Study area; Malarious area of Bandar Abbas southern Iran was selected for experiments (Fig.1)
The mixture of two insecticide, chlothianidin and deltamethrin (Fludora® Fusion) is provided by Bayer company. Fludora® Fusion (WP-SB 562.5 g/kg) containing 500g/kg clothianidin (CTD) + 62.5 g/kg deltamethrin (DLT), in 100g water soluble bags.
Preparation of the different hut trail rooms with different surfaces
Initially, different hut rooms including cement, plaster, clay and wood surfaces selected in the appropriate village. These rooms is treated with insecticide while four untreated control containers have been maintained .
Insecticide was sprayed using a compression sprayer recommended by WHO for the IRS which is equipped with a pressure gauge and HSS-8002 nozzles tips with regulator set at 24–55 PSI. Pesticide is dissolved in 10 liters of water in compression sprayer tanks. The sprayer discharge rate was set to 755 to 780 ml/min. The spray duration was adjusted to spray 19 m2 in one minute. The operation is done by an expert under supervision
Mosquito species tested
Anopheles stephensi larvae have been collected from Hormoodar village (27°19'14.72"N, 56°19'14.80"E), in the south of Bandar Abbas city during and were transferred to the insectary. The larvae is reared into F1 generation for subsequent tests.
Adult susceptibility tests
According to the guideline developed by WHO, diagnostic dose of insecticides was used against female adult of An. stephensi mosquitoes. A total of four replicates were used as exposure (20-25 mosquitos per test, totally 100 specimens) for each insecticide and two replicates as control (totally 50 specimens). Test kits and insecticide-impregnated papers were purchased from the WHO collaborative center in University Sains Malaysia (USM), Penang, Malaysia. Bendiocarb 0.1%, permethrin 0.75% lambda-cyhalothrin 0.05 % and deltamethrin 0.05% were used against adult mosquitoes. The control test mosquitoes were exposed to papers impregnated only with the appropriate carrier oil for pyrethroids and carbamates that was without insecticide.Tests were performed on the F1 progeny of wild-caught adult females of filed population, 3–5 days old fed with sugar. The mosquitoes were exposed to different insecticides by 60-minutes exposure time and 24 hours recovery period. A pad of a cotton wool soaked in 10% sugar solution was provided as feeding source of mosquitoes during the recovery period. Tests were carried out in an insectary maintained at (27±2)°C temperature and (75±10)% relative humidity, 14:10 light: dark]. Mortality rate for each test was estimated at the end of tests. Results showed resistance to both pyrethroid and carbamate insecticides with mortality rates of (84.31±2.62)%, (89.66±2.34)%, (82.27±5.20)% and (86.10±3.61)% to lambda-cyhalothrin, deltamethrin, permethrin and bendiocarb, respectively. An. stephensi showed resistance to both pyrethroid and carbamate insecticide groups that used in this study (lambda-cyhalothrin, deltamethrin, permethrin, and bendiocarb).
The bioassay tests were carried out for evaluation of residual effect of insecticide by WHO cones. The cones was fitted on different treated surfaces using rubber band. About 10–12 sugar-fed, 3-5 days old female mosquitoes was gently released into each cone at the vertical position. The mosquitoes have been exposed for 30 minutes to each treated surfaces in five different replicates. The same procedures were carried out for control container. At the end of exposure time, the adults transferred into clean cups with cotton wool pad containing 10% sucrose solution and were kept in the insectary for 24h recovery period, the time for recording the mortality rate. The mortality followed after 48 and 72 hours as well (WHO, 2020). (Figure. 2)
Minimum safety instructions and protective measures must be observed at all times. The operators will ensure that the insecticide formulation is safely and correctly applied. Standard Operating Procedures (SOPs) as well as WHO manual prepared for field staff working in national vector control programmes (WHO, 2003) applicable for Phase II trials must be available on site (e.g. safe use of compression sprayers, handling and spraying techniques, protective clothing and visors). The hut walls will be sprayed to attain dosages as per manufacturer’s recommendation. The spray men will be carefully supervised during spraying. Products will be applied via Indoor Residual Spraying (IRS) with the maximum application rate amounting to 40 (50) mL spray/m2. Only one round of spraying will be done. Calibration of the spray pumps will be done to obtain uniform and good quality spraying for the targeted dose. Protective clothing, goggles, gloves, etc. are provided to all spray men for their general safety.
Sachets and waste water will be disposed of according to WHO guidelines (WHO, 2007).
Data obtained from different replicates were collected for each surface. The mortality rate under 80% is considered as threshold level. Tests with control mortality rate between 5 and 20%, were corrected using Abbott’s formula.
Calculation of mortality rate
Mortality rate in plaster, mud cement, wood surfaces has been calculated and plot the diagram.
Application equipment and other materials
One Hudson® Expert compression sprayer fitted with a constant flow valve (CFV), Safety equipment: overalls, boots, helmet, visor, gloves, chemical mask.WHO testing cones (complete kit with cones and aspirators), Consumables including: cotton wool, paper cups, marker pen, masking tape, gloves, glucose, elastic bands, netting material, shoe tacks, tetramin, cerelac, filter papers, pipettes (with rubber teat), distilled water, 30cm by 30cm by 30cm mosquito cages, mouth aspirators, stop watch, weather station (thermometers, hygrometer, etc.), tiny tags/labels, mosquitoes (charged per organism from the insectary), cool boxes and equipment to transport mosquitoes to insectary, cement,whitewash,sand , plywood/ceiling board , grass thatch, sheets galvanized iron, mud daub, insecticides, nails.