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1 J Vector Borne Dis 53, June 2016, pp Spatial distribution and insecticide susceptibility status of Aedes aegypti and Aedes albopictus in dengue affected urban areas of Rawalpindi, Pakistan Ali Arslan 1, Hamayun Rashid Rathor 1, Muhammad Uzair Mukhtar 1, Shumaila Mushtaq 1, Adil Bhatti 1, Muhammad Asif 2, Israr Arshad 3 & Jam Farooq Ahmad 4 1 Department of Medical Entomology and Disease Vector Control (MEDVC), Health Services Academy, Quaid-e-Azam University, Islamabad; 2 University of Veterinary and Animal Sciences, Lahore; 3 Agriculture Department, Pest Warning and Quality Control of Pesticides, Government of Punjab; 4 Department of Anthropology, Quaid-e-Azam University, Islamabad, Pakistan ABSTRACT Key words Background & objectives: Dengue is one of the most common arthropod-borne viral diseases which is transmitted mainly by two vector species, Aedes aegypti (Linnaeus 1762) and Ae. albopictus (Skuse, 1894) worldwide. As there is no effective medicine and vaccine available, vector control remains the most effective measure to prevent its transmission and outbreak. The aim of the study was to confirm the co-occurrence of Aedes aegypti and Ae. albopictus populations in the different localities of Rawalpindi, Pakistan and examine their susceptibility status against different groups of insecticides. Methods: Ovitraps were randomly placed in the study localities. The number of eggs from all the ovitraps were counted and incubated for hatching in Medical Entomology and Disease Vector Control (MEDVC) insectarium for rearing up to adult stage. The adults were then identified by using the pictorial keys. Spatial distribution and aggregation of both Ae. aegypti and Ae. albopictus populations was determined by using Index of dispersion or variance to mean ratio and k values of the negative binomial distribution. The susceptibility status of both the species against different insecticides was assessed by using the World Health Organization (WHO) standard bioassay tests. Results: The results showed that there was coexistence among Ae. aegypti and Ae. albopictus populations and the aggregation of their eggs was also observed in all the localities studied in Rawalpindi. Larval bioassays of both the populations exhibited incipient resistance against temephos while adult susceptibility testing results showed that both the species were resistant to DDT, malathion, bendiocarb and permethrin. Interpretation & conclusion: The results suggested that all the field populations of Ae. aegypti and Ae. albopictus existed together and showed qualitative changes in their susceptibility status. Resistance against deltamethrin and lambdacyhalothrin was not confirmed and further investigation was recommended to confirm the change in their susceptibility status. This study could help public health authorities to apply simultaneous control activities on both species due to their coexistence and also resistance management strategies should be formulated to slow down the process of development of resistance. Aedes aegypti; Aedes albopictus; dengue; insecticide susceptibility; spatial distribution INTRODUCTION Dengue has become a major concern for public health in the last decade in Pakistan. It is an arthropod borne viral disease. The dengue virus comprises of five serotypes, DEN-1 to 4 1 and DEN-5 2. In spite of the fact that all the five serotypes are antigenically similar, they vary in their response to produce cross- immunity for only a few months, after infection by any one of them 3. Dengue virus is transmitted through the bites of two main vector species namely Aedes aegypti (Linnaeus 1762) and Ae. albopictus (Skuse 1894) of Family: Culicidae 4. The disease is a major threat to public health in tropics and subtropics of the world, where about million cases are reported per year with severe cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) reaching to a level of 500,000, which makes it the most common mosquito-borne viral disease in the world 5. According to a recent estimate of 2015, there are about 390 million dengue infections per year and around 3.9 billion people in 128 countries are reported to be at risk 6. In Asia, the first outbreak of DHF occurred in Thailand and Philippines in 1950s. But, in the next 20 yr, the disease spread throughout Southeast Asia 7. Dengue fever epidemics were common in Asia and Pacific throughout the 20th century 7. In Pakistan, 40,987 cases of dengue fever were reported with 490 deaths during the year In August 2013, dengue outbreak occurred in Khyber Pakhtunkhwa province, infecting >7000 people including 26 deaths 7. In Rawalpindi district, there were about 1100 and 1406 confirmed cases of dengue fever in the year 2013 and 2014 respectively. The first confirmed

