Dengue is the most important mosquito-borne viral disease in the world . Dengue is also a significant public health problem in Indonesia since the first reported dengue outbreak in 1968. All four dengue virus (DENV, Flavivirus) serotypes are detected, suggesting hyperendemicity in all of 34 provinces in Indonesia [2, 3]. Dengue virus is transmitted by Aedes aegypti, a mosquito that has also been linked with the transmission of other major arboviral diseases including chikungunya (CHIKV) and Zika (ZIKV). The virologically confirmed chikungunya was initially reported during an outbreak in Jambi in 1982 . After nearly 20 years of absence, twenty-four distinct outbreaks of probable chikungunya re-emerged in South Sumatera, Aceh and West Java in the early 2000s . To date, there has been no recorded fatality related to CHIKV infection in Indonesia . Zika virus (ZIKV, family Flaviviridae) has become one of the global public health threats because of its association with Guillain-Barré syndrome and microcephaly. Until recently, most evidence for Zika virus infection reported in Asia, including in Indonesia, has been serological .
Indonesia has made dengue a notifiable disease. On average, 136,670 DENV infections and 1112 deaths were reported annually from 2013 to 2016, an incidence of about 54 dengue fever or dengue hemorrhagic fever cases per 100,000 population and a case fatality rate of approximately 1% . Dengue cases in Indonesia are expected to be under-reported due to poor disease surveillance and a low level of reporting . The surveillance database likely covers dengue probable cases with supportive dengue serology or with epidemiologic linkage, and dengue confirmed cases with confirmatory laboratory criteria [8, 9]. Dengue infections deprived of diagnostic tests may not end up in the surveillance database . Despite the level of uncertainty on total case number, dengue visibly results in a considerable cost to the health sector, and a heavy economic and social impact is likely.
Indonesia has made progress in many areas of dengue prevention and control. In August 2016, Indonesia became the sixth country to approve the first licensed dengue vaccine, Dengvaxia® [10, 11]. To date, the vaccine so far has been approved in 20 countries and launched in 11, including Indonesia . However, in December 2017, the National Agency of Drug and Food (Badan POM) suspended the use of dengue vaccine in Indonesia over safety concerns  and later in February 2018, the BPOM revised recommendation that the use of vaccine be limited for people between 9–16 years of age who had a dengue infection prior to vaccination and that the vaccine should not be administered to people who have never had a dengue infection before . There is no rapid, reliable test for previous dengue infection, so the Dengvaxia® vaccine cannot be widely used . Nevertheless, effective vector control methods are still essential, targeting Ae. aegypti in its immature and adult stages. It has been argued that even if the current vaccine is highly targeted and low-cost, sustained mosquito control will remain cost-effective .
The mainstay of the current vector control programme in Indonesia is environmental management, which in recent years, has emphasized community participation to reduce container breeding sites [2, 16]. The country is renowned for its 3M plus campaigns, aimed at covering and cleaning water containers and burying discarded water containers, complemented with biological approaches using natural predators or pathogens as alternatives for vector control [2, 16]. The efficacy of community-based approaches is measured by a larvae-free index (percentage of houses free from Ae. aegypti larvae and pupae infestation); unfortunately, data from the last five years showed a larvae-free index ranging between 24–80%, less than the target of 95% .
Chemical insecticides still play a central role in dengue vector control in Indonesia. Vector control during outbreaks depends primarily on chemical insecticides to effect a rapid reduction in the number of infected mosquitoes and to break the dengue transmission cycle [2, 16–18]. Since the 1970s, the organophosphates malathion and temephos have been widely used to control dengue, and starting in the 1980s, dengue vector control has been highly reliant on pyrethroids. Pyrethroids are also widely used in public health for prevention and control of other mosquito-borne diseases such as malaria and filariasis, and as agricultural insecticides . Pyrethroids are also approved for household protection against dengue [20, 21]. The abundant and prolonged use of pyrethroids has led to the development of resistance in Ae. aegypti populations in many countries including Indonesia. Resistance to pyrethroids, based on bioassay data, has been reported in some areas of Indonesia [22–26].
Two main mechanisms for pyrethroid resistance have been identified in Ae. aegypti, metabolic resistance and target site resistance. Metabolic resistance occurs when increased levels or modified activities of one or more detoxifying enzymes result in a more rapid detoxification of the insecticide, preventing the insecticide from reaching its target in the nervous system. Metabolic resistance involves three groups of enzymes, i.e. esterases, multi-function oxidases P450 and glutathione s-transferases (GST) [27–29]. Limited studies about metabolic resistance based on biochemical assays are available for Indonesian dengue vectors [26, 30–32].
