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 41 and DEN-52. 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 them3. Dengue virus is trans- mitted through the bites of two main vector species namely Aedes aegypti (Linnaeus 1762) and Ae. albopictus (Skuse 1894) of Family: Culicidae4.
The disease is a major threat to public health in tropics and subtropics of the world, where about 50–100 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 world5. 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 risk6.
In Asia, the first outbreak of DHF occurred in Thai- land and Philippines in 1950s. But, in the next 20 yr, the disease spread throughout Southeast Asia7. Dengue fe- ver epidemics were common in Asia and Pacific through- out the 20thcentury7. In Pakistan, 40,987 cases of den- gue fever were reported with 490 deaths during the year 2006–116. In August 2013, dengue outbreak occurred in Khyber Pakhtunkhwa province, infecting >7000 people including 26 deaths7. In Rawalpindi district, there were about 1100 and 1406 confirmed cases of dengue fever in the year 2013 and 2014 respectively. The first confirmed
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
41Department of Medical Entomology and Disease Vector Control (MEDVC), Health Services Academy, Quaid-e-Azam University, Islamabad;
2University of Veterinary and Animal Sciences, Lahore; 3Agriculture Department, Pest Warning and Quality Control of Pesticides, Government of Punjab; 4Department of Anthropology, Quaid-e-Azam University, Islamabad, Pakistan
ABSTRACT
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.
Key words Aedes aegypti; Aedes albopictus; dengue; insecticide susceptibility; spatial distribution
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 wit- nessed an increase in dengue fever cases as 361 cases were reported in 2015 while in Lahore the total reported cases of dengue fever were 1468.
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 disease9. Chemical control is the most common method employed worldwide which targets the immature stages of the Aedes mosquito, breeding in artificial con- tainers 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 sites10–11. Latin American countries also employ chemical insecticides as a major control method in limiting the transmission of DF12–13. Resistance to insecticides has been recorded in Aedes species in Asia including Thailand14–15, Cambo- dia16, Malaysia17 and India18 and South America19–20 .
In Pakistan, DDT and malathion were used as the most important insecticides in malaria control programme in the year 1969–79 and 1984–96 respectively21. Accord- ing to WHO pesticide evaluation scheme (WHOPES), organophosphates and pyrethroids are the most commonly used insecticides against malaria and dengue vectors in Pakistan22. Resistance to various insecticides against Anopheles mosquitoes and Ae. albopictus has been re- ported in Pakistan23–24.
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 resistance25. This study focuses on the spatial distribution, abundance and aggregation of Ae. aegypti and Ae. albopictus populations and their susceptibility status to WHO recommended di- agnostic concentrations of various insecticides, in locali- ties with differentiated urban characteristics where den- gue 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 dwellings26–27 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 mu-
nicipalities. Ethical approval was obtained from the In- stitutional Ethics and Disciplinary Committee, Depart- ment of Medical Entomology and Disease Vector Con- trol (MEDVC), Health Services Academy (HSA), Islamabad, Pakistan (No. E & DC-149-HSA/2014). These municipalities reported several cases of dengue fever dur- ing August to December 2014 and 2015. 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 prevalence28.
Study period
The study was conducted from August to December 2014.
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 vari- ance to mean ratio as per the following equation29:
I = S2/m^
Whereas, I is for index of dispersion; S2 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 arrangement29. Criteria for evaluating the k-values of the negative bino- mial distribution in order to analyze the aggregation index29 was as follows: (a) negative k-values exhibit uniform dis- tribution; (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 Medi- cal 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 (24 × 36 × 6 cm) containing water for hatch- ing. Larvae were reared in steel trays and fed on steril- ized broiler chicken liver diet to get the adult population.
Pictorial keys of Rueda30 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
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 rec- ommended diagnostic dosages of 0.02 mg/l for temephos (50% E.C. Temeguard®) and adult bioassays at WHO rec- ommended diagnostic dosages using control and test pa- pers 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 resis- tance to DDT (4%), malathion (5%), bendiocarb (0.1%), permethrin (0.75%), deltamethrin (0.05%), and lambda- cyhalothrin (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 photope- riod of 12 : 12 h32.
