The Susceptibility of Aedes aegypti in Dengue Endemic Areas, Tegal, Central Java Indonesia Kerentanan Aedes aegypti di Wilayah Endemis Dengue, Tegal, Jawa Tengah, Indonesia

Tegal district is a dengue-endemic area. One of the strategies to control Ae. aegypti is the use of insecticides. The determination of insecticide resistance in a dengue-endemic area is useful for supporting policies for Ae. aegypti control program. The aim of this study is to determine the susceptibility of Ae. aegypti in Tegal district, Central Java. Aedes aegypti larvae were collected from June to July 2018. Susceptibility bioassay of Ae. aegypti larvae against temephos and Ae. aegypti female against permethrin were conducted refers to the WHO protocol. The susceptibility of Ae. aegypti was interpreted based on WHO protocol as well. The mortality of Ae. aegypti larvae were at 0.025 ppm by 90%. The LC50 at 0.0005 ppm, and LC99 at 1.1037 ppm, respectively. The mortality rate of Ae. aegypti against permethrin was 26%. The LT50 at 6611.636 minutes, and LT99 at 5958807.272 minutes, respectively. The susceptibility of Ae. aegypti larvae were possible resistant but adult Ae. aegypti was resistant.


INTRODUCTION
Dengue fever (DF) is transmitted by Aedes aegypti and caused by Dengue Virus. In Indonesia, the incidence rate of Dengue Haemorrhagic Fever (DHF) was 77.96 cases per 100,000 people in 2016. 1 DHF cases has been reported in various regions of Indonesia throughout the country, including Tegal district. In 2017, the number of DHF cases in Tegal was 261 cases with a morbidity rate of 18.2 per 100,000 population. In Tegal district, DF cases still occur and the cases tend to fluctuate annually. 2 Nowadays, Tegal district is an endemic area for DHF. 2 One of the strategies to control Ae. aegypti is the use of insecticides, 3 because of quick action and high efficiency. 4 Temephos, an organophosphate group, is currently still recommended as an insecticide for killing Ae. aegypti larvae. 5 In the adult stage, permethrin, a pyrethroid group, is chosen to control Ae. aegypti due to its low toxicity against mammals and low price. 6 However, if insecticides are used continuously for a long time, this may create an insecticidal-resistant generation of Ae. aegypti. 7 Various studies showed that Ae. aegypti population has been resistant against these insecticide groups in Indonesia. A study in Padang of West Sumatra reported that Ae. aegypti larval resistance occurs in the Ka Koto and Gu Pangilun populations. 8 Resistance to permethrin in Ae. aegypti was reported in Banjarmasin populations with low mortality due to mutation in the voltage-gated sodium channel gene. 9 Aedes aegypti has been resistant against malathion, temephos, cypermethrin, deltamethrin, lambdacyhalothrin, and tolerant against alphacypermethrin in Tegal municipality. 10 DHF cases are still reported, although temephos and cypermethrin of pyrethroid group have long been applied to control Ae. aegypti. 2 Also, insecticide resistance has become a global concern, and a challenge faced by policymakers in vector control. 1 Meanwhile, insecticide resistance may affect the effectiveness of vector control program.
Detection and monitoring of insecticide resistance in a DHF endemic area is useful for supporting vector control policy. Currently, limited data has been reported regarding the susceptibility of Ae. aegypti against temephos and permethrin in Tegal district of central Java Indonesia. Therefore, the aim of this study is to determine the susceptibility of Ae. aegypti against temephos and permethrin.

Aedes aegypti Samples
Aedes aegypti larvae were collected in field surveillance site from 100 houses by systematic sampling in Pangkah village, Tegal district from June to July 2018. Larvae were reared into adults in insectarium at the Department of Parasitology, Faculty of Medicine, University of Indonesia. The larvae were fed with commercial larval food. After eclosion, the adult females were fed the blood through an artificial membrane feeding, as Costa-da-Silva had previously demonstrated with some modifications 11 and then cotton pads with 10% sugar solution were used to feed the adult females. F3 generation of larvae were used for temephos bioassay and F3 generation of adult females were used for permethrin bioassay. This study was approved by the Ethics Committee of Faculty of Medicine UI with a reference number; 0633/UN2.F1/ETIK/2018.

