J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 185 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Assessment the Changing Trend of Susceptibility to Two Insecticides Among Field-Population Culex quinquefasciatus Compared with the Same Population Undergoing to Multiple Colonization Atieh Shemshadian 1 ; *Mohammad Reza Abai 1,2 ; *Hassan Vatandoost 1,2 ; Navid Dinparast- Djadid 3 ; Mohammad Ali Oshaghi 1 ; Abdolrasoul Mojahedi 1,4 1 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3 Malaria and Vector Research Group, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran 4 Provincial Health Center, Bandar Abbas University of Medical Sciences, Bandar Abbas, Iran (Received 25 Apr 2019; accepted 08 June 2020) Abstract Background: During the past decade, rapid development of insecticide resistance have been reported among many species of mosquito vectors against four main categories of insecticides worldwide. The aim of the research was to assess the variation trend of susceptibility levels of Culex quinquefasciatus to two insecticides separately for the field population compared with subsequent generations of the same sample after multiple colonization. Methods: Larvae and pupae of Cx. quinquefasciatus were collected from house sewages and reared to adult which blood-fed on roosters. Ten percent sucrose fed female mosquitoes aged 2–3 days were used for susceptibility tests with DDT and deltamethrin. Susceptibility levels was assessed in the adult stage of field stran Cx. quinquefasciatus against DDT 4.0% and deltamethrin 0.05% and continued up to next six generations undergoing multiple rearing at insectary condition. Results: The susceptibility levels to DDT 4.0% did not change compared to the field with the lab population to six generations. Regarding deltamethrin 0.05%, no significant difference was shown between field strain (58.3%) and 3 rd generation (52.7%) compared to the 6 th one (33.8%). Conclusion: This finding may reflect the role of the kdr gene in resistance to organochlorine which has cross-re- sistance with pyrethroid insecticides. The results of this study clearly showed the irreversible trend of p yrethroid re- sistance among colonized mosquitoes. This is the first study of the resistance status of Cx. quinquefasciatus in Iran. Keywords: Culex quinquefasciatus; Variation; Susceptibility; Insecticides multiple generations Introduction Culex quinquefasciatus Say, commonly known as the southern house mosquito, is widely distributed in the tropical and subtrop- ical zones (1). It is also found at high densi- ties in similar climates southern Iran. The bi- ologic behavior of this species brings mos- quitoes to the adjacent premises, providing conditions for easy transmission of arboviral diseases to humans as well as to domestic and wild animals, including West Nile fever, Saint Louis encephalitis, Western equine enceph- alitis, Rift Valley fever, avian malaria and lymphatic filariasis. This mosquito also plays an important role in the transmission of the nocturnal periodic form of Wuchereria bancrofti (2) and is a ma- jor cause of acute and chronic morbidity, af- fecting all ages and both sexes throughout the tropical and sub-tropical areas of the world (3). Moreover, Cx. quinquefasciatus is iden- *Corresponding authors: Mr Mohammad Reza Abai, E-mail: abaimr@tums.ac.ir, Dr Hassan Vatan- doost, E-mail: hvatandoost1@yahoo.com https://en.wikipedia.org/wiki/Avian_malaria mailto:abaimr@tums.ac.ir J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 186 http://jad.tums.ac.ir Published Online: June 30, 2020 tified as a potential vector of Zika virus (4). The major strategy for controlling this mos- quito is application of insecticides. Addition- ally, this species can become quickly resistant to most insecticides compared to other mos- quitoes (5) and resistant to different ones in most countries in the world (6). Resistance to DDT in Cx. quinquefasciatus was report- ed in West Africa for the first time (7). There are several reports of resistance to different groups of insecticides from West African re- gions including Coˆte d’Ivoire, Burkina Faso, Benin and Ghana (8-10). According to these reports, resistance to the organochlorine in- secticides has developed by releasing pesti- cide residues into wastewater in major cities (11). Resistance of Cx. quinquefasciatus to pyrethroid was first reported from California (12). Resistance to organophosphates was re- ported in a strain of this vector in Sri Lanka