J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 178 http://jad.tums.ac.ir Published Online: January 05, 2016 Original Article Irritability Levels of Field and Laboratory Population of Culex pipiens Complex in Tehran to Different Groups of Insecticides Sara Rahimi 1, *Hassan Vatandoost 1,2, *Mohammad Reza Abai 1, Ahmad Raeisi 3, Ahmad Ali Hanafi-Bojd 1, Fatemeh Rafi 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3National Program Manager for Malaria Control, Center for Communicable Diseases, Ministry of Health and Medical Education, Tehran, Iran (Received 6 Sep 2014; accepted 11 Jan 2015) Abstract Background: The irritant effect of some insecticides can cause a proportion of mosquitoes to leave the sprayed rooms before acquiring a lethal dose, so the repeated contact al sub-lethal dose may lead to extent the resistance. Methods: Larvae and pupae of Culex pipiens complex were collected in mass from open canals of waste water in capital city Tehran and reared to obtain the first generation at laboratory. Sugar-fed 2–3 days female mosquitoes were used for the experiments and compared with laboratory strain. The irritability tests of insecticides impregnated papers were measured in plastic conical exposure chambers placed which implemented at controlled conditions ac- cording to the method described by WHO .Number of take-offs were counted during 15 minutes of exposure time. Results: DDT had the most irritancy effect against field population of Cx. pipiens. DDT, permethrin and deltamethrin was moderately irritable against laboratory strain, whereas, addition to three previous insecticides, malathion, cyfluthrin and propoxur should be also considered as moderately irritable insecticides for field population of. Irritability level of etofenprox, fenithrothion, bendiocarb, and lambdacyhalothrin did not differ from control group. Conclusion: The irritability response of mosquitoes may have a negative impact on control measures. Periodical execution of irritability tests with insecticides that routinely used in vector control program is highly recommended. Keywords: Irritability, Insecticides, Culex pipiens, Tehran, Iran Introduction Culex pipiens complex has a great medical importance due to transmission of arbovirus and zoonotic diseases including Dirofilaria immitis (Azari et al. 2006). Also its biting and nuisance causes the severe allergies in humans and other hosts, and this led to discomfort es- pecially in urban areas (Vinogradova 2000). The Cx. pipiens are known as a complex species including Cx. pipiens, and Culex quinquefaciatus are important members of Cx. pipiens complex (Azari et al. 2007, Harbach 2011). Culex pipiens pipiens are distributed in both temperate and tropical areas whereas Cx. quinquefasciatus in the tropical regions of the world (Harbach 1988, Smith and Fonseca 2004, Azari et al. 2010). Culex pipiens are spread in most parts of Iran, whiles Cx. quinquefasciatus are scattered in the southern of country (Zaim and Cranston 1986, Zaim 1987, Azari and Harbach 2009, Azari-Hamidian et al. 2010). Vectors control is facing many problems especially occur- rence of insecticide resistance. Five major mechanisms have evolved to overcome toxi- *Corresponding authors: Prof Hassan Vatandoost, Email: hvatandoost1@yahoo.com, Mr Mohammad Reza Abai, Email: abaimr@sina.tums.ac.ir J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 179 http://jad.tums.ac.ir Published Online: January 05, 2016 cants such as reduces penetration, metabolic resistance, Target site insensitivity, excretion and behavioral change. In the behavioral change the mosquitoes are better able to resist the toxic effects of the insecticides, these changes, gradual resistance, gradually and inevitability increases the resistance genes in the population until all individuals are physiologically resistant (Thomson 1947, Vatandoost and Borhani 2004). The use of carbamate and phosphorus insecticides, es- pecially in agricultural fields, was led to the development of this type of resistance to in- secticides (Vatandoost et al. 2005a). Change of vector behaviors that occurs in different forms, so that it is possible to change the relaxing habits in the indoor and because of irritancy-repellency phenomenon due to some insecticides, which makes the insect does not rest long enough on spraying surface and thus, it does not receive a lethal dose. There- fore, it led to change the movement pattern of the mosquitoes into the houses and so they bitten out of the houses, avoids the insecticide- treated surfaces, occurrence of the exophagic and exophilic tendency and finally affected the results of vector control programs (Vatandoost and Borhani 2004). The mosquitoes could be avoided the spraying surfaces due to natural tendency and rest outside of