Interpretation of larval susceptibility tests
The criterion of Davidson and Zahar33 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 expo- sure tubes lined with insecticide impregnated papers and 50 in two control tubes with oil impregnated papers) sepa- rately against WHO recommended diagnostic dosages of each insecticide for one hour. The four replicates of each
vector species containing 25 female mosquitoes per repli- cate were set up simultaneously for each insecticide. Con- trol 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 ± 2oC and relative humidity of 80% ± 10oC34. 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’s35 formula, if needed, was used for its correction.
Interpretation of adult susceptibility tests
Adult susceptibility tests were evaluated by follow- ing the WHO recommended criteria as: (a) mortality in the range of 98–100% indicates susceptibility; (b) mor- tality of < 98% is suggestive of the development of resis- tance 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 confirmed34.
RESULTS
Of the 240 ovitraps used in the study, 62.5% were positive for eggs (5192), and rearing resulted in emer- gence of 3484 adults (46% Ae. aegypti and 54% Ae.
albopictus). The number of positive ovitraps and the per- centage 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) val- ues for the eggs of Ae. aegypti and Ae. albopictus were significantly >1 thus, exhibiting highly aggregated dis- tribution 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 locali- ties 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 81.25 and 83.12 for Rawal Town, 83.96 and 86.67 for Pothohar Town, 86.87 and 88.12 for Satellite Town and 82.5 and 84.37 for Cantonment area respectively. Table 3 presents the susceptibility status of Ae. aegypti and
Ae. albopictus larvae collected from different localities of Rawalpindi against WHO suggested diagnostic con- centration of temephos (0.02 mg/l). The results of diag- nostic 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 Zahar33.
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 popu- lations of Ae. aegypti (92 to 96) and Ae. albopictus (90 to 96). Abbot’s formula was not needed as control mortali- ties were less than 5%. Percent mortality and insecticide susceptibility status of both species against various in- secticides 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 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 92.03 1.19
Pothohar Town 78.22 0.63
Satellite Town 64.47 1.52
Cantonment area 85.16 0.96
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 480 120 390 3 81.25 IR*
Ae. albopictus 480 120 399 2 83.12 IR*
Pothohar Town Ae. aegypti 480 120 403 4 83.96 IR*
Ae. albopictus 480 120 416 2 86.67 IR*
Satellite Town Ae. aegypti 480 120 417 3 86.87 IR*
Ae. albopictus 480 120 423 3 88.12 IR*
Cantonment area Ae. aegypti 480 120 396 2 82.5 IR*
Ae. albopictus 480 120 405 5 84.37 IR*
MEDVC Lab- Ae. aegypti 480 120 478 0 99.58 S
Pakistan population Ae. albopictus 480 120 478 0 99.58 S
S: Susceptible, if 98–100% observed mortality; IR*: 80–97% observed mortality suggests incipient resistance; WHO diagnostic concentration of (0.02 mg/l).
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 60/41 1493 1012 68 591 311 0.53 421 215 0.51 1 : 0.71
Pothohar Town 60/37 1231 869 71 306 185 0.60 563 311 0.55 0.98 : 1
Satellite Town 60/32 1046 698 67 282 156 0.55 416 201 0.48 0.68 : 1
Cantonment area 60/40 1422 905 63 435 245 0.56 470 254 0.54 0.93 : 1
a rural background but the increase in its population in major dengue affected urban areas was an alarming fac- tor observed in this study. Franco-Road and Crag Jr36 re- ported difficulty in controlling the spread of Ae. albopictus population possibly due to vast distribution of its natural and artificial breeding habitats. Chiaravalloti et al37 ob- served the frequent occurrence Ae. albopictus in periph- eral urban areas. Braks et al38 reported similar distribu- tion 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 aggre- gation of Ae. aegypti and Ae. albopictus eggs were simi- lar to that observed by Fantinatti et al39, Prophiro et al40 and Gomez et al41.
