung Sokor and Kampung Sahom showed possible resistance while Kampung Sg. Lui strain demonstrated confirmed resistance to malathion (Table 4).

Table 4 Insecticide susceptibility test of Anopheles larvae from selected knowlesi malaria endemic areas in Peninsular Malaysia (24 h exposure).

Table 5 LC50 values of insecticide against Anopheles larvae from selected knowlesi malaria endemic areas in Peninsular Malaysia (ng/L).

With regards to RR and LC50, both species indicated a low RR (less than 5) for larvicidal activity. However, all strains exhibited relatively high LC50as compared to the laboratory strain, indicating that all field populations are possibly susceptible towards malathion and temephos (Table 5).

3.2.3. Biochemical enzyme assay

3.2.3.1. MO enzyme assay

The analysis of MO enzyme activity showed that, among all the strains ofAn. cracensmosquito tested, Kampung Sahom strain exhibited the significantly highest MO activity of 30.837 µmole product/min/µg protein which was significantly higher than all other strains with RR of 2.9 fold higher than RR of laboratory strain.An. maculatusof the same strain also showed the highest MO activity of 17.467 µmole product/min/µg protein. Its RR was 1.7 fold higher than that of the laboratory strain and significantly higher than strains of Kampung Sokor, Mersing and Gerik (Table 6).

Table 6 MO activity for Anopheles species from selected knowlesi malaria endemic areas in Peninsular Malaysia.

3.2.3.2. NSE enzyme assay

The analysis of NSE enzyme activity showed thatAn. cracensof Kampung Sahom strain demonstrated the highest activity (0.868µmole α-napthol/min/µg protein) which was significantly higher than all other strains and RR that was 2.1 folds higher than the RR of the laboratory strain. As forAn. maculatus, the Kampung Sg.Lui strain was found to be the strain with the highest NSE enzyme activity of 0.800 µmole α-napthol/min/µg protein and RR that was 1.8 folds higher than RR of the laboratory strain and significantly higher than all other strains (Table 7).

Table 7 NSE activity for Anopheles species from selected knowlesi malaria endemic areas in Peninsular Malaysia.

3.2.3.3. GST enzyme assay

The analysis of GST enzyme activity showed that, allAn. cracensandAn. maculatusstrains showed relatively low GST enzyme activity. In fact when compared to laboratory strainAn. cracens,strains of Kampung Sokor, Lata Cemerung, and Mersing showed GST enzyme activity that were significantly lower than laboratory strain. As forAn. maculatus, all strains demonstrated significantly lower GST enzyme activity than laboratory strain (Table 8).

Table 8 GST activity for Anopheles species from selected knowlesi malaria endemic areas in Peninsular Malaysia.

3.2.3.4. AChE enzyme assay

Comparing the findings to the laboratory strain,An. cracensof Kampung Sahom strain showed the highest AChE enzyme activity of 169.7% and RR which was 1.7 fold higher than that of the laboratory strain.An. maculatusof the same locality was also the strain that showed the highest AChE enzyme activity of 116.1%and RR which was 1.16 fold higher than that of the laboratory strain (Table 9).

Table 9 AChE activity for Anopheles species from selected knowlesi malaria endemic areas in Peninsular Malaysia.

Based on analysis of all enzymes, it was found thatAn. cracensof Kampung Sahom strain exhibited the highest level of enzyme activity for MO, NSE and AchE.An. maculatusfrom the same locality also exhibited the highest enzyme activity for MO and AchE. The highest activity of NSE was shown byAn. maculatusstrain from Kampung Sg. Lui. As far as GST enzyme activity is concerned, all field strains ofAn. cracensandAn. maculatusexhibited low level of GST enzyme activity as compared to laboratory strain.

4. Discussion

In this study, the insecticide susceptibility status and resistance mechanism ofAn. cracensandAn. maculatuscollected from knowlesi malaria endemic areas in Peninsular Malaysia were determined using the standard WHO adult bioassay, larval bioassay and biochemical enzyme assay.