buildings and houses (exophilic behavior). The irritation let to reduce to contact of mosquitoes with the sprayed surfaces, as well as absorption of enough dose of insecticides (Quinones and Suarez 1989). The different species of anopheline vectors have a different irrita- bility level, some stay long time on insec- ticide-treated surfaces, while others moti- vated and escape immediately. Some vectors may be hyper-irritable e.g Anopheles gambiae that has 40–50 take-off during 15 minute’s obligatory contact with DDT, whereas some other species may have hypo-irritable such as An. albimanus that has just 2–3 take-off in similar time, and some species are moderate- irritable, this phenomenon limits the residual spraying properties of the residential places against irritable species (Thomson 1947, Coluzzi 1963). The first studies on the irri- tability of Anopheles mosquitoes in Iran was conducted against An. maculipennis against DDT in Isfahan Province (Eshghy 1972). The other studies on the irritability of An. stephensi and An. culicifacies was determine against different insecticides was conducted in Sistan and Baluchistan Province (Vatandoost and Borhani 2004). It has been shown that re- sistant strains such as: Anopheles gambiae and Cx. quinquefasciatus demonstrate the different levels of irritability to the pyrethroids (Hougard and Duchon 2003). Reasonable and proper use of insecticides depends on the knowledge an awareness about sensitivity and irritability of the vectors to these insec- ticides. Many studies aimed to better under- standing and effective application of the control methods of both larval and adult of Cx. pipiens complex (Oshaghi et al. 2007). Long-term use of insecticide may increase the resistance level of mosquitoes popula- tions due to behavioral habits of mosquitoes and diversion from indoor to outdoor resting places (Thomson 1947). The irritability prop- erties may change the mosquitoes behavior and led the leaving the indoor to outdoor which have been observed during the resid- ual spraying of the villages with deltamethrin in endemic parts of malaria foci at southern east of Iran where the An. culicifacies and An. stephensi acts as main vectors (Abai et al. 1999, Alipour et al. 2005). Irritability is a first reaction of mosquito towards the sprayed environment, which initially increases move- ments of mosquitoes and finally led to away from the sprayed area, So repellency effect of insecticides is due to their irritability property (WHO 1970). The Cx. pipiens com- plex is main prevalent species at southern part of capital city of Tehran and easily bred in the high density at the sewage water as well as rice fields especially in the south parts where it reaches to this plateau (Vatandoost J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 180 http://jad.tums.ac.ir Published Online: January 05, 2016 et al. 2005). Different insecticides are being used for household and agricultural pest control in the capital city of Iran, Tehran which may lead to enhanced tolerance of house mosquito to different Insecticides (Vatandoost et al. 2005). Due to rare studies on irritability of Cx. pipiens complex this study was designed to understand the irritability level of field strain of Capital City Tehran compared with laboratory TEH-SPH strain to different insecticides. Materials and Methods Different ages of larvae as well as pupae were collected using standard dippers from rice field and margin of open sewage canals where it reached to the plateau at the south part of the capital city of Tehran. The larval colony was transported to the insectary of School of Public Health (SPH), bred the immature using the habitat water and a few amount of flake fish food added daily to each enameled pan until adults emerged. La- boratory strain of Cx. pipiens (TEH-SPH) which was used as a reference in this study originally collected from capital city of Teh- ran and had been colonized in SPH insectary from 1960 for more than 50 years and never exposed to any insecticides. Mosquitoes were maintained at 28–30 °C and 55–75 % relative humidity (RH). The light intensity in test-room was 5–8 foot- candles using two 40 W florescent lamps without any natural light and the reflected light reduced to a minimum. The tests were carried out between 9.0 AM and 5.0 PM. The emerged adults were fed with 5 % su- crose solution until 2–3 age mosquitoes which used for the tests. The irritability tests were carried out ac- cording the instruction of World Health Or- ganization (WHO) and the irritability test kit (WHO 1970). Due to exhibition of more take-offs of female mosquitoes than males (Rutledge et al. 1999), so only sugar-fed fe- male mosquitoes were individually tested for the irritability level using both TEH-SPH strain and field