High Levels of Resistance in A Culex quinquefasciatus Population to the Insecticide Permethrin in Filariasis Endemic Areas in Central Java

Resistance among Culex mosquitoes to various insecticides has been reported in many countries. However, there have been no studies of the resistance status of Culex in Indonesia. There is a need for such studies to develop a database for use in vector control management. This study aimed to investigate the insecticide resistance status of C. quenquefasciatus, which is the primary vector of filariasis, to aid the planning of a vector control management program. In the present study, Culex quinquefasciatus larvae were collected from five districts/municipalities where filariasis is endemic in Central Java. The larvae were reared to adult stage, and insecticide susceptibility testing was then conducted according to standard bioassay procedures of the World Health Organization (WHO). The results of the bioassays showed that C. quinquefasciatus had a high level of resistance against 0.75% permethrin, with mortality rates ranging from 4.8 to 21.6%. The lowest resistance was found among mosquitoes collected from Grobogan district. This may be explained by the district’s remote geographical location. The high level of resistance found in the present study may be caused by exposure to local insecticides, which have been applied for many years as part of a dengue vector control program. These insecticides may also have contaminated the breeding sites of C. quinquefasciatus mosquitoes. Better vector control management is needed to help prevent the development and spread of resistance. Such management should include routine insecticide surveillance and insecticide alternation.


High Levels of Resistance in A Culex quinquefasciatus Population to the High Levels of Resistance in A Culex quinquefasciatus Population to the Insecticide Permethrin in Filariasis Endemic Areas in Central Java Insecticide Permethrin in Filariasis Endemic Areas in Central Java
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Introduction
Mosquitoes belonging to the genus Culex are wide spread in tropical, subtropical, and temperate climates worldwide, especially in urban areas [1]. Culex pipiens and C. quinquefasciatus are the main vectors of filariasis in many regions of the world [2][3][4], with the distribution of Culex mosquitoes in various countries, including Indonesia, directly associated with the incidence of filariasis [5]. As shown by a survey of vector density, mosquitoes belonging to the genus Culex predominate in Malaysia and Indonesia [6][7][8]. One study reported that the density of C. quinquefasciatus was 5.91 mosquitoes/person/hour indoors and 4.75 mosqui toes/person/hour outdoors [9]. Research also showed that the infectivity of Culex microfilariae was higher than that of microfilariae from other genera [10]. The existence of habitats suitable for C. quinque fasciatus is associated with the incidence of filariasis [11].
Vector control is an important element of any strategy aimed at controlling vector borne diseases, and chemical control is the most widely used approach [12]. Research has demonstrated the effectiveness of insecticides in reducing the incidence of vector borne diseases, including malaria [13]. However, more frequent use of insecticides in the last decade has been directly linked to the development of resistance genes and increased resistance among Culex. Data are lacking on vector resistance to insecticides, as many countries do not perform routine susceptibility testing [14].
Many countries have reported that Culex mosquitoes are resistant to various insecticides. For example, research conducted in India indicated that C. quinquefasciatus was highly resistant to dichloro-diphenyl-trichloroe thane, more commonly known as DDT, and malathion [15]. A study conducted in Zambia demonstrated that Culex was resistant to pyrethroids, permethrin, and deltamethrin [16]. In Kuala Lumpur, larvae of C. quinquefasciatus were resistant to malathion and permethrin [17], and they were resistant to pyrethroids in Zanzibar [18]. The insecticide resistance status of Culex has not been reported in Indonesia. Therefore, this study aimed to investigate the insecticide resistance status of C. quenquefasciatus, which is the primary vector of filariasis, to aid the planning of a vector control management program.

Materials and Methods
Study sites and mosquito collection. A larval survey of stagnant water, puddles, sewage, and excavation sites was first conducted based on the latest filariasis incidence data for 2014 [unpublished result from authorized provincial health department]. The survey included a special exhaust deliberately in shape by local people around filariasis patients over which being a breeding place of the vector. A modified larval collector tool was used to collect the larvae. The survey was conducted during February-April 2015 in five districts/ municipali ties in Central Java known to be filariasis endemic areas: Semarang (Sendangguwo), Demak (Wonosari), Jepara (Lebak), Grobogan (Bangsri), and Pekalongan (Wiroditan) (Figure 1). Permission to conduct mosquito and larval collection in households was given by the provincial and district/municipality authorities following recommendations by the relevant district and municipal health departments C. quinquefasciatus larvae were reared in the laboratory to imago stage (3-5 days after hatching). Post-hatching, the mosquitoes were collected in containers and subjected to susceptibility tests, as described below. Additionally, the method to identify the species was based on our previous publication [19].
Insecticide susceptibility tests. All insecticide suscep tibility tests were performed using standard WHO bioas say procedures, using diagnostic test kits and nitrocel lulose paper impregnated with one of several pyreth roids or organophosphate compounds [20]. To assess cross-resistance to these compounds, the test papers included 0.75% permethrin.
In each test group, 125 female mosquitoes from each specified location of district/municipal were divided into five tubes, and each tube contained 25 adults. Four replicates of each treatment compound were established, in conjunction with a matched control. The test was repeated three times for each insecticide, as recommended by WHO protocols [20]. Temperature and humidity during the test were maintained at ranges of 25 ± 2°C and 80% ± 10%, respectively. The following day (after 24 h), the numbers of dead mosquitoes were calculated to determine the resistance status. The resistance status of the mosquitoes was defined as follows: resistant if mortality was < 90%, tolerant if mortality was 90-98%, and susceptible if mortality was > 98% [21][22]. The resistance ratio was calculated using probit analysis, as described previously [23][24].