Bioassays with larvicide temephos suggested incipi- ent 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 now42. Arslan et al also reported moderate type of temephos resistance in Ae.
aegypti from three localities of Rawalpindi43. Prolonged use of temephos and lack of resistance management strat- egies 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-
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 300 150 67.66 R 300 150 53 R
Malathion 300 150 77.67 R 300 150 76 R
Bendiocarb 300 150 71 R 300 150 71 R
Deltamethrin 300 150 93.33 PR 300 150 95 PR
Permethrin 300 150 75.33 R 300 150 74 R
Lambdacyhalothrin 300 150 95 PR 300 150 96 PR
Pothohar Town DDT 300 150 64 R 300 150 43 R
Malathion 300 150 74 R 300 150 73 R
Bendiocarb 300 150 68 R 300 150 65 R
Deltamethrin 300 150 91.67 PR 300 150 90.33 PR
Permethrin 300 150 74 R 300 150 71 R
Lambdacyhalothrin 300 150 92 PR 300 150 94 PR
Satellite Town DDT 300 150 66 R 300 150 47 R
Malathion 300 150 79 R 300 150 73 R
Bendiocarb 300 150 70 R 300 150 64 R
Deltamethrin 300 150 92 PR 300 150 91 PR
Permethrin 300 150 77 R 300 150 74 R
Lambdacyhalothrin 300 150 96 PR 300 150 93 PR
Cantonment area DDT 300 150 59 R 300 150 37 R
Malathion 300 150 64 R 300 150 61 R
Bendiocarb 300 150 66 R 300 150 67 R
Deltamethrin 300 150 92 PR 300 150 92 PR
Permethrin 300 150 67 R 300 150 62 R
Lambdacyhalothrin 300 150 93 PR 300 150 90 PR
MEDVC Lab– DDT 300 150 94 PR 300 150 94 PR
Pakistan Malathion 300 150 100 S 300 150 100 S
population Bendiocarb 300 150 100 S 300 150 100 S
Deltamethrin 300 150 100 S 300 150 100 S
Permethrin 300 150 100 S 300 150 100 S
Lambdacyhalothrin 300 150 100 S 300 150 100 S
S: Susceptible, if 98–100% 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.
ity varied from 90 to 96%. Khan et al24 also reported mod- erate to high level of resistance to carbamates, organo- phosphates 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 al23. 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 Warn- ing and Quality Control of Pesticides, Punjab, the major insecticides used in agriculture are organophosphates, car- bamates and pyrethroids44. Moreover organophosphates, carbamates and pyrethroids are commonly used for the control of cotton insect pests in Punjab, Pakistan45. There might be a spillover of insecticide resistance from agri- cultural sector as insecticide residues have the ability to drift into mosquito breeding sites after pesticide treatments in agricultural crops46. Vigorous carpet fogging and in- tense indoor residual spraying with pyrethroids in case of emergency case response, as standard operating proce- dures (SoPs), might also be causing the increase in resis- tance in major dengue vectors in Rawalpindi47. Resis- tance to pyrethroids has generally been associated with DDT as reported by Ranson et al48. Brogdon et al49 also reported cross resistance between pyrethroids (deltamethrin) and organophosphates (fenitrothion) in An.
albimanus which might endanger the future vector con- trol programmes. The resistance pattern of Ae. aegypti and Ae. albopictus populations against various insecti- cides has also been previously reported in Cambodia16, Venezuela50, Italy51, Malaysia52–55, Thailand14–15, 56–57
and India18, 58–59.
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 dis- trict, Pakistan. There is a need for continuous surveil- lance and vector control activities as dengue outbreaks have become more common in Pakistan in the past de- cade. This study revealed resistance in both Ae. aegypti and Ae. albopictus populations against various insecti- cides in Rawalpindi, Pakistan. The observations could help public health authorities to formulate appropriate mea- sures to counter reductions in effectiveness of vector con- trol 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, bio- chemical and molecular studies are also recommended to assess the processes involved in development of insecti- cide resistance among arthropod borne viral disease vec- tors in Pakistan.
ACKNOWLEDGEMENTS
The authors are grateful to Ms. Sadia and Ms.
Mehwish, District Entomologists of Rawalpindi for pro- viding logistic support for field activities. The authors are also thankful to the former Executive District Officer (Health), Rawalpindi, Dr Zafar Iqbal Gondal for provid- ing manpower for conducting field activities. The authors are thankful to insectary technicians of MEDVC, Mr.
Shaheen Akhtar and Mr. Syed Nawaz Husain Bukhari.
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