population and exposed 15 min to WHO’s impregnated papers inside the WHO’s cone and the number of take-off was counted and mean and standard error was assessed as a criteria for irritability level. Due to direct correlation of mosquitoes take-off with temperature (Kaschef 1968), the tests were done at insectary condition with 29–30 °C and 55–75 % of RH and the light only provided with two 40 W florescent lamps and the intensity varied between 5 to 8 foot candle for laboratory environment without any natural light. The following in- secticide impregnated papers of WHO were employed, DDT 4 %, bendiocarb 0.1 %, propoxur 0.1%, malathion 5 %, fenitrothion 1.0 %, permethrin 0.75 %, deltamethrin 0.05 %, lambdacyhalothrin 0.05 %, etofenprox 0.5 % and cyfluthrin 0.15 % and the oil im- pregnated papers were used for the control group. The mean of take-off was analyzed using ANOVA and independent t-test was separately for the treated and control groups according to field population and TEH-SPH strain under SPSS ver. 18.0. Results The irritability levels of were presented in Table 1,2 and Figs 1, 2,3 using TEH-SPH and field strains of Cx. pipiens complex according to different insecticides. The mean and standard error of take-off for DDT 4.0 % as an organochlorine pesticide was 11.2±1.5 and 2±0.2 respectively in both field and TEH-SPH strains. This insecticide should be regarded as a discriminative for the detection of irritability among mosquitoes population. From the organophosphate, fenitrothion 1.0 % and malathion 5.0 % were assessed and number of take-off per minute were 4.6±1.6 and 2.8±1.0 for the field strain and 1.3±0.7 J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 181 http://jad.tums.ac.ir Published Online: January 05, 2016 and 2.2±0.9 for TEH-SPH compared to 1.7± 0.3 for the control group. From carbamate, propoxur 0.1 % and bendiocarb 0.1 % were assessed for the irritability level against field and TEH-SPH strains of Cx. pipiens complex. The number of take-off per minute of propoxur 0.1 % and bendiocarb 0.1 % were 4.0±1.1 and 2.8±0.2 for the field strain and 3.6±0.2 and 2.9±0.2 compared to 1.3±0.7 for TEH-SPH strain. The irritability of both type I (permethrin 0.75%) and type II (deltamethrin 0.05 %, cyfluthrin 0.15% and lambdacyhalothrin 0.05%) as well as pseudo-pyrethroid (etofenprox 0.5%) were assessed. The irritability level for deltamethrin 0.05 %, cyfluthrin 0.15 %, lambdacyhalothrin 0.05 % were respectively 6.7±0.3, 4.1±0.2, 2.3±0.2 for the field strain and 3.5±1.4, 2.1±1.0 and 2.1±0.9 compared to 1.3±0.7 and 5.4±0.4 for TEH-SPH strain. The irrita- bility caused by etofenprox 0.5 % as pseudo- pyrethroid was 2.0±0.2 and 1.5±0.8 for field and TEH-SPH strains. The number of take- off per minute for permethrin 0.75 % as pyrethroids type I was 6.1±0.3 and 4.1±1.5 for the field and TEH-SPH strains. Table 1. Irritability levels of Culex pipiens (Tehran field strain) to different insecticides at laboratory conditions using WHO’ kit and the impregnated papers Irritability levelStandard Error (SE) Mean of take off in 15 minutes Number of Take off Insecticides Moderately-irritable0.511.2223DDT 4.0% Moderately-irritable0.36.7133Deltamethrin 0.05% Moderately-irritable0.36.1121Permethrin 0.75% Moderately-irritable0.34.895Malathion 5.0% Moderately-irritable0.24.181Cyfluthrin 0.15% Moderately-irritable0.23.676Propoxur 0.1% Hypo-irritable0.22.957Bendiocarb 0.1% Hypo-irritable0.12.549Fenitrothion 1.0% Hypo-irritable0.22.345Lambdacyhalothrin 0.05% Hypo-irritable0.22.039Etofenprox 0.5% Non-irritable0.31.9538Control Table 2. Irritability levels of Culex pipiens (TEH-SPH strain) to different insecticides at laboratory conditions using WHO kit and impregnated papers Irritability levelStandard devi- ation (SE) Mean of take off in 15 minutes Number of Take off Insecticides Moderately-irritable0.94.4132DDT 4.0% Moderately-irritable0.94.1123Permethrin 0.75% Moderately-irritable0.83.5106Deltamethrin 0.05% Hypo-irritable0.52.265Malathion 5.0% Hypo-irritable0.52.163Cyfluthrin 0.15% Hypo-irritable0.52.164Lambdacyhalothrin 0.05% Non-irritable0.21.544Etofenprox 0.5% Non-irritable0.21.338Fenitrothion 1.0% Non-irritable0.21.340Bendiocarb 0.1% Non-irritable0.21.338Propoxur 0.1% Non-irritable0.21.3165Control J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 182 http://jad.tums.ac.ir Published Online: January 05, 2016 J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 183 http://jad.tums.ac.ir Published Online: January 05, 2016 J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 184 http://jad.tums.ac.ir Published Online: January 05, 2016 J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 185 http://jad.tums.ac.ir Published Online: January 05, 2016 Fig. 1. Irritability levels of the field population compared with the