2 Arslan et al: Spatial distribution and insecticide susceptibility status of dengue vector mosquitoes in Rawalpindi, Pakistan 137 case of dengue fever in Rawalpindi in the year 2015 was reported on August 9; while from August till December 31 around 3900 confirmed cases including seven deaths were reported from the district. Multan district also witnessed an increase in dengue fever cases as 361 cases were reported in 2015 while in Lahore the total reported cases of dengue fever were The best plan of action to prevent the disease from further spread is to control the vector populations as there is no vaccine and specific treatment available to combat this disease 9. Chemical control is the most common method employed worldwide which targets the immature stages of the Aedes mosquito, breeding in artificial containers near human habitations. Among chemical control methods, chemical larvicides are most commonly used to target the larval populations of Ae. aegypti and Ae. albopictus in their potential breeding sites Latin American countries also employ chemical insecticides as a major control method in limiting the transmission of DF Resistance to insecticides has been recorded in Aedes species in Asia including Thailand 14 15, Cambodia 16, Malaysia 17 and India 18 and South America In Pakistan, DDT and malathion were used as the most important insecticides in malaria control programme in the year and respectively 21. According to WHO pesticide evaluation scheme (WHOPES), organophosphates and pyrethroids are the most commonly used insecticides against malaria and dengue vectors in Pakistan 22. Resistance to various insecticides against Anopheles mosquitoes and Ae. albopictus has been reported in Pakistan In order to carry out proper entomological activities, it is very essential to examine the biotic behaviour of these vector species and how they develop the resistance 25. This study focuses on the spatial distribution, abundance and aggregation of Ae. aegypti and Ae. albopictus populations and their susceptibility status to WHO recommended diagnostic concentrations of various insecticides, in localities with differentiated urban characteristics where dengue is prevalent. MATERIAL & METHODS Study area Ovitraps, each containing 0.5 L of 10% hay solution in glass jar (opening 7.8 cm, base diam 6.5 cm, height 9 cm) were randomly placed near human and animal dwellings at potential breeding spots of Aedes larvae in each of the four localities of Rawal Town, Pothohar Town, Satellite Town and Cantonment of Rawalpindi, Pakistan. Consent was taken from household owners prior to the placement of ovitraps in all the municipalities. Ethical approval was obtained from the Institutional Ethics and Disciplinary Committee, Department of Medical Entomology and Disease Vector Control (MEDVC), Health Services Academy (HSA), Islamabad, Pakistan (No. E & DC-149-HSA/2014). These municipalities reported several cases of dengue fever during August to December 2014 and Ovitraps were randomly placed at a distance of 850 m from each other for a period of one week in the residential neighbourhood of urban areas with established evidences of dengue or Aedes species prevalence 28. Study period The study was conducted from August to December Criteria for the study of Aedes population Aedes population was analyzed to estimate spatial distribution, randomness and aggregation of the eggs per ovitrap, calculated by using index of dispersion or variance to mean ratio as per the following equation 29 : I = S 2 /m^ Whereas, I is for index of dispersion; S 2 for variance; and m^ for mean. The criteria for evaluating the variance to mean ratio is as follows: (a) values < 1 exhibit uniform/regular spatial arrangement; (b) values equivalent to one exhibit random spatial arrangement; and (c) values substantially higher than one exhibit highly aggregated arrangement 29. Criteria for evaluating the k-values of the negative binomial distribution in order to analyze the aggregation index 29 was as follows: (a) negative k-values exhibit uniform distribution; (b) low and positive k-values (k < 2) exhibit highly aggregated arrangement; (c) k-values ranging from two to eight exhibit moderate aggregation; and (d) k-values above eight (k > 8) exhibit random arrangement. Mosquito sampling The ovitraps were brought to the insectary of Medical Entomology and Disease Vector Control (MEDVC), Department at Health Services Academy, Islamabad, Pakistan where the eggs of each ovitrap were counted (no species identification yet) and placed one by one in steel trays ( cm) containing water for hatching. Larvae were reared in steel trays and fed on sterilized broiler chicken liver diet to get the adult population. Pictorial keys of Rueda 30 were used to identify the adults of Ae. aegypti and Ae. albopictus. The emerging adults of each species were kept separately in cages and fed on white albino mice to acquire the F1 generation to be used