due to increased esterase activity (13). There are also reports on resistance to a variety of insecticides from Thailand, Pakistan, France, and Saudi Arabia (14-16). However, there is no report of the resistance status of Cx. quin- quefasciatus to insecticides in southern Iran. The aim of the research was to assess var- iation trend of susceptibility levels of Culex quinquefasciatus to two insecticides separate- ly for field population compared with subse- quent generations of the same sample achieved after multiple colonization. Materials and Methods Mosquito collection The larval and pupae sampling of Cx. quin- quefasciatus were done in Suru County, Ban- dar-Abbas District, Hormozgan Province in 27.168142° N and 56.252175° E at an ele- vation of 3m above sea level from March to April 2016 (Fig. 1). In order to provide labor- atory colonies of mosquitoes, the larvae and pupae of Cx. quinquefasciatus were collect- ed from breeding places in the wastewater of the houses running outside near the seaside using the dipping method (Fig. 1). Moreover, immature mosquitoes were collected from abandoned boats where the polluted water could penetrate. The eggs, larvae and pupae stages of Cx. quinquefasciatus along with their breeding places, water, and herbs were transported to an insectary located in Bandar- Abbas Training and Research Station. The mosquitoes were reared at 30±5 °C with 65– 80% relative humidity. Female mosquitoes aged 2–3 days were fed on 10% sucrose so- lution and used for susceptibility tests. Insecticides Susceptibility tests was done using with both DDT 4.0% impregnated-papers with im- pregnation date July 2013, expiry date July 2018 with Batch No under code: DD 188 and deltamethrin impregnated-papers with impreg- nation date July 2016, expiry date July 2017 with Batch No under code: DE 332. The im- pregnated-papers to insecticides was purchased from the Vector Control Research Unit, School of Biological Sciences, 11800 Minden, Pe- nang, Malaysia. Susceptibility tests Adult susceptibility tests were conducted using WHO standard kits provided by WHO (17). Twenty-five female mosquitoes aged 2–3 days fed on 10% sucrose solution during past night were exposed to DDT 4.0% and deltamethrin 0.05% impregnated papers for 60 minutes. The solvent-impregnated papers were used for the control group. There were 10 replicates for the treatment group and two replicates for the control one. After ending the exposure time, both mosquito groups were allowed to recover in holding tubes with a piece of cotton containing 10% sucrose solu- tion on the top of tube for 24 hours and then the number of dead and live mosquitoes was counted. The susceptibility tests were sepa- rately repeated in the 3 rd and 6 th generations of Cx. quinquefasciatus in the insectary. J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 187 http://jad.tums.ac.ir Published Online: June 30, 2020 Mosquito colonization For the first time, a success was gained for establishment and colonization of Cx. quin- quefasciatus in the water containing intact Ber- muda grass “Cynodon dactylon” a perennial plant with spreading rhizomes and stolon. The plant was used directly in the immature breeding pans at natural state and without any chopping (Fig. 2). The adult mosquitoes were easily mated in a wooden netted cages dimensioned 30x 30x 30cm. The mosquitoes were blood-fed on a rooster, why the protein sources needed maturing the eggs. Statistical analysis The mortality rate of Cx. quinquefasciatus after 24 hours recovery was calculated for each insecticide and generation and correct- ed using the Abbott’s formula if the mortal- ity rate was 5–20% in the control group. The interpretation of the resistance level in Cx. quinquefasciatus was adopted from the WHO criteria and the terms resistant, suggestive of the existence of resistance and further inves- tigation required, and susceptible were used for mortality rates <90%, 90–98% and >98%, respectively (17). Univariate analysis of var- iance (ANOVA) was applied after Arcsin transformation of the mortality rate to deter- mine differences in mosquito mortality rates among generations. Significant differences of the mean of mortality rate were compared using Tukey or Games-Howell tests depend- ing on the Post Hoc Test result. The signifi- cant difference in the mortality rate of Cx. quinquefasciatus between different generations was calculated using following formula: SE= √pq/n, where: “p” is the mortality rate, “q” is equal