Insecticide Bioassay
Temephos bioassay to Ae. aegypti larvae were carried out according to the World Health Organization (WHO) protocol. 12 We used four temephos concentrations (0.05, 0.025, 0.0125, 0.00625 ppm) to estimate the lethal concentration LC50, and the LC99. The concentration determination was based on an approach to the WHO resistance cut-off value (the LC99 = 0.02 ppm). To obtain these bioassay solution concentrations, we weighed commercial temephos powder (Abate). Briefly, 1 mg temephos powder was diluted in 200 ml water to produce 0.05 ppm bioassay solution. Following this, we formulated other concentrations of bioassay solution under the same conditions: 0.5 mg for 0.025 ppm, 0.25 mg for 0.0125 ppm, and 0.125 mg for 0.00625 ppm, respectively. For each concentration, four replicates were used and each replicate consists of twenty-five larvae. The control solution was 200 ml distilled water. As the control larvae, we used Ae. aegypti larvae from insectarium of the Department of Parasitology, Faculty of Medicine, University of Indonesia. The larvae mortality was counted at the end of a 24 hours exposure. If there was a mortality of 5-20% on the control larvae, the result should be corrected by the Abbot formula. 12 Permethrin Bioassays against Ae. aegypti were conducted using WHO test tubes containing permethrin (0.25%) insecticide papers obtained from the University of Sains Malaysia's Vector Control Research Unit. Briefly, we performed four repetitions. One repetition consists of four test tubes (red tube) and one tube as control (yellow tube). As the control mosquitoes, we used Ae. aegypti from the Department of Parasitology insectarium, Faculty of Medicine, University of Indonesia. In each tube, a total of 25 individuals Ae. aegypti was needed. Twenty-five individuals with a minimum of 4 days old and feedingsugar female Ae. aegypti were introduced into a green tube without insecticide paper for 30 minutes. Aedes aegypti were then placed into a red tube with permethrin 0.25% paper and into a yellow tube as a control for 60 minutes. Ae. aegypti were then transferred into paper cups with absorbed cotton of 10% sugar solution and stored at room temperature. 13 Aedes aegypti mortality was counted at 1, 6, 12, 24 hours period, respectively. The formula used to determine Ae. aegypti mortality rate was a total number of dead mosquitoes/total sample size at each exposure time x 100%. The susceptibility of Ae. aegypti was interpreted based on WHO protocol (≥ 98% mortality: susceptible, 90-97% mortality: possible resistance; < 90% mortality: resistance) 13 , where the cut-off value for larvae was 0.025 ppm. Probit analysis was performed to calculate the LC50 and LC99 values, and the LT50 and the LT99 value using SPSS 20 (IBM, USA).

RESULTS
The temephos bioassay results for larvae are presented in Figure 1. The mortality of Ae. aegypti larvae were at a concentration over 0.025 ppm, as indicated by the average mortality rate at 0.025 ppm was 90%. Probit analysis result was y = 0.701x + 7,296. Based on this equation, the estimated Probit for LC50 value was obtained 0.0005 ppm and LC99 value was obtained 1.104 ( Figure 2).
The permethrin bioassay results are presented in Figure 3. Aedes aegypti population from Pangkah showed mortalities less than 90%. Probit analysis result was y = 0.787x -3.008. Based on this equation, the estimated Probit for LT50 value was obtained 6611,636 minutes and LT99 values were obtained 5958807,272 minutes ( Figure 4).