3 138 J Vector Borne Dis 53, June 2016 in insecticide susceptibility bioassays. Aedes mosquito populations were provided 10% glucose solution and kept in insectaries maintained at constant temperature of 27 ± 2 C and relative humidity of 80 ± 10% 31. Insecticides Larval bioassays were carried out by using WHO recommended diagnostic dosages of 0.02 mg/l for temephos (50% E.C. Temeguard ) and adult bioassays at WHO recommended diagnostic dosages using control and test papers of different insecticides, namely organochlorine (DDT), organophosphorus (malathion), carbamates (bendiocarb) and pyrethroids (permethrin, deltamethrin and lambdacyhalothrin). Insecticide impregnated papers of various diagnostic dosages procured from University Sains Malaysia were utilized for determination of resistance to DDT (4%), malathion (5%), bendiocarb (0.1%), permethrin (0.75%), deltamethrin (0.05%), and lambdacyhalothrin (0.05%). Larval bioassays Larval bioassays were conducted separately for both Ae. aegypti and Ae. albopictus by transferring 30 late III or early IV instar larvae with the help of droppers in small disposable test cups which contain 99 ml of water and 1 ml of temephos diagnostic concentration of 0.02 mg/l. Each bioassay was comprised of four replicates and one control group. Mortality was estimated after 24 h of temephos exposure. Larvae were considered dead when they were incapable of reaching to the water surface after being touched. Each bioassay was repeated four times on separate days and was conducted at a temperature of 27 ± 2 C, relative humidity of 80 ± 10% and a photoperiod of 12 : 12 h 32. Interpretation of larval susceptibility tests The criterion of Davidson and Zahar 33 was used to evaluate the qualitative modifications in susceptibility status of studied populations. A percent mortality > 98% against the diagnostic concentration indicates susceptible status; mortality between 80 and 98% indicates incipient resistance status; while percent mortality < 80% confirms the resistance status. Adult bioassays About 150 active female mosquitoes of each species were transferred to six exposure tubes (100 in four exposure tubes lined with insecticide impregnated papers and 50 in two control tubes with oil impregnated papers) separately against WHO recommended diagnostic dosages of each insecticide for one hour. The four replicates of each vector species containing 25 female mosquitoes per replicate were set up simultaneously for each insecticide. Control replicates were also held concurrent to each test. After exposure for one hour the mosquitoes were transferred to six holding tubes for recovery. During this recovery time period, the holding tubes were kept in cool, dark and shady places immediately, at room temperature of 27 ± 2 o C and relative humidity of 80% ± 10 o C 34. Cotton pads soaked in 10% glucose solution were provided as supplementary food during recovery time period for 24 h. The percent mortalities were computed by calculating the dead and alive mosquitoes after 24 h of recovery time period and Abbott s 35 formula, if needed, was used for its correction. Interpretation of adult susceptibility tests Adult susceptibility tests were evaluated by following the WHO recommended criteria as: (a) mortality in the range of % indicates susceptibility; (b) mortality of < 98% is suggestive of the development of resistance and further investigation is needed; (c) if the observed mortality (corrected if necessary) is between 90 and 97%, the presence of resistant genes in the vector population must be confirmed; and (d) if mortality is < 90%, resistance is confirmed 34. RESULTS Of the 240 ovitraps used in the study, 62.5% were positive for eggs (5192), and rearing resulted in emergence of 3484 adults (46% Ae. aegypti and 54% Ae. albopictus). The number of positive ovitraps and the percentage emergence of adult males and females of both species in different localities of Rawalpindi district are shown in Table 1. The index of dispersion (variance to mean ratio) values for the eggs of Ae. aegypti and Ae. albopictus were significantly >1 thus, exhibiting highly aggregated distribution in each of the localities analyzed (irrespective of the species identified). Likewise the k-values acquired from the negative binomial distribution also exhibited aggregated distribution of egg samples in all the localities investigated and their values were always positive ranging between zero and two as shown in Table 2. Larval bioassays The results of larval bioassays showed that percent mortality of Ae. aegypti and Ae. albopictus varied from and for Rawal Town, and for Pothohar Town, and for Satellite Town and 82.5 and for Cantonment area respectively. Table 3 presents the susceptibility status of Ae. aegypti and