TEH-SPH strain of Culex pipiens to 10 insecti- cides, Tehran, Iran Fig. 2. Comparison of irritability mean of Culex pipiens (field strain) to different insecticides, Tehran, Iran J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 186 http://jad.tums.ac.ir Published Online: January 05, 2016 Fig. 3. Comparison of irritability means of Culex pipiens (TEH-SPH strain) to different insecticides, Tehran, Iran Discussion In Iran, many pesticides have been ap- plied for the control of medically important arthropods in both the private and public health sectors. Various vector control measures including residual spraying (IRS), larviciding using Bacillus thuringiensis (BTi), impreg- nation of bed nets (ITNs) and long-lasting impregnated nets (LLITNs) have been em- ployed in malaria foci. Currently the deltamethrin is one of the most commonly insecticide used in public health against malaria vectors in Iran since 1994. In recent years, the new evidence of pyrethroids re- sistance has been indicated with the increas- ing trend of tolerance among different species of the mosquitoes in Iran (Vatandoost and Hanafi 2012). The resistance history to insec- ticides has been notified during past years. Susceptibility tests which carried out during July-August 2000, the Cx. pipiens was highly resistant to DDT 4.0 % with LC50 value reported 6.8 % and the resistance range had been varied at both north and south parts of Tehran (Nazari and Janbakhsh 2000). Expert committees of WHO has been classified the irritability level of mosquitoes to insecticides as hypo-irritable (2–3 take-off per minute), moderately irritable (3–30 take- off per minute) and hyper-irritable (30–40 take-off per minute) (WHO 1964). In this study, for the better statistical analysis and reduction of bias, the irritability tests were carried out at uniform conditions including fixed time (9 AM–5.0 PM), controlled tem- J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 187 http://jad.tums.ac.ir Published Online: January 05, 2016 perature (29–30 °C), humidity (55–75%) and light intensity (5–8 fc) as well as the tested mosquitoes were sugar-fed, 2–3 ages and the first generation of the field population hav- ing definite strains of female Cx. pipiens were used. The locality of immature sam- pling was fixed in south part of Tehran dur- ing the mass collection. A full series of irritability tests were car- ried out using 10 groups of insecticides in- cluding organochlorine, organophosphate, carbamate and pyrethroids against field pop- ulation of Cx. pipiens in comparison with a THE-SPH strain. The finding concerning DDT-irritability tests revealed the moderate- ly irritable level both for the field population (11.2±0.5) and the TEH-SPH strain (6.4±0.2). The take-off mean for DDT was recorded as the highest value in field strain and statisti- cally differed from irritation caused by other insecticides (P< 0.05). Furthermore DDT, a series of irritability tests was carried out on the organophosphate (malathion 5.0%), one carbamate (propoxur 0.1%) and three pyrethroids (permethrin 0.75%, deltamethrin 0.05% and cyfluthrin 0.15%) which assessed moderately-irritable with the take-off means ranged between 3.5 % and 6.7 % against both insectary and field population of Cx. pipiens. The results of statistical analysis of irritability tests were shown in Fig. 1 and Fig. 2. DDT, permethrin and deltamethrin induced significantly more take-offs than other insecticides but mean of take-off for three latter insecticides did not differ from each other against both strains of Cx. pipiens (Fig. 2). Irritability tests for malathion 5.0 %, cyfluthrin 0.15 and propoxur 0.1 % also revealed the moderately irritable level. The irritability of remaining insecticides includ- ing bendiocarb 0.1 %, fenithrothion 1.0 %, lambdacyhalothrin 0.05 % and etofenprox 0.5 % did not significantly differed from control group and so assessed as non-irrita- ble level. DDT has two types of toxic effect on mosquitoes-an initial rapid knock down ef- fect, followed by a lethal effect as well as an excito-repellent effect is shown (Hodjati et al. 2003). Irritability tests with the ʺbig-sized mosquitoʺ An. maculipennis showed that the mean of take-off for DDT 4.0 % was 26.4 and 19.4 for An. superpictus (Eshghi 1972). Under the large cage conditions DDT- resistant An. stephensi were more readily irritated by the insecticide than a susceptible strain of the same species (Eshghy 1977). Also the similar work with another big-sized mosquitoes An. sacharovi showed that DDT had the most and deltamethrin the least irritancy effect. The average number of take offs/fly/minutes were recorded 0.8±0.2, 0.7± 0.2, 0.5±0.2, 0.5±0.3, and 0.2±0.1, for DDT, permethrin, lambdacyhalothrin, cyfluthrin and deltamethrin respectively (Vatandoost