Insecticides	Total tested		Mortality rate (%) after 24h maintenance		Resistance status
	WHO	CDC	WHO	CDC	WHO	CDC
Malathion	86	296	100	99.7	* S	S
Propoxur	103	270	90.3%	66.2	** T	*** R
Permethrin	99	104	100	100	S	S

* Susceptible, ** Tolerance, *** Resistance,
Table 1: Susceptibility data recorded according to both WHO and CDC methods against An.stephensi

Insecticide	Susceptibility method	A	B±SE	LT50, 95% C.I. (Min)	LT90, 95% C.I. ( Min )	X2(df)	P-value
Malathion- 5%	WHO	-2.3423	3.4211 ± 0.869	1.1187 4.8381 7.8334	7.2247 11.4629 176.2323	11.345 (3)	0.05
Propoxur 0.1%	WHO	-5.3064	3.6879± 0.327	24.5860 27.4713 30.4591	53.2056 61.1502 73.3081	5.024 (2)	0.05
Permethrin 0.75%	WHO	-0.1205	2.6649± 0.204	0.9737 1.1098 1.2531	2.8496 3.3586 4.1274	18.475 (6)	0.01

Table 2: Susceptibility data recorded according to both WHO methods against An. stephensi

Figure 1: Distribution of An.stephensi in Iran

Figure 2:WHO protocol (Insecticide impregnated papers) for evaluation of insecticide resistance in mosquitoes

Figure 3: CDC protocol for evaluation of insecticide resistance in mosquitoes

Figure 4: Mortality rate and regression analysis of bioassays of An.stephensi exposed to Malathion using WHO and CDC methods

Figure 5: Mortality rate and regression analysis of bioassays of An. stephensi exposed to Propoxur using WHO and CDC methods

Figure 6: Mortality rate and regression analysis of bioassays of An. stephensi exposed to Permethrin using WHO and CDC methods

Introduction

Malaria is a mosquito-borne protozoan disease that remains one of the public health concerns in the world [1,2]. Despite many global efforts to eradicate it, malaria still causes many deaths in the world.

Malaria is one of the important infectious diseases in Iran [3]. More than 80% of malaria cases in Iran are reported from three provinces' southern and southeastern areas of the country [4,5]. Human migration and movement across eastern borders contribute to the spread of malaria in Iran [6].

Some species of Anopheles mosquitoes are vectors of malaria in different parts of the world. Anopheles stephensi is an important vector of malaria with a geographical range from the Middle East to India and China [7]. In its geographic distribution, this is an important vector for both Plasmodium falciparum and P.vivax [8-10]. Recent studies on Anopheline mosquitoes in Iran have reported the presence of 31 Anopheles species including genotypes and sibling species. Eight of them are involved in malaria transmission and among vectors of malaria, An.stephensi in Iran is considered as the main vector of malaria in southern Iran [11-13] (Figure 1). Vector control is the main approach to reduce malaria transmission at the community level and in many parts of the world, it is considered the most effective measure for eradicating malaria. It has been reported to be the only measure that can reduce malaria transmission from very high levels to close to zero [14,15]. Resistance to insecticides in vectors of malaria is one of the most important growing concerns in many countries and bioassays allow for the detection and characterization of insecticide resistance in a vector population [16,17]. Pesticide resistance is a decrease in a pest population’s susceptibility to the mode of action of a pesticide, causing the pesticide to no longer control the pest population as efficiently. Pesticide resistance is not new or uncommon. It has been a side effect of insect vector control programs since 1914, and insect disease vectors in over 45 countries are resistant to at least one pesticide class. Consequently, there is a risk of pesticide resistance developing in any pest population anywhere. There are several mechanisms of resistance in insects including reduced penetration, target site insensitivity, enzymatic resistance, behavioral change and excretion.