4 Arslan et al: Spatial distribution and insecticide susceptibility status of dengue vector mosquitoes in Rawalpindi, Pakistan 139 Table 1. Aedes aegypti and Ae. albopictus collected with ovitraps in the localities of Rawal Town, Pothohar Town, Satellite Town and Cantonment area of Rawalpindi, Pakistan Localities Ovitraps Eggs Adults Emergence Aedes Female Sex Aedes Female Sex Ae. aegypti and installed/ (No.) (No.) (%) aegypti ratio albopictus ratio Ae. albopictus positive ratio Rawal Town 60/ : 0.71 Pothohar Town 60/ : 1 Satellite Town 60/ : 1 Cantonment area 60/ : 1 Table 2. Index of dispersion and k-values of negative binomial distribution exhibiting spatial oviposition arrangement of Aedes species in the different localities of Rawalpindi district Name of localities Index of dispersion k-value Rawal Town Pothohar Town Satellite Town Cantonment area Ae. albopictus larvae collected from different localities of Rawalpindi against WHO suggested diagnostic concentration of temephos (0.02 mg/l). The results of diagnostic dose tests revealed that larvae of Ae. aegypti and Ae. albopictus collected from all the localities of Rawalpindi showed incipient resistance according to the criterion of Davidson and Zahar 33. Adult bioassays After 24 h post-exposure, all the field populations of Ae. aegypti showed resistance to DDT, malathion, bendiocarb and permethrin with percent mortality rates ranging from 59 to 67.66, 64 to 79, 66 to 71 and 67 to 77 respectively, while percent mortality results of Ae. albopictus field populations ranged from 37 to 53 for DDT, 61 to 76 for malathion, 64 to 71 for bendiocarb and 62 to 74 for permethrin. Percent mortality rates in case of deltamethrin suggested probable resistance in all the field populations of Ae. aegypti (91.67 to 93.33) and Ae. albopictus (90.33 to 95). Probable resistance was also detected against lambdacyhalothrin in all the field populations of Ae. aegypti (92 to 96) and Ae. albopictus (90 to 96). Abbot s formula was not needed as control mortalities were less than 5%. Percent mortality and insecticide susceptibility status of both species against various insecticides are shown in Table 4. DISCUSSION This study provided an update on the current spatial distribution and insecticide resistance status of Ae. aegypti and Ae. albopictus. Both the species coexisted and the relative prevalence of Ae. aegypti and Ae. albopictus in all the localities of Rawalpindi was observed to be 46% and 54% respectively. Ae. albopictus was thought to have Table 3. Insecticide susceptibility status of the late III or early IV instar of Aedes aegypti and Ae. albopictus against temephos in different localities of Rawalpindi, Pakistan Name of localities Species No. of larvae exposed No. of larvae dead Average Susceptibility Test Control Test Control mortality (%) status Rawal Town Ae. aegypti IR * Ae. albopictus IR * Pothohar Town Ae. aegypti IR * Ae. albopictus IR * Satellite Town Ae. aegypti IR * Ae. albopictus IR * Cantonment area Ae. aegypti IR * Ae. albopictus IR * MEDVC Lab- Ae. aegypti S Pakistan population Ae. albopictus S S: Susceptible, if % observed mortality; IR * : 80 97% observed mortality suggests incipient resistance; WHO diagnostic concentration of (0.02 mg/l).

5 140 J Vector Borne Dis 53, June 2016 Table 4. Insecticide susceptibility status of the adults of Aedes aegypti and Ae. albopictus in different localities of Rawalpindi, Pakistan Name of locality Insecticide papers Ae. aegypti Mortality Susceptibility Ae. albopictus Mortality Susceptibility used in percent mosquitoes exposed of Ae. status mosquitoes exposed of Ae. status concentration aegypti albopictus Test Control (%) Test Control (%) Rawal Town DDT R R Malathion R R Bendiocarb R R Deltamethrin PR PR Permethrin R R Lambdacyhalothrin PR PR Pothohar Town DDT R R Malathion R R Bendiocarb R R Deltamethrin PR PR Permethrin R R Lambdacyhalothrin PR PR Satellite Town DDT R R Malathion R R Bendiocarb R R Deltamethrin PR PR Permethrin R R Lambdacyhalothrin PR PR Cantonment area DDT R R Malathion R R Bendiocarb R R Deltamethrin PR PR Permethrin R R Lambdacyhalothrin PR PR MEDVC Lab DDT PR PR Pakistan Malathion S S population Bendiocarb S S Deltamethrin S S Permethrin S S Lambdacyhalothrin S S S: Susceptible, if % observed mortality; PR: Probable resistance, if 90 97% observed mortality suggests the possibility of resistance that needs to be further confirmed; R: Resistant, if < 90% observed mortality. a rural background but the increase in its population in major dengue affected urban areas was an alarming factor observed in this study. Franco-Road and Crag Jr 36 reported difficulty in controlling the spread of Ae. albopictus population possibly due to vast distribution of its natural and artificial breeding habitats. Chiaravalloti et al 37 observed the frequent occurrence Ae. albopictus in peripheral urban areas. Braks et al 38 reported similar distribution of both the species in the outskirts of Rio