Based on result of the WHO adult bioassay, it was clear that severalAn. cracensstrains tested have developed some degree of resistance towards 5% malathion namely strain of Kg Ular in Pahang and Kg Sahom in Perak (“possible resistance”) and to 4% DDT namely strain of Kampung Sg. Mela in Pahang, Kampung Sokor in Kelantan dan also Kampung Sahom in Perak (also “possible resistance”). In addition, strain of Kampung Sg Ular in Pahang indicated “confirmed resistance” to 4% DDT.An. maculatusstrain also demonstrated development of some degree of resistance not only to 5% malathion (“possible resistance” by Kampung. Sg. Lui strain in Selangor) and 4% DDT (“possible resistance” by Gerik strain in Perak, “confirmed resistance” by Kampung Sokor strain in Kelantan and Kampung Sg. Lui strain in Selangor) but to 0.05%deltamethrin as well (“possible resistance” by Kampung Sokor strain in Kelantan) which was not observed in theAn. cracensstrains tested during this study. With regards to theAn. maculatusstrain that demonstrated development of some degree of resistance to 4% DDT, for example the Kampung Sg. Lui strain in Selangor,the mosquito was collected from rubber estate, oil palm plantation and fruit orchard where agricultural pesticides are being frequently used. Cross resistance may be one of the reasons accounting for the development of resistance towards DDT for this species in this particular location. Interestingly Rohaniet al.[16] showed that the mortality ofAn. maculatusin three localities in Selangor, namely Sungai Congkak, Sungai Tamu and Kuang were 93.3%, 87.5%and 86.7% respectively indicating low resistance towards DDT has been detected much earlier in Selangor.

These results were also in line with the study done in Betau,Pahang wherebyAn. maculatusof Betau strain was reported as“confirmed resistance” towards DDT with mortality of 86.7%using WHO adult bioassay[17]. Similar study conducted in three other localities also in Pahang, namely Temerloh, Senderut and Pos Lenjang however were in contrast to these results[18].

Apart fromAn. maculatusof Kampung Sg Lui, Selangor,An.maculatusof Kg Sokor, Kelantan was also detected as “confirmed resistance” towards 4% DDT in the current study. These finding however were not in agreement with study conducted in the same state but involving different areas namely Gua Musang and Jeli[18].In that particular study the same anopheline species exhibited only“possible resistance” towards 4% DDT with mortality of 90.7%and 96.7%, respectively. Nonetheless it is not impossible that the resistance status in Kelantan has changed from possible resistance to confirmed resistance.

It is also interesting to note thatAn. maculatusfrom Kampung Sokor, Kelantan was found having “possible resistance” (96% of mortality) against 0.05% deltamethrin. The fact that Kelantan is sharing its border with Thailand, this result was found in agreement to the study by Chaumeauet al[19]. In that studyAn. maculatuscollected from Thailand-Myanmar border also demonstrated“possible resistance” towards deltamethrin and permethrin (85%and 97% mortality respectively)[19]. In another study onAn.maculatusfrom Northern Thailand 74% to 92% mortality towards methyl parathion was indicated[20]. These studies together with result obtained from the current study strongly suggest that resistance has developed in the area around the Thailand border.

In addition to susceptibility test, biochemical enzyme assay was also conducted in the adult and larval stages of these mosquitoes.Development of physiology resistance to insecticide is accounted for increased detoxification activity and over expression of enzyme. Chareonviriyaphapet al.[21] reportedly showed that oxidases are the major enzyme contributors to pyrethroid resistance in metabolic mechanism of resistance inAnopheles minimusof susceptible and resistant colonies in Thailand. This is in accordance with the current findings where “possible resistance”or “confirmed resistance” detected were associated with elevation of enzyme activity as shown byAn. cracensof Kampung Sahom strain that exhibited highest level of enzyme activity for MO, NSE and AchE. SimilarlyAn. maculatusof Kampung Sg. Lui strain(having high MO and AchE enzyme activity).

Based on the evaluation criteria of WHO[22], it is concluded that most of the field strains tested remain susceptible towards DDT, malathion and deltamethrin, except strains from Kampung Sungai Ular (Pahang), Kampung Sg. Lui (Selangor) and Kampung Sokor (Kelantan). Further investigation is needed to confirm the insecticide susceptibility ofAn. maculatusof the neighbouring areas. On the other hand, this is the first report of insecticide susceptibility ofAn. cracensand biochemical detection of resistance mechanism inAn. maculatusandAn. cracensin Malaysia. The data will serve as important reference for further monitoring and planning of counter measures to ensure continued effectiveness of chemical insecticides used in malaria vector control.

Conflict of interest statement

We declare that we have no conflict of interest.