Biochemical Characterization of Insecticide Resistance and Exposure in Aedes Aegypti Population From Wonosobo, Central Java, Indonesia

Resistance to insecticides mainly occurs due to changes in insect metabolic enzyme. Increased metabolism is often caused by qualitative or quantitative changes of esterase and glutathione S-transferase. Susceptibility test and biochemical assay to detect organophosphate and synthetic pyrethroid resistance were conducted on Aedes aegypti from Wonosobo (new highland Dengue endemic area). The test were performed on F1 generation of Ae.aegypti field caught mosquitoes which aimed to determine the resistance mechanisms regarding two detoxifying enzymes i.e. esterase and glutathione S-transferase. Susceptibility test showed 23.4 and 46.7% mortalities after exposure to 0.8% malathion and 0.05% cypermethrin. The biochemical assay result suggested that esterase, monooxygenase and glutathione s-transferase activity tend to increase in Ae.aegypti in Wonosobo. Interview and questionaires conclude that synthethic pyrethroid was the only insecticide type used in vector control program by Wonosobo Health Office and was the most frequent insecticide type to be used in household by Wonosobo society to control Ae.aegypti population. Knowledge of localized resistance and underlying mechanisms helps in making rational decisions in selection of appropriate and effective insecticides in the event of a dengue outbreak.

Dengue haemorrhagic fever is still one of the major PRVTXLWR ERUQH GLVHDVHV LQ ,QGRQHVLD ZLWK Aedes aegypti as the principal vector. Aedes aegypti is closely associated with humans and human habitation. The female is predominantly an indoor day-biter that feeds almost exclusively on humans. $GXOW IHPDOH XVXDOO\ H[SORLWV DUWL¿FLDO FRQWDLQHUV DV sites to deposit her eggs. Aedes aegypti PRVTXLWRHV are abundant and endemic dengue virus transmission usually occurs in low-elevation areas. 1 But recently a large proportion of the human population lives in high-altitude cities. On the other side, due to the rising global temperature, more areas of the world become favourable for the survival of tropical insects like Ae. aegypti. This could lead to the spread of this insect towards more high elevation areas thus supporting the expansion of dengue endemic areas. 2 Wonosobo is one of dengue endemic district in Central Java Province, is a mountainous area with altitude ranges from 250 m to 2,250 m above sea level. Dengue fever was reported in Wonosobo since 2007. 3 Vector control by reducing Ae.aegypti population is at present the only viable option available to control the disease.
Especially during epidemics of the disease, the use of insecticides is needed. For example, application of temephos for larval control, thermal fogging or ULV sprays of certain organophosphates such as malathion and synthethic pyrethroids such as cypermethrin for adult control. Fogging with cypermethrin was DOZD\V GRQH LQ WKH ODVW ¿YH \HDUV E\ WKH JRYHUQPHQW as an effort to reduce Dengue vectors in Wonosobo. 4 Insecticide resistance could develop and would be a major problem in controlling the vectors and other pest insects.

Study area
Wonosobo regency is a mountainous area with an altitude between 250 m to 2,250 m above sea level, including the type of young mountains with steep valley. Dengue cases were found in this regency since 2007 and distributed in some municipalities ( Figure  1). The altitude of these municipalities ranges from 300 to 900 m above sea level. Daily temperature ranges from 24 o C -30 o C. The study was conducted in January-October 2014, included three Dengue endemic municipalities located in Wonosobo Regency i.e. Wonosobo, Selomerto and Leksono.

Mosquito Samples
Stock of Ae.aegypti RULJLQDWHG IURP ¿HOG FROOHFWHG HJJ from three Dengue endemic municipalities located in Wonosobo Regency i.e. Wonosobo, Selomerto and Leksono. These eggs were maintained in the cloth cages in an insectary at 28 °C and 80% RH with 12:12 day:night. )HPDOHV ZHUH LQGXFHG WR OD\ HJJV DQG VXEVHTXHQW colonies were reared through to adults. F1 progeny were divided into subsamples according to the number emerged. One subsamples was stored in deep freezer (-80 °C) to be used for biochemical analysis while the other subsamples were used for susceptibility assay.