Pyrethroids act on the insect nervous system, targeting the voltage-sensitive sodium channel (VSSC). They modify the gating kinetics of the channel by slowing both the activation and the inactivation, stimulating the nerve cells to produce repetitive discharge that lead to paralysis and death of insects, an effect known as knockdown. Target site resistance is caused by point mutations in the VSSC gene, resulting in amino acid substitutions that affect pyrethroid binding sites, being known as kdr (knockdown resistance) mutations [33, 34]. A total of 12 point mutations in the VSSC of Ae. aegypti have been identified to be associated with kdr resistance to pyrethroids. Only five of these mutations have been functionally confirmed to reduce the sensitivity of mosquito sodium channels to pyrethroids, i.e. S989P, I1011M, V1016G/I, F1534C, and recently V410L . The mutations L982W, G923V, I1011M and V1016G were the first reported sodium channel mutations in permethrin/DDT-resistant populations of Ae. aegypti from various countries , located in the domain IIS5 (for L982W and G923 mutations) and IIS6 (for I1011M and V1016G mutations) of the VSSC . Further studies have reported novel mutations, including I1011V and V1016I in Latin American populations . In Asian countries, the F1534C mutation (in domain IIIS6) was detected in Ae. aegypti mosquitoes from Thailand  and Vietnam . The S989P mutation, located in linker between domains IIS5–S6, was first reported in Ae. aegypti populations in Thailand , the D1763Y mutation (in linker IVS5-S6) was reported in a Taiwan population , and mutation T1520I (in domain IIS6) was detected in an Indian population . In 2017, the mutation V410L was first identified in Brazilian strains of Ae. aegypti , and in 2018, a novel mutation V419L was found in populations from Colombia  (both mutations are located in domain IS6).
Three of these mutations, V1016G, F1534C and S989P, are widely distributed and detected in pyrethroid-resistant populations in Southeast Asian countries including Thailand, Indonesia, Malaysia, Singapore, Vietnam, Cambodia and Laos . Co-occurrence of kdr mutations has been a common phenomenon observed and, for some combinations, has been shown to confer a higher level of resistance than singly occurring mutations . At least three patterns of mutational associations have been identified in Ae. aegypti from Southeast Asia: V1016G/F1534C, V1016G/S989P and V1016G/F1534C/S989P. Co-occurrence of V1016G and F1534C was reported in populations from Thailand , Myanmar , Malaysia  and Indonesia [49–52] and co-occurrence of V1016G and S989P point mutations was detected in Thailand , Myanmar , Indonesia, [49, 50, 52] and Papua New Guinea . In addition, co-occurrence of triple mutations V1016G/F1534C/S989P in heterozygous form has been identified commonly in Ae. aegypti from Thailand , Myanmar  and Indonesia [49, 50, 52]. However, co-occurrence of triple homozygous point mutations (homozygous mutation for each of V1016G, F1534C and S989P) is very rare having only been reported from Myanmar at a frequency of 0.98%  and in Indonesia in one individual (at a frequency of 0.34%) 
Despite all of the strategies implemented in Indonesia, the existing methods of controlling dengue have limited success. The development of a novel strategy of vector control that can be incorporated into the existing vector control strategy is essential. One of the innovative approaches to preventing transmission of dengue virus involves introduction of strains of the bacterium Wolbachia into Ae. aegypti, which has both life-shortening effects on the mosquito and direct transmission-blocking effects on dengue virus. This new strategy, together with more traditional approaches for vector control including insecticide application, may provide promising results to reduce dengue transmission. With the support of communities and approval from regulators, Indonesia’s first field trial of Wolbachia-infected mosquitoes began in Yogyakarta in 2014 . The first field trial in two areas in the outer city of Yogyakarta has yielded results that point to local invasion  and release of Wolbachia-infected mosquitoes on a broader scale in the inner city areas of Yogyakarta has since been initiated in August 2016 .
To assist the spread of Wolbachia, insecticide resistance in the strain of Ae. aegypti being released, needs to match that of the background population to ensure that released individuals persist and reproduce, allowing a Wolbachia invasion to take place . In the inner city of Yogyakarta, resistance is expected because of the local heavy application of chemical insecticides. It is possible that insecticide usage is higher in these areas than in the outer rim of Yogyakarta, as inner city areas have more dengue cases  and are more densely populated . In a previous study, we screened samples from Yogyakarta outer areas for kdr mutations and obtained a high frequency of kdr alleles in samples collected from the outer area of Yogyakarta . The most common kdr co-occurrence was V1016G homozygous mutant/F1534 wild type, combined either with S989P heterozygous, or S989P wild type, or S989P homozygous mutant, respectively. This co-occurrence was more common in the surviving than in the dead mosquitoes  and a strong association between the V1016G mutation with pyrethroid resistance (type I and type II) was confirmed. In contrast, F1534C mutant homozygotes were rare and there was only a weak association between heterozygote individuals for the F1534C mutation and resistance to a type I pyrethroid. The S989P mutation, in addition to the V101G mutation, was found to have an additive effect in resistance to Type II pyrethroids . In this study, we have screened Yogyakarta inner city areas for kdr mutations, based on a total of 1314 individuals collected from 27 localities (Fig. 1, Table 1) and aim to compare frequency and occurrence of kdr mutations between the inner and outer city.