In the Iran region, the WHO insecticide susceptibility test is the most common method for assessing resistance status. (CDC) bottle bioassay is another tool for detecting resistance to insecticides that are not widely used in Iran [18]. CDC bottle bioassay relies on time mortality data, which are measures of the time it takes an insecticide to penetrate a vector, traverse its intervening tissues, get to the target site, and act on that site [19]. It can measure the efficacy of an insecticide formulation. The aim of this bioassay is to measure the mortality of a population at a given dose of insecticide. In WHO test, mosquitoes are exposed to known concentrations of an insecticide for a fixed period at the end of which the number of mortality is recorded [20]. In this study, we compared the WHO tests with the CDC bottle bioassay. Pesticide resistance occurs in a pest, resistance testing must be performed on individuals from the population. For mosquitoes, these assays include the Centers for Disease Control (CDC) bottle bioassay for adults and the World Health Organization’s (WHO) susceptibility bioassay. WHO method requires more mosquitoes than CDC method, the comparison between the results of both methods is clear. When the WHO susceptibility kit is not readily available, bottle bioassays can be used to determine insecticide resistance status of mosquito populations. WHO bioassays utilize cylinder plastic tubes whereas CDC bottles bioassays use 250 ml Wheaton bottles which are made of glass. World Health organization (WHO) papers do not need to be treated by oneself before their utilization because they are ordered in the impregnated form. Conversely, CDC bottles need to be coated with insecticide by oneself before each bioassay. In fact, the shelf life and reuse of prepared bottles are still not well documented or studied in laboratory conditions.

Figure 1

Methods

Study area

The larvae were collected from artificial ponds in the urban area of Chabahar Port of Sistan and Baluchistan province and colonized at the insectary of the School of Public Health (SPH), Tehran University of Medical Science.

Insecticide materials

The insecticide-impregnated papers were purchased with a WHO representative in Penang, Malaysia. The technical active ingredient of malathion, permethrin, and propoxur was provided from the Eco-toxicology Laboratory, School of Public Health, Tehran University of Medical Sciences.

Adult susceptibility test

Adult susceptibility tests were followed according to the current WHO and CDC protocols on unfed female mosquitoes aged 2–5 days old, reared from the larval collections. For each insecticide mortality rate at various times was calculated and then the regression line to each insecticide was plotted using Microsoft Excel (version. 2013). All susceptibility tests were conducted in the (SPH) insectary at 25±−2°C and 70 - 80% relative humidity.

WHO protocol (Insecticide impregnated papers)

The principle of the WHO bioassay is to expose insects to a given dose of insecticide for a given time to assess susceptibility or resistance. The standard WHO discriminating dosages were used [21]. In this study, three insecticides were tested including malathion 5%, permethrin 0.75%, and propoxur 0.75%. An aspirator was used to introduce 20 to 25 unfed female mosquitoes aged 2–5 days from batch into five WHO holding tubes (four tests and one control) that contained untreated papers. They were then gently blown into the exposure tubes containing the insecticide-impregnated papers. After one hour of exposure, mosquitoes were transferred back into holding tubes and provided with cotton wool moistened with a 10% sucrose solution. The number of mosquitoes “knocked down” at 60 minutes and mortalities at 24 hours was recorded following the WHO protocol [21] (Figure 2).

Figure 2

CDC protocol

CDC bottle bioassay is a surveillance tool for detecting resistance to insecticides in vector populations. The CDC bottle bioassay relies on time mortality data. The diagnostic dose and diagnostic time that was applied in the present study recommended by the CDC. The solutions were prepared and the bottles were coated according to the CDC protocol [19].15 to 30 unfed female mosquitoes aged 2–5 days were fed with 10% sucrose solution were introduced into each test bottle coated with insecticide and one control bottle coated with acetone only. The number of dead or alive mosquitoes was monitored and count at different times (15, 30, 45, 60, 75, 90, 105, and 120 minutes). This allowed us to determine the total percent mortality (Y-axis) against time (X-axis) for all replicates using a linear scale (Figure 3)

Figure 3

Statistical analysis and data interpretation

The resistance status determined according to the latest WHO criteria (20) as follows:

• When 98%–100% mortality at the recommended diagnostic time indicates susceptibility;

• When 90%–97% mortality at the recommended diagnostic time suggests the possibility of resistance that needs to be confirmed;

• When < 90% mortality at the recommended diagnostic time suggests resistance.

The resistance status of mosquito samples was tested by the CDC method determined according to the CDC criteria [19]. The susceptibility thresholds at the diagnostic time of 30 minutes for all insecticides are:

• Mortality rate = 100%: the population is fully susceptible

• Mortality rate < 100%: the population is considered resistant to the tested insecticides.

Results were analyzed by using of Probit program (Finney 1971) [22]. Error bars for each mortality were calculated based on the statistical method at α=5%. The lethal Time for 50% and 90% mortality (LT₅₀ and LT₉₀) values and their 95% confidence interval also. Probit regression line parameters were estimated and then the regression line of all Insecticides was plotted using Microsoft Excel (version. 2013).

Results

The results of 24 hours mortality recorded after 60 minutes of exposure of mosquitoes to impregnated papers of malathion, Probit analysis and the LT50 and LT90 values and confidence interval (95%) were calculated for each of the insecticides. (Table 2).