Insecticide Exposure
The usage of household insecticide can trigger the occurrence of resistance because active ingredients in household insecticides are mostly derived from WKH V\QWKHWLF S\UHWKURLG $ TXHVWLRQQDLUH VXUYH\ RI 100 households was conducted to obtain information on usage of household insecticide in each village in :RQRVRER ,QWHUYLHZ ZLWK 3XEOLF +HDOWK 2I¿FHU was also conducted to collect information about the usage of insecticide in vector control activities by 5HJHQF\ +HDOWK 2I¿FH

Susceptibility assay
Adult insecticide susceptibility bioassays were carried out using WHO test kits for Malathion and Cypermethrin. 14 The insecticides impregnated and control (risella oil) papers were obtained from Vector Control Research Unit (VCRU), University Sains Malaysia, Malaysia.
7ZHQW\ ¿YH QRQ EORRG IHG DGXOW IHPDOH Ae.aegypti aged 2-3 days old of Wonosobo strains were exposed to the diagnostic concentrations of cypermethrin 0.05% and malathion 0.8% impregnated paper respectively for 1 hour. 14 The mortality was recorded at the end of 24 hours holding period. Four replicates were conducted with controls exposed only to risella oil.

Biochemical test
1LQHW\ PRVTXLWRHV ZHUH KRPRJHQL]HG LQGLYLGXDOO\ LQ 200 µl distilled water. Each of 20 µl of the homogenate was used for esterase and monooxygenase assay. The protocol for each assay followed WHO. 15  Monooxygenase assay. A hundred microlitres of sodium phosphate buffer pH 7.2 was added to the DOLTXRWV RI PRVTXLWR KRPRJHQDWHV DQG J RI 3,3,5',5'-tetramethhyl benzidine (TMBZ) solution (0.01 g of 3,3,5',5'-tetramethhyl benzidine in 5 ml of absolute methanol, mixed with 0.25M sodium DFHWDWH EXIIHU S+ ZDV DGGHG 7ZHQW\ ¿YH microlitres of 3% hydrogen peroxide was added and the mixture was left for two hours at room temperature. The oxidase enzyme activity was then read at 630 nm. The microplate assay for monooxygenase were interpreted in correspondence with the experimental evidence for absorbance value RI WKH ¿QDO FRORU LQWHQVLW\ RI WKH HQ]\PDWLF UHDFWLRQV obtained by Matowo and, that was as follows: (1) monooxygenase reactions which were colorless/faint blue were read at AV < 0.165 and esterase reactions which were deep blue were read at AV > 0.165. 17

Data analysis
1XPEHU RI PRVTXLWR PRUWDOLW\ LQ VXVFHSWLELOLW\ WHVW DEVRU bance value data from each microtitre plate and the usage of household insecticide were shown in percentage.

Susceptibility test
The adults of control group showed zero mortality to risella paper. While adults treated group were exposed to 0.8% malathion , 0.05% cypermethrin, the mortalities of 23.3 % and 46.7% were observed (Table 1). Susceptibility test result showed that Ae. aegypti population in Wonosobo was resistant toward Malathion and Cypermethrin.

Biochemical test
The results of the biochemical assay showed elevated activity of esterase (12.2%) and monooxygenase (10.4%). Elevation of enzyme activities were determeined from absorbance value exceeding the cut off point.  Table 3 showed that synthethic pyrethroid was the only insecticide type used in vector control programme E\ :RQRVRER +HDOWK 2I¿FH WR FRQWURO Ae.aegypti population. This insecticide has been used for more than 5 years in Wonosobo. Wonosobo is new Dengue endemic area. Ae.aegypti population in Wonosobo might consist of various strains from other endemic areas which migrated to Wonosobo along with rising temperature caused by global warming. These migrant populations could bring malathion resistant character which came from their original habitat. Afterwards, the resistant character was more increasing with intra strain cross mating in Wonosobo. Several studies elsewhere have examined this phenomenon. 20,21 Unfortunately, data about sibling species in Ae.aegypti population from Wonosobo is not available.

Household Insectiside
7KH VXVFHSWLELOLW\ WHVW RI DGXOW PRVTXLWRHV WR GLDJQRVWLF concentration for cypermethrin 0.05% impregnated paper also showed a potential resistance development at 24 hours holding period. Ae.aegypti PRVTXLWR populations in Wonosobo are under heavy pressure from synthethic pyrethroid compounds through indoor house spraying of cypermethrin by local Public Health 2I¿FH 7DEOH ,Q DGGLWLRQ Ae.aegypti is also exposed to a range of synthethic pyrethroid compounds used LQ KRXVHKROG LQVHFWLFLGH 7DEOH 0RVTXLWR FRLO FRQWDLQHG V\QWKHWKLF S\UHWKURLG ZDV WKH PRVW IUHTXHQW insecticide type to be used in household by Wonosobo society to control Ae.aegypti population. According to WKH TXHVWLRQQDLUH UHVXOW WKLV LQVHFWLFLGH KDV EHHQ XVHG for more than 1 year in Wonosobo.

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household insecticidal product in the world with sales exceeding 45 to 50 billion coils used by two billion people worldwide each year. 22 The popularity of coils is due to their low cost, their ability to be XVHG ZLWKRXW HOHFWULFLW\ RU HTXLSPHQW DQG FXOWXUDO acceptance, because smoke is used in many cultures WR GULYH DZD\ PRVTXLWRHV 23 These products present a great opportunity for public health, because such products could provide a means of disease control that is already proven highly acceptable to end-users and has undergone stringent safety testing. 24 7KH PRVW IUHTXHQW SURGXFW IRXQG LQ VWXG\ DUHD ZDV WUDQVÀXWKULQ PRVTXLWR FRLOV 7UDQVÀXWKULQ LV a highly effective fast-acting pyrethroid insecticide used extensively in household and hygiene products, PDLQO\ DJDLQVW À\LQJ LQVHFWV VXFK DV PRVTXLWRHV DQG ÀLHV 7KH :+2 KDYH FDUULHG RXW DQ HYDOXDWLRQ RI WKH H[WHQVLYH WR[LFLW\ OLWHUDWXUH DYDLODEOH RQ WUDQVÀXWKULQ DQG FRQFOXGHG WKDW WUDQVÀXWKULQ LV ³XQOLNHO\ WR present acute hazard in normal use". 25 7UDQVÀXWKULQ has light turbid color until browny, and easy to dilute in organic solvent such as hexane, dicloromethane and toluene, with dissolved level at 200000 mg/l. This compound is contact and stomach toxic that has better knockdown power and residue. 24 Aedes aegypti from Wonosobo showed increased levels of esterase activity (12.2%). This percentage was lower than report form Pekalongan and Yogyakarta. This suggested that the possibility of a resistance mechanism in Ae. aegypti population from Wonosobo to Malathion was not only based on enzymatic UHDFWLRQV EXW DOVR LQÀXHQFHG E\ PHFKDQLVPV IURP other pathways. However, esterases might have a role in resistance mechanism toward malathion in :RQRVRER (VWHUDVHV DUH WKH GHWR[L¿FDWLRQ HQ]\PHV involved mainly in insecticide resistance. In 0DOD\VLD /HH KDG FRQ¿UPHG E\ XVLQJ ELRFKHPLFDO tests that a major factor resulting to resistance in Cx. quinquefasciatus was due to elevated levels of esterases which correlated directly with malathion (OP) resistance. 26 Besides this, Yu et al also had found that resistance development to malathion was highly associated with increased esterase activity which LQGLFDWHG WKDW PHWDEROLF GHWR[L¿FDWLRQ ZDV OLNHO\ the major resistance mechanism in insects other than PRVTXLWRHV VXFK DV SODQW EXJ Lygus lineolaris. 27 Monooxygenases also showed increased level in Ae.aegypti population in Wonosobo (10.4%). Monooxygenases (P450) are an extremely important metabolic system because of their involvement in regulating the titers of endogenous compounds such as hormones, fatty acids and steroids, and in the catabolism and anabolism of xenobiotics such as drugs, pesticides and plant toxins. 9