to 1 minus the mortality rate, and “n” is the sample size. Results Mass colonization For the first time, a success was gained for establishment and colonization of Cx. quin- quefasciatus in the water containing intact Bermuda grass “Cynodon dactylon” a peren- nial plant with spreading rhizomes and sto- lon. The plant was used directly in the im- mature breeding pans at natural state and with- out any chopping (Fig. 2). The adult mosqui- toes were easily mated in a wooden netted cage dimensioned 30x 30cm. The mosquitoes were blood-fed on a rooster why the protein sources needed maturing the eggs. Bioassays Two-three days old female mosquitoes was exposed to DDT 4.0% and mortality rate of the field strain was 12.1%, compared to 9.5% and 10.5% in the 3rd (F3) and 6th (F6) genera- tions. The mortalities between different gen- erations exposed to DDT was not differed with each other, but with significant difference com- pared with control group (F= 2.036, p< 0.05) (Fig. 3). The Cx. quinquefasciatus were revealed 58.3%, 52.7%, and 33.8% of mortality, when exposed 60min to deltamethrin 0.05% re- spectively for field strain, F3, and F6 gen- erations. It was shown significant differences F3 compared to field strain and F6 mosqui- toes and the mortality in control group showed significant differences with the treatment group (Fig. 4). The evidence showed that the sub- sequent mass colonization of a field strain, the susceptibility level will be unchangeable compared with the lab-colonized of the same population which undergone several mass- colonization which established the pyrethroid resistance. J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 188 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 1. A. Satellite map of Suru location, Bandar-Abbas Port, Hormozgan Province, Persian Gulf littoral, Iran b. Ground landscapes of the breeding places of Culex quinequefaciatus in study area Fig. 2. Collection of immature Culex quinquefasciatus from natural breeding places with floating Bermuda grass and subsequent establishment in the insectary of Bandar-Abbas Health Training and Research Station, Persian Gulf littoral, Iran Fig. 3. Comparison of susceptibility level of Culex quinquefasciatus exposed to DDT 4.0% among field population and subsequent generations gained from same population A B J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 189 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 4. Comparison of susceptibility level of Culex quinquefasciatus to deltamethrin 0.05% on field population compared with lab strain achieved after mass colonization up to the six generations Discussion Despite the confirmed role of Cx. quinque- fasciatus as an arboviral vector in the subtrop- ical climate, little attention has been paid to its bioecological characteristics in Iran. This is while huge densities of this species at lar- val and adult stages were observed at the ur- ban area and extensive larval habitats could be found around the houses which its water originated from houses sewage. Moreover, pools and superficial wells at agricultural lands are potential larval habitats in the south and southeast Iran. The susceptibility levels of female Cx. quin- quefasciatus were tested according to a meth- od recommended by the WHO. The results showed that this species was highly resistant to DDT and deltamethrin at diagnostic dose and 60min of exposure time. After the mass colonization of Cx. quinquefasciatus, the re- sults of susceptibility tests were compared between field population and lab generations up to six generations achieved from the same field population to shown stability of re- sistance both for DDT and deltamethrin de- spite mass colonization up to six generations in the insectary condition. In this study, the susceptibility testing was followed from the latest WHO’s instruction (17), which indicat- ed a 60min exposure time for all mosquito species and for specified diagnostic concen- trations of insecticides, but according to the newly published WHO’s guidelines, the ex- posure time for DDT and Culex species were changed to 240min at the same discriminat- ing concentration (18). Resistance-related stud- ies of Cx. quinquefasciatus have been carried out for numerous insecticides around the world, from Asia to Europe, West Africa, and Amer- ica. A high level of resistance to pyrethroids was found in Coˆte d’Ivoire and Burkina Fa- so, West Africa (8). There is a report of the resistance of Cx. quinquefasciatus to DDT