Table 1

Table 2

Figure 4

Figure 5

Figure 6

Discussion

To evaluate the susceptibility level of An.stephensi to insecticides, two methods of bioassay testing were used. The WHO standard method is major and widely used, and recently, the CDC Bottle Bioassay method was also used.

Our information about the susceptibility of the vectors to insecticide is essential for chemical interventions, so routine testing of these tests is an integral part of the control of the vectors and is very practical. In this study, we compared two important methods that were used in the world to perform susceptibility tests Malaria vector, An.stephensi that colonized at the insectary of School of Public Health (SPH), Tehran University of Medical Science.

The reason for using this strain is previous reports of resistance or tolerance to several types of insecticides [24,25]. WHO and CDC methods indicate that An.stephensi is susceptible to malathion. Approximately in all previously conducted studies on An. stephensi in Iran, susceptibility to malathion has been reported [26-28].

Permethrin shows significant protection against mosquito bites. This insecticide can be used to protect people from mosquito bites and reduce diseases transmitted by mosquitoes [29].

WHO recommended insecticides for indoor residual spraying against malaria vectors are: DDT, Malathion, Fenitrothion, Pirimiphos- methyl, Bendiocarb, Propoxur, Alpha-cypermethrin, Bifenthrin, Cyfluthrin, Deltamethrin, Etofenprox, Lambdacyhalothrin, Clothianidin [30].

WHO insecticide susceptibility test is the most common method for assessing resistance status in Iran [18]. WHO method requires more mosquitoes than the CDC method. When the WHO susceptibility kit is not readily available, bottle bioassays can be used to determine the insecticide resistance status of mosquito populations. WHO bioassays utilize cylinder plastic tubes whereas CDC bottles bioassays use 250 ml Wheaton bottles which are made of glass. WHO papers do not need to be treated by oneself before their utilization because they are ordered in the impregnated form. Conversely, CDC bottles need to be coated with insecticide by oneself before each bioassay. The shelf life and reuse of preprepared bottles are still not well documented or studied in laboratory conditions [31]. however, in field conditions, the studies of Perea et al (2009) showed that bottles treated with 10 μg a.i deltamethrin per bottle could be stored for at least 14 days and reused on three occasions [32]. The major advantages of the bottle assays are that any concentration of a custom insecticide (pure or formulated) may be evaluated and the technique is simple, rapid, and economical. One of the disadvantages of the WHO method is the transfer of mosquitoes to the tubes, which can damage the mosquitoes and cause an error in the test results. This problem is partly resolved in the CDC method Because CDC bottles bioassays do not need mosquitoes to be transferred from the exposure bottle. In WHO susceptibility tests mosquitoes must remain in the recovery period (stable conditions of temperature and relative humidity) during the 24 hours after exposure to insecticide-treated paper. The environmental conditions that mosquitoes have in the recovery period can affect the test results, which is one of the disadvantages of this method. In the CDC bottle bioassays method, this problem has been solved. the CDC bottles need to be clean, dry, and coated with insecticide by oneself before each bioassay that takes a long time. If the bottles are contaminated before the coating, there is an error in the test results and this is one of the disadvantages of the CDC bottle bioassays method. The current study emphasizes that the results of the two bioassays methods (WHO and CDC) were almost similar and there was no significant difference between the two methods. Each of the two methods has some specificity. CDC bottle bioassay can be used as part of a broader insecticide resistance monitoring program, which may include the World Health Organization (WHO) paper-based bioassay, and biochemical and molecular methods. . According to this problem that concentrations that proposed by the WHO and CDC is not local, to increase quality of testing and save time and cost. it is recommended that concentrations for the most important vectors of malaria in malarious area in our country is distinction.It is suggested to carry out tests CDC Bottle bioassay used to bottles with narrow openings until when transmitting mosquitoes into the bottle, do not escape into outer space. Similar studies on important species Culex and Aedes to be done.To evaluate the efficacy of CDC Bottle bioassay in field conditions and compared with WHO method. If the CDC bottle bioassay is to be used for routine insecticide susceptibility surveillance, the following conditions should be noted: the procedures detailed in the CDC guidelines should be strictly adhered to; in particular, those procedures relating to the use of the recommended insecticide solvents (ethanol or acetone) and the bottle treatment protocols;

Declarations

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

The research is financially supported by the Tehran University of Medical Sciences under code umber 95-03-27-32784, 18984.

Authors' contributions

All authors were involved

Competing of Interests

The authors declare that there are no competing interests.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Acknowledgments

Tehran University of Medical Sciences for financial support

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