Genetic Analysis of Aedes aegypti using Random Amplified Polymorphic DNA (RAPD) Markers from Dengue Outbreaks in Pakistan
AbstractBackground: Keeping in view the havoc situation of dengue fever in Pakistan, the current study was designed to demonstrate the genetic variations, gene flow and rate of migration from Lahore and Faisalabad.Methods: The larvae were collected from both natural and artificial breeding places from each collection site. The adult mosquitoes were collected by means of sweep net and battery-operated aspirator. DNA extraction was performed using TNE buffer method. Ten GeneLink-A series RAPD primers were used for PCR amplification and the data was analyzed through POPGENE.Results: The number of amplification products produced per primer varied from 8-12, ranging from 200 to 2000 bp with an average of 10.0 bands per primer. The percentage of polymorphic loci amplified by each primer varied from 22.5 to 51%. The UPGMA dendrogram demonstrates two distinct groups from Faisalabad and Lahore populations. The genetic diversity ranged from 0.260 in Faisalabad to 0.294 in Lahore with a total heterozygosity of 0.379. The GST value for nine populations within Lahore was 0.131 (Nm= 3.317), whereas for nine populations in Faisalabad GST value was 0.117 (Nm= 3.773). The overall genetic variation among eighteen populations showed GST= 0.341 and Nm= 1.966.Conclusion: The genetic relatedness and Nm value show that Ae. aegypti populations exhibit intra-population gene flow both in Faisalabad and Lahore. Although, both cities show a distinct pattern of genetic structure; however, few areas from both the cities show genetic similarity. The gene flow and the genetic relatedness in few populations of Lahore and Faisalabad cities need further investigation.
Apostol BL, Black 4th WC, Reiter P, Miller BR (1996) Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico. Heredity. 76: 325–334.
Ashfaq M, Hebert PDN, Mirza JH, Khan AM, Zafar Y, Mirza MS (2014) Ana- lyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Bar- coding. PLoS ONE. 9(5): 1–12.
Ashraf HM (2013) Studies on the comparative genetic analysis of Aedes aegypti pop- ulations from Lahore and Faisalabad using molecular DNA markers. MPhil thesis, Department of Zoology, Wild- life and Fisheries, Government Col- lege University Faisalabad, Pakistan.
Ayres CFJ, Melo-Santos MAV, Sole-Cava AM, Furtado AF (2003) Genetic dif- ferentiation of Aedes aegypti (Diptera: Culicidae), the major dengue vector in Brazil. J Med Entomol. 40: 430–435.
Ayres CFJ, Romao TPA, Melo-Santos MAV, Furtado AF (2002) Genetic diversity in Brazilian populations of Aedes al- bopictus. Mem Inst Oswaldo Cruz.97(6): 871–875.
Ballinger-Crabtree ME, Black WC, Miller B.R (1992) Use of genetic polymor- phisms detected by the random-am- plified polymorphic DNA polymerase chain reaction (RAPD-PCR) for dif- ferentiation and identification of Ae- des aegypti subspecies and popula- tions. Am J Trop Med Hyg. 47: 893–901.
Beebe NW, Whelan PI, Hurk AVD, Ritchie SA, Cooper RD (2005) Genetic diver- sity of the dengue vector Aedes ae- gypti in Australia and implications for future surveillance and mainland in- cursion monitoring. Commun Dis In- tell. 29: 299–304.
Bibi M, Zahoor MK, Zahoor MA, Ashraf HM, Majeed HN, Nasir S, Rasool B, 2015
Genetic analysis of mosquitoes from rural and urban areas of Sialkot, Paki- stan. Int J Agric Biol. 17(4): 809-814.
Bosio CF, Harrington LC, Jones JW, Sithiprasasna R, Norris DE, Scott TW (2005) Genetic structure of Aedes ae- gypti populations in Thailand using mitochondrial DNA. Am J Trop Med Hyg. 72: 434–442.
Bracco J, Capurro M, Lourenço-DE-Oliveira R, Sallum MA (2007) Genetic varia- bility of Aedes aegypti in the Ameri- cas using a mitochondrial gene: evi- dence of multiple introductions. Mem Inst Oswaldo Cruz. 102: 573–580.
Caldera SM, Jaramillo MC, Cochero S, Pé- rez-Doria A, Bejarano EE (2013) Ge- netic differences between populations of Aedes aegypti from municipalitiesin northern Colombia, with high and low dengue incidence. Biomédica. 33:89-98.
Cologna R, Armstrong PM, Rico-Hesse R (2005) Selection for virulent dengue viruses occurs in humans and mos- quitoes. J Virol. 79: 853–859.
Costa-da-Silva, ALD, Capurro ML, Bracco JE (2005) Genetic lineages in the yel- low fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Pe- ru. Memórias do Instituto