and deltamethrin in Thailand caused by con- tinuous use of insecticides for dengue vector control (19). In northeastern India, a study showed resistance to DDT and deltamethrin in Cx. quinquefasciatus collected from army cantonments and neighboring villages (20). There is a report of the resistance of Cx. quinquefasciatus to organochlorines, organ- J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 190 http://jad.tums.ac.ir Published Online: June 30, 2020 ophosphates, and pyrethroids in La Réunion Island, France (21). According to recent find- ings, resistance to DDT and pyrethroid in this species subsequently may lead to the ineffi- cacy of the long-used insecticide-treated nets (10). This high resistance can be caused by sev- eral factors in the study area, one of which could be long-term use of DDT for combat- ting malaria vectors, especially in the south of Iran that goes back to half a century ago. Another factor may be the tendency of female gravid adults to lay eggs at sewage enriched with organic materials. Because of the blood- feeding tendency of Cx. quinquefasciatus to- wards the poultries and wild birds, which are considered as potential reservoirs of arboviral agents, the risk of the transmission of these diseases to humans increases in the subsequent blood-feeding sessions. This vector is exposed to the chemicals that may be present in the ur- ban wastewaters. Cross-resistance should al- so be considered as another factor for inacti- vation of pyrethroid insecticides. The target of organochlorines (DDT) and synthetic py- rethroids is the sodium channels of the nerve sheath (22). Therefore, due to the similar tar- get site, cross-resistance may develop and re- sistance to one of these insecticides may make mosquitoes resistant to the other one. The find- ings of present study may reflect the role of the kdr gene in resistance to organochlorine which has cross-resistance with pyrethroid in- secticides. Furthermore, another finding is ir- reversible and progressive trends of resistance intensity to the pyrethroids among the colo- nized mosquitoes reared for several genera- tions. This is the first study of the resistance status of Cx. quinquefasciatus in Iran. Conclusion The findings indicated the stability of DDT resistance in the field population of Cx. quiq- uefasciatus as well as among lab-bread of same population. The deltamethrin resistance showed progressive trend in the lab compared with wild population after multiple coloniza- tion in the insectary. Hence, precise control management should be spotted for efficacy of control operations against Culex vectors. Acknowledgements The authors wish to thank Dr K Holakouie- Naieni, Head of Bandar-Abbas Research and Training Station (BRTS), School of Public Health, Tehran University of Medical Sci- ences for his sincere cooperation during field works. Moreover, the authors are grateful to Hosein Shabkhiz, the internal manager of BRTS for his continuous assistance during this research. This study was funded and supported by Tehran University of Medical Sciences. This study was a part of MSc dissertation funded and supported by Tehran University of Med- ical Sciences; Grant No 921126016. The au- thors claim no conflicts of interest. References 1. Harbach RE (1988) The mosquitoes of the subgenus Culex in southwestern Asia and Egypt (Diptera: Culicidae). Contri- butions of the American Entomological Institute (Gainesville). 24(1): 1–240. 2. Manimegalai K, Sukanya S (2014) Biology of the filarial vector, Culex quinque- fasciatus (Diptera:Culicidae). Int J Curr Microbiol App Sci. 3(4): 718–724. 3. Njenga SM, Muita M, Kirigi G, Mbugua J, Mitsuiy Y, Fujimai Y, Aoki Y (2000) Bancroftian filariasis in Kwale district, Kenya. East Afr Med J. 77(5): 245–249. 4. Xiao-Xia G, Chun-Xiao L, Yong-Qiang D, Dan X, Qin-Mei L, Qun W, Ai-Juan S, Yan-De D, Wu-Chun C, Cheng-Feng Q, Tong-Yan Z (2016) Culex pipiens quin- quefasciatus: a potential vector to trans- mit Zika virus. Emerg Microbes Infect. 5(9): 1–5. J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 191 http://jad.tums.ac.ir Published Online: June 30, 2020 5. Norris LC, Norris DE (2011) Insecticide resistance in Culex quinquefasciatus mosquitoes after the introduction of insecticide-treated bed nets in Macha, Zambia. J Vector Ecol. 36(2): 411–420. 6. Yadouléton A, Badirou K, Agbanrin R, Jöst H, Attolou R, Srinivasan R, Pa- donou G, Akogbéto M (2014) Insecti- cide resistance status in Culex quin- quefasciatus in Benin. Parasit Vectors. 