and Abai 2012) All the above-mentioned three species showed variability in their irritability to different pyrethroids especially deltamethrin. Results from DDT irritability assays also showed that organochlorine insecticides can induce behavior-modifying actions, such as contact irritancy and spatial repellency, which reduces man-vector contact, despite evidence of insecticide resistance within the test pop- ulation (Achee et al. 2009). The irritability to DDT shown in some populations of An. albimanus and An. nuneztovari may reduce the effectiveness of residual applications of this insecticide by causing the mosquitoes to seek untreated surfaces and/or leave the house to rest outdoors and thus avoid a lethal dose (Quinones and Suarez 1989). It has been shown that An. hyrcanus had a low irritability to insecticides but high resistance to DDT and moderate resistance to propoxur, while the susceptibility to malathion and fenitrothion remained complete. It is sup- posed that the nature of adaptation to insecti- cide pressure is, to a great extent, determined by mosquito endophily or exophily (Sorokin J Arthropod-Borne Dis, June 2016, 10(2): 178–191 S Rahimi: Irritability Levels of Field … 188 http://jad.tums.ac.ir Published Online: January 05, 2016 and Mingaleva 1992). The behavioral avoid- ance to insecticides may play a significant role in reducing the selection pressure and thus occurrence and spread of insecticide resistance (Chareonviriyaphap et al. 2013). The other irritability experiments which carried out in Kahnooj district, southeastern Iran, showed that DDT and permethrin had the most irritancy effect on An. stephensi and An. dthali. In contrast with the our re- sults, DDT and deltamethrin showed the least irritancy effect against An. stephensi with 0.42+0.08 and 0.77+0.12 take-offs min/adult, respectively. However lambdacy- halothrin had the least irritancy effect against An. dthali with 0.096+0.02 take-offs/min/adult. The mean number of take-offs/min/adult with permethrin showed significant differ- ence to DDT, lambdacyhalothrin, cyfluthrin and deltamethrin (Vatandoost et al. 2005b). Also in the similar area, Jiroft district, south- eastern Iran, the low take-off for deltamethrin and the higher value for DDT were shown. The average number of take-offs per min per adult was 2.09± 0.13 for DDT, 0.581±0.05 for dieldrin, 1.85±0.08 for permethrin, 1.87± 0.21 for lambda-cyhalothrin, 1.53±0.13 for cyfluthrin, and 1.23±0.1 for deltamethrin (Abai et al. 2009). The irritability tests were carried out in a malaria–prone area, Iranshahr and Nikshahr districts which similar values were recorded for An. stephensi exposed to permethrin, deltamethrin, cyfluthrin and lambdacyhalothrin as 6.64±1.04, 3.11±0.67, 2.73±0.61 and 2.57±0.67, and An. culicifacies were 2.24±0.37, 1.44±0.38, 1.59±0.35 and 1.46±0.5, respectively (Vatandoost and Borhani 2004). The results of another investigation with An. culicifacies showed the most irritancy for DDT and lowest for permethrin and cyfluthrin in Iranshahr and Sarbaz district, southeastern Iran (Vatandoost et al. 2011). The other study with An. stephensi showed the most irritancy effect for lambdacyhalothrin, the moderate for cyfluthrin and the less irritancy for deltamethrin (Vatandoost 2001). Behavioral resistance appears more rapidly in endophilic species than exophilic ones (Muirhead 1960) and the An. stephensi has more endophilicity habit than An. culicifacies and so it is assumed that they are under insecticides pressure selection (Bhatia and Deobhankar 1963, Abai et al. 1999, Alipour et al. 2005). Irritability studies of resistant and susceptible strains indicated that the resistant strain is 2.8 times less irritable to permethrin in comparison with susceptible one (Vatandoost 2000) and the value of irritability may be differed with mosquito strains (Brown 1958). In conclusion, based on the finding, con- joint implementation of irritability and sus- ceptibility tests in problematic area recom- mended for assessment the possible chang- ing of mosquitoes behavior such as diversion to untreated indoor shelters or resting at out- doors and in long term, reinforcement of exophilic population at the areas under in- secticide pressure whether indoor residual spraying or impregnation of bed nets opera- tions. The behavioral avoidance to insecti- cides may play a significant role in occur- rence and spread of insecticide resistance. Conclusion Periodically experiments on irritancy of different insecticides against mosquito will provide a clue for Integrated Vector Man- agement Acknowledgements We acknowledge of Dr A Latifi, PhD student for his kind assistance during the mass collection of immature stages of Culicidae in Tehran. 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