and Florida river. Ae. aegypti and Ae. albopictus exhibited coexistence in all the localities of Rawalpindi district. Our results of spatial distribution, abundance, randomness and aggregation of Ae. aegypti and Ae. albopictus eggs were similar to that observed by Fantinatti et al 39, Prophiro et al 40 and Gomez et al 41. Bioassays with larvicide temephos suggested incipient resistance in dengue vectors in all the four localities of Rawalpindi. This might be due to the fact that temephos is the only larvicide used in malaria and dengue control programmes since 1969 up till now 42. Arslan et al also reported moderate type of temephos resistance in Ae. aegypti from three localities of Rawalpindi 43. Prolonged use of temephos and lack of resistance management strategies in the vector control programme in Pakistan might be the limiting factors in increasing tolerance against this larvicide in its resistance. The adult bioassays indicated that all the field populations of Ae. aegypti and Ae. albopictus of Rawalpindi were resistant to DDT, malathion, bendiocarb and permethrin. Resistance against deltamethrin and lambdacyhalothrin was not confirmed and further investigations are needed as percent mortal-

6 Arslan et al: Spatial distribution and insecticide susceptibility status of dengue vector mosquitoes in Rawalpindi, Pakistan 141 ity varied from 90 to 96%. Khan et al 24 also reported moderate to high level of resistance to carbamates, organophosphates and pyrethroids in field populations of Ae. albopictus in Lahore, Sargodha and Faisalabad. Major classes of insecticides of agricultural and public health importance have already been subjected to high level of resistance in Anopheles culicifacies and An. stephensi as reported by Rathor et al 23. This study reports the first known occurrence of high level of resistance in major dengue vector species, Ae. aegypti and Ae. albopictus against insecticides of public health importance. According to the Directorate General of Pest Warning and Quality Control of Pesticides, Punjab, the major insecticides used in agriculture are organophosphates, carbamates and pyrethroids 44. Moreover organophosphates, carbamates and pyrethroids are commonly used for the control of cotton insect pests in Punjab, Pakistan 45. There might be a spillover of insecticide resistance from agricultural sector as insecticide residues have the ability to drift into mosquito breeding sites after pesticide treatments in agricultural crops 46. Vigorous carpet fogging and intense indoor residual spraying with pyrethroids in case of emergency case response, as standard operating procedures (SoPs), might also be causing the increase in resistance in major dengue vectors in Rawalpindi 47. Resistance to pyrethroids has generally been associated with DDT as reported by Ranson et al 48. Brogdon et al 49 also reported cross resistance between pyrethroids (deltamethrin) and organophosphates (fenitrothion) in An. albimanus which might endanger the future vector control programmes. The resistance pattern of Ae. aegypti and Ae. albopictus populations against various insecticides has also been previously reported in Cambodia 16, Venezuela 50, Italy 51, Malaysia 52 55, Thailand14 15, and India 18, CONCLUSION This is the first study to examine the coexistence and susceptibility status of Ae. aegypti and Ae. albopictus against different groups of insecticides in Rawalpindi district, Pakistan. There is a need for continuous surveillance and vector control activities as dengue outbreaks have become more common in Pakistan in the past decade. This study revealed resistance in both Ae. aegypti and Ae. albopictus populations against various insecticides in Rawalpindi, Pakistan. The observations could help public health authorities to formulate appropriate measures to counter reductions in effectiveness of vector control efforts that may lead towards an emerging problem of insecticide resistance. Due to coexistence of Ae. aegypti and Ae. albopictus populations, constant vector control measures and resistance management strategies should be applied on both dengue vector species. Further, biochemical and molecular studies are also recommended to assess the processes involved in development of insecticide resistance among arthropod borne viral disease vectors in Pakistan. ACKNOWLEDGEMENTS The authors are grateful to Ms. Sadia and Ms. Mehwish, District Entomologists of Rawalpindi for providing logistic support for field activities. The authors are also thankful to the former Executive District Officer (Health), Rawalpindi, Dr Zafar Iqbal Gondal for providing manpower for conducting field activities. The authors are thankful to insectary technicians of MEDVC, Mr. Shaheen Akhtar and Mr. Syed Nawaz Husain Bukhari. REFERENCES 1. Dengue and severe dengue: Fact sheet. 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