8(7): 1–6. 7. Jones CM, Machin C, Mohammed K, Majambere S, Ali AS, Khatib BO, Mcha J, Ranson H, Kelly-Hope LA (2012) Insecticide resistance in Culex quin- quefasciatus from Zanzibar: implica- tions for vector control programs. Par- asit Vectors. 5 (78): 1–9. 8. Chandre F, Arriet F, Darder M, Cuany A, Doannio JMC, Pasteura N, Guillet P (1998) Pyrethroid resistance in Culex quinquefasciatus from West Africa. Med Vet Entomol. 12: 359–366. 9. Corbel V, Guessan RN, Brengues C, Chan- dre F, Djogbenou L, Martin T, Akogbe- to M, Hougard JM, Rowland M (2007) Multiple insecticide resistance mecha- nisms in Anopheles gambiae and Cu- lex quinquefasciatus from Benin, West Africa. Acta Trop. 101: 207–216. 10. Kudom AA, Mensah BA, Froeschl G, Rinder H, Boakye D (2015) DDT and pyrethroid resistance status and labora- tory evaluation of bio-efficacy of long lasting insecticide treated nets against Culex quinquefasciatus and Culex de- cens in Ghana. Acta Trop. 150: 122–130. 11. Agrawal A, Pandey RS, Sharma B (2010) water pollution with special reference to pesticide contamination in India. J Water Res Prot. 2: 432–448. 12. Priester TM, Georghiou GP (1978) In- duction of high resistance to permethrin in Culex pipiens quinquefasciatus. J Econ Entomol. 71(2): 197–200. 13. Peiris H, Hemingway J (1990) Mechanisms of insecticide resistance in a temephos selected Culex quinquefasciatus (Dip- tera: Culicidae) strain from Sri Lanka. Bull Entomol Res. 80(4): 453–457. 14. Yanola J, Chamnanya S, Lumjuan N, Som- boon P (2015) Insecticides resistance in the Culex quinquefasciatus popula- tions from northern Thailand and pos- sible resistance mechanisms. Acta Trop. 149: 232–238. 15. Shah RM, Alam M, Ahmad D, Waqas M, Ali Q, Binyamin M, Ali Sarfraz (2016) Toxicity of 25 synthetic insec- ticides to the field population of Culex quinquefasciatus say. Parasitol Res. 115 (11): 4345–4351. 16. Amin AM, Peiris HTR (1990) Detection and selection of organophosphate and carbamate resistance in Culex quinque- fasciatus from Saudi Arabia. Med Vet Entomol. 4: 269–273. 17. World Health Organization (2016) Test procedures for insecticide resistance monitoring in malaria vector mosqui- toes -2 nd ed. WHO Document Production Services, Geneva, Switzerland. Available at: http://apps.who.int/iris/bitstream/1066 5/250677/1/9789241511575-eng.pdf. 18. World Health Organization (2014) Pesti- cide evaluation scheme. Discriminating concentrations of insecticide for adult mosquitoes. World Health Organization, Geneva: Available at: https://www.who.int/neglected_diseas es/vector_ecology/resources/en/Diagn ostic_concentrations.pdf?ua=1 19. Sathantriphop S, Paeporn P, Supaphath- om K (2006) Detection of insecticides resistance status in Culex quinquefas- ciatus and Aedes aegypti to four major groups of insecticides. Trop Biomed. 23(1): 97–101. 20. Sarkar M, Bhattacharyya IK, Borkotoki A, Goswami D, Rabha B, Baruah I, Srivastava RB (2009) Insecticide re- https://www.ncbi.nlm.nih.gov/pubmed/?term=Sarkar%20M%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Bhattacharyya%20IK%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Borkotoki%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Borkotoki%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Goswami%20D%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Goswami%20D%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Baruah%20I%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=Srivastava%20RB%5BAuthor%5D&cauthor=true&cauthor_uid=19493193 J Arthropod-Borne Dis, June 2020, 14(2): 185–192 A Shemshadian et al.: Assessment the … 192 http://jad.tums.ac.ir Published Online: June 30, 2020 sistance and detoxifying enzyme activ- ity in the principal bancroftian filariasis vector, Culex quinquefasciatus, in north- eastern India. Med Vet Entomol. 23(2): 122–131. 21. Tantelya ML, Pablo Tortosab, Alouta H, Berticata C, Berthomieua A, Ruteec A, Dehecqc JS, Makoundoua P, Labbéa P, Pasteura N, Weilla M (2010) Insecti- cide resistance in Culex pipiens quin- quefasciatus and Aedes albopictus mos- quitoes from La Réunion Island. Insect Biochem Mol Biol. 40(4): 317–324. 22. Brogdon WG, McAllister JC (1998) In- secticide resistance and vector control. Emerg Infect Dis. 4(4): 605–613. http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff2 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff3 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff3 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/S0965174810000342#aff1