Results and Discussion
In the morphological analysis, all the samples were identified as C. quinquefasciatus. Adult C. quinquefas ciatus mosquitoes have dappled black and white across its abdominal segment, with a black head and white color at the edges. On the thorax, there are two curved white lines. White arches at the back section, characteristics typical of C. quinquefasciatus, were also oserved.
In the susceptibility tests, after exposure to the insec ticide for 24 h, the mortality rates ranged from 4.8% (n = 6, Semarang city) to 21.6% (n = 27, Grobogan district), with average mortality of 9.6% (n = 12) and a standard deviation of 8.515 9 (Table 1). These results indicate that mortality among this C. quinquefasciatus population was less than 80%. Figure 2 showed the proportion of mosquitoes that knocked down before 24 h holding time. After holding time, it can be confirmed whether the mosquitoes is truly susceptible or resistance.
The results of the probit analysis indicated that the KDT50 (i.e., time to knock down 50% of the total population) of C. quinquefasciatus after exposure to the insecticides ranged from 49.67 to 975 min, whereas the KDT95 (i.e., time to knock down 95% of the population) ranged from 86.3 to 1815 min (Fig. 3). Figure 4 showed the relationship between KDT95 with mortality. Grobogan had the lowest KDT95 and resulted in high mortality.
As shown by the results of the bioassay of resistance in C. quinquefasciatus samples collected in Central Java, the mosquito population showed a high level of resistance to 0.75% permethrin., with mortality rates ranging from 4.8% to 21.6%. The KDT50 was also high (range of 49.67 to 975 min) (Fig. 3). These findings differ from those reported in previous studies, which found that the KDT50 for mosquitoes exposed to 0.75% permethrin ranged from 40 to 50 min [25] in Malaysia and 33.1 min in Thailand [26]. The high resistance of C. quinquefasciatus to permethrin in Central Java is in contrast to reports in other countries, which found that Culex was vulnerable to 0.75% permethrin [25][26][27]. In the present study, the mortality rates of C. quinquefasciatus in the insecticide susceptibility tests were relatively higher than those reported for isolates sampled in Pathum thani, Nonthaburi, and Bangkok in Thailand, with studies reporting mortality rates following exposure to permethrin of 67.4 ± 2.4%, 72.4 ± 0.4%, and 80.6 ± 1.7%, respectively [28]. Studies conducted in Thailand reported high levels of resistance in C. quinquefasciatus to 0.75%   [29] and resistance of 10.1% found in Baan Suan [30].
Resistance among C. quinquefasciatus populations in Indonesia has not been reported previously. The high rate of resistance among C. quinquefasciatus in Central Java has important implications for insecticide-based vector control programs. In line with this study, previous studies of the distribution of vector resistance reported increased resistance among Aedes mosquitoes in Indonesia [31,32]. High resistance among Aedes was also reported in Central Java, with mortality ranging from 1.6 to 15.2% and the KDT50 ranging from 120.2 to 1337.7 min [33].
In the present study, the distribution of insecticide resistance to pyrethroids varied, depending on the region, with a very high level of resistance in four districts (Semarang, Pekalongan, Demak, and Jepara) but lower resistance in Grobogan. Geographically, Grobogan is a much more remote area than the other regions. Nevertheless, the level of resistance was still relatively high.
The high level of resistance may be caused by exposure of C. quinquefasciatus to local insecticides as part of a dengue vector control program, which has been ongoing for many years, especially in urban areas (Semarang, Demak, Pekalongan and Jepara). Moreover, breeding sites of C. quinquefasciatus mosquitoes may have been contaminated with insecticides [34]. Other than insecticides, which are widely employed in Central Java, household insecticides and repellants containing active compounds (pyrethroids) are increasingly used [33]. Interestingly, resistance to insecticides may develop faster in C. quinquefasciatus than in other mosquitoes [35]. Given the close interrelatedness between the development of resistance and intensity of insecticide exposure, the use of insecticides in vector control programs needs to be evaluated. Page: 152 In addition, programs are needed to educate the public about the effects of household insecticide use.

Conclusions
In conclusion, long-tem and intensive use of insecticides have led to the development of insecticide-resistant phenotypes. Selective pressure resulting from continuous insecticide use favors the development of resistance among mosquitoes [36]. The frequency of these mutants may dramatically increase, and they may become widespread within a population [37], as demonstrated in a recent study. Despite the rapid evolution of resistant mosquitos, as indicated in recent laboratory studies [38][39], the "resistance" background can rapidly revert back to susceptible when selective pressure is relaxed [40]. Routine surveillance is needed in mosquito vector control management programs based on the application of insecticides to avoid the spread of resistance.