DISCUSSION
As a result, Ae. aegypti larvae were classified as possible resistant since the larvae mortality rate was 90% at 0.025 ppm, according to WHO criteria. 14 Temephos resistance can occur due to changes in the work of enzymes in the larval body. These changes make it difficult for larvicides to bind to targets or to interfere in the larval body. 15 Furthermore, one of the factors contributing to Ae. aegypti larvae resistance is varying the application of temephos with various frequencies of implementation. 16 Since the use of temephos in Mexico varies between urban and rural applications, the larval eradication program fails and risks causing resistance to Ae. aegypti larvae. 17 For the permethrin 0.25% bioassay, this result revealed that the Pangkah population was resistant to permethrin. Various studies regarding the permethrin resistant mechanism have been widely reported. Resistance against permethrin could be due to decreased sensitivity to sodium channels 18 and increased metabolic detoxification mainly through the P450 enzyme. 15 In Jakarta, Ae. aegypti population, the key mechanism of permethrin resistance through the V1016G gene mutation which plays a role in decreasing sodium channel sensitivity. 3 Meanwhile, resistance is mainly associated with mutations in Cys 1,534 gene which play a role in the sodium channel in Jamaican Ae. aegypti population. 19 This study revealed different results compared to other similar studies. These could be due to differences in the study's geographical conditions. Tegal district is known to have a tropical climate, with temperatures ranging from 24 to 28ºC. Glunt et al reported that the ambient temperature influences on the efficacy and toxicity of insecticides used in public health such as applications for Anopheles spp. 20 Temperature changes are known to affect permethrin effectiveness. Permethrin perform effectively within a temperature range of 30-32ºC and perform poorly in a temperature range of 32-34ºC. 21 Pyrethroid and organophosphate resistance studies have been carried out worldwide including Indonesia since most dengue-endemic countries are currently using pyrethroids and organophosphates in their vector control programs. Similar to this study, Ae. aegypti larvae of Padang strain are possible resistant against temephos, where the mortality is 86.7%. 22 The LC50 value (0.0005 ppm) obtained in this study was lower than the LC50 value obtained from the Bekasi strain (0.001 ppm), but the LC99 value (1.104 ppm) was higher than the LC99 value of the Bekasi strain (0.018 ppm) reported by Sinaga et al. 23 However, based on the probit analysis, the LC99 value exceeds 0.02 ppm, precisely at 1,104 ppm. These results indicate that there has been resistant of Ae. aegypti larvae against temephos. Ikawati et al revealed that insecticide resistance to Ae. aegypti may be due to cross-resistance in the same group of insecticides and long-term use of the same group of insecticides. 24 After the discovery of DDT resistance in mosquitoes in 1976, insecticide resistance has become a major issue in vector control. Moreover, if this issue is not addressed immediately, long-term resistance is likely to occur and the vector control system may fail while insecticides are very useful as vector control weapons. 18 As mentioned previously, temephos and cypermethrin have long been used for controlling Ae. aegypti in Tegal district. Based on this fact, it seems urgent to immediately modify the insecticide control method that can be combined with other approaches such as the biological control method.
Biological control could be carried out using organisms that prey, parasitize, or compete with target species with the aim to reduce population. Biological control against Ae. aegypti can use Gambusia affinis fish, Toxorhynchites mosquitoes larvae, or cepopods especially mesocyclops and macrocyclops species. 25 Also, the use of plant extracts could be an alternative to kill Ae. aegypti larvae 26 and the adult stage. 27

CONCLUSION
In Pangkah village, Tegal, Ae. aegypti larvae is possible resistant against temephos, while adult Ae. aegypti has been resistant to permethrin 0.25%.

RECOMMENDATION
Insecticides have been confirmed resistance should be replaced or rotated with the susceptible insecticides for application in the field.

AUTHOR CONTRIBUTION
In this article, NEF and HW are the main contributors who conduct conceptualisation, investigation, methodology, validation, supervision. IK, BFA, HA, VAA, RW, RS, and LS as co-contributors who conduct field studies, data curation, formal analysis and writing-review and editing.

ACKNOWLEDGMENT
We would like to thank Pangkah villagers who volunteered to assist us in collecting larvae in their neighborhoods. This work was supported by Directorate of Research and Community Engagement University of Indonesia (DRPM UI) with award number 4541/UN.2.R3.1/PPM.00/2018.