The study was approved by the Ethics Committee from the Faculty of Medicine, the University of Indonesia (No.KET-055/UN2.F1.D1.2/PDP/Riset-2/2019).
The study used two insecticides from the organophosphate group, malathion (C10H19O6PS2) and temephos (C16H20O5P2S3), and two from the pyrethroid group, cypermethrin (C22H19Cl2NO3) and deltamethrin (C22H19Br2NO3). All the insecticides were purchased from chemical shops in Jakarta. These insecticides are registered in Indonesia as follows: malathion: MEGATHION 1200 UL, PT Citra Sari Kimia, No. Reg. RI 0809120103771; deltamethrin: DELFOX 25 EC, PT Indo Pest Biochem, No. Reg. RI 06090120093424; cypermethrin: CYNOFF, PT Bina Guna Kmia, However, liquid temephos was purchased from the Directorate of Vector-Borne and Zoonotic Diseases, Ministry of Health Indonesia, Jakarta, Indonesia.
Cx. quinquefasciatus larvae collection sites
Cx. quinquefasciatus larvae were collected from five fields of Jakarta; 1) Kampung Gedong (East Jakarta), 2) Johar Baru (Central Jakarta), 3) Marunda (North Jakarta), 4) Cengkareng (West Jakarta), and 5) Setu Babakan (South Jakarta) as seen in Fig 1. Using a dipper, the larvae were collected from polluted stagnant water bodies, sewers, or drains surrounding houses, i.e., their natural habitat. They were washed with tap water in 2000 mL plastic containers before being placed in 1000 mL plastic containers containing tap water. All of the specimens were subsequently identified at the Entomology Laboratory of the Department of Parasitology, University of Indonesia. Only third and fourth instars larvae were used in the larval bioassays.
In each study site, Research Team recorded the use of insecticides by the health center, natural breeding sites for Cx. quinquefasciatus mosquito, and household insecticides in supermarkets. The number of cases of malaria, DHF, and filariasis in Jakarta was obtained from the annual report (2019) of the Jakarta Health Office .
Larval Susceptibility tests
In this study, the larval susceptibility test was divided into two parts. First, the larval susceptibility test to temephos used the WHO standard kits (1.25, 6.25, 31.25, and 156.25 ppm) [13,14]. In the second part, we used malathion (0.5 ppm), cypermethrin (0.25 ppm), and deltamethrin (0.35 ppm) concentrations by modifying the temephos concentration of the WHO standard kits [13,15].
Cx.quinquefasciatus larvae bioassay was carried out by the WHO protocol . In the control groups, a total of 25 healthy Cx. quinquefasciatus larvae were exposed only to tap water in a 200 mL plastic cup; five replicates were conducted using a total of 125 larvae. In the experimental treatment groups, 25 Cx. quinquefasciatus larvae per 200 mL plastic cup were exposed to five different concentrations of each of the tested four insecticides (a total of 625 larvae). After 24 h of exposure in each experimental group, the number of dead and live larvae were recorded.
Detoxifying enzyme activity
In this study, the number of larvae for examination of detoxification enzyme activity was 50 Cx.quinquefasciatus larvae from the control, temephos, malathion, cypermethrin, and deltamethrin groups respectively. So, the total larvae used were 250 larvae. Cx.quinquefasciatus larvae were used for the enzyme activity examination. The same samples were used to examine AChE, GST, and oxidase activity.
The AChE activity was assayed as previously described [23,24]. Dead larvae collected from the bioassays after the 24 h exposure to tested insecticides were homogenized in 1.0 mL 0.25 M KPO4 (pH 7.2). At room temperature, 100 µL aliquots of the test sample homogenates were loaded into triplicate ELISA microplate wells. Similarly, 100 µL of the control (healthy Cx.quinquefasciatus larvae) solutions were added to triplicate microplate wells. Acetylcholine iodide (ACTH) and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) were then added to every well (100 µL of each per well) and the absorbance at 414 nm was immediately read with an ELISA reader (T0) and again after 10 minutes (T10). The unit of AChE activity is absorbance per minute or Abs/min. Elisa reader made in Finland, Thermo Fisher, ScientificTM, Cat number 51119000.
GST activity was assayed as previously described [23.24]. The present study, the dead Cx. quinquefasciatus larvae collected from the bioassay after 24 h exposure to tested insecticides were homogenized in 1.0 mL 0.25 M KPO4 (pH 7.2). Triplicate 100 µL aliquots of each homogenate were loaded into ELISA microplate wells at room temperature; similar wells were prepared using 100 µL of the control group (healthy Cx.quinquefasciatus larvae). Aliquots (100 µL) of reduced glutathione solution (Sigma G4251) and 1-chloro-2,4’- dinitrobenzene (cDNB) were added and the plates were read immediately at 340 nm (T0) with an ELISA reader and again at 5 min (T5). The unit of GST activity is absorbance per minute or Abs/min.
Oxidase was assayed as previously described [23,24]. The dead Cx. quinquefasciatus larvae were collected from the bioassays after 24 h exposure to tested insecticides. The dead Cx. quinquefasciatus larvae were homogenized with 1000 µL 0.25 M KPO4 (pH 7.2). The following positive controls were also prepared: (i) 1:55 (22 µL stock, µL 1.2 mL KPO4 buffer) and (ii) 1:110 (11 µL cytochrome stock, 1.2 mL KPO4 buffer). Triplicate aliquots (100 µL) of the test sample homogenates were added to ELISA microplate wells, and 100 µL KPO4 was added to the negative and positive control wells. The cytochrome-C positive control (cytochrome-C bovine heart) was added (100 µL), followed by a 200 µL TMBZ solution. One drop of 3 % hydrogen peroxide (H2O2) was added to each well and incubated for 5min. The plates were immediately read (T0) at 620 nm with an ELISA reader. The unit of oxidase activity is absorbance per minute or Abs/min.
Toxicity of OP and PYR
A routine histopathological technique was employed as previously described . In total, 150 dead Cx. quinquefasciatus larvae were examined; these consisted of 25 larvae from the negative control group and 125 larvae from the treatment groups (25 larvae from each insecticide treatment). All of these specimens were fixed with 10% formalin and then dehydrated using a series of increasing alcohol concentrations (70%, 80%, 90%, 95%, and 100%). Afterward, the specimens were embedded in xylene 1, xylene 2, and xylene 3 solutions and in paraffin blocks. The blocks were cut to thicknesses of 5 µm using a manual microtome (Model 320, No. 17664, USA) and feather microtome blades (Feather, S35, Japan). Finally, the sections were stained with hematoxylin and eosin and the stained specimens were observed under a light microscope and imaged using a digital microscopic mounted camera (Zeiss Axiocam ERC 5s, Germany).
The present study used TEM to evaluate the damage of midgut cells including cell membrane, mitochondria, and others. The samples were processed according to Ma et al , with a slight modification of the fixation liquid. The whole bodies of the treated Cx. quinquefasciatus larvae were pre-fixed in 2.5% glutaraldehyde at 4oC for a minimum of 2 days and then washed three times with cacodylate buffer for 15 min each time. The samples were fixed in 2% osmium tetroxide and 2.5% K3Fe(CN)6 in the buffer for 2 h, and then rinsed in cacodylate buffer as described in the previous step. The samples were then dehydrated in an ethanol series in ascending order (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min each. After dehydration, the samples were infiltrated using absolute ethanol and propylene oxide in specific ratios (2:1, 1:1, 1:2, v/v) for 30 min each. The samples were embedded in Spurr resin. The prepared samples were cut using an ultramicrotome (Leica UC6, Wetzlar, Germany) and observed using TEM (JEOL JEM 1010, Japan).
Data were analyzed using Statistical Package for Social Science (SPPS) version 26.0 The susceptibility response of the Cx.quinquefasciatus larvae to the insecticides was determined based on criteria as previously described . At 24 h, a 100% death rate indicated that Cx.quinquefasciatus larvae were completely susceptible to the insecticide, a 99-90 % death rate indicated moderate susceptibility, and a < 90% death rate indicated low susceptibility .
Data from AChE (0 min to 10 min), GST (0 min to 5 min), oxidase (0 min to 5 min) activities were not a normal distribution, so the Wilcoxon Signed Ranks Test (nonparametric) was used . The purpose of the Wilcoxon tests was to determine whether there was a difference in the mean AChE activity from 0 min to 10 min, GST activity from 0 min to 10 min, and oxidase activity from 0 min to 5 min.
The digestive system cytotoxicity of tested insecticides was measured based on damage to the midgut of Cx.quinquefasciatus larvae including the breakdown of the epithelial cells, microvilli, peritrophic membranes, and food boluses. Because the midgut damage data were not normally distributed, the Kruskal-Wallis test was used . The purpose of the Kruskal-Wallis test was to determine whether there was a significant difference in midgut damage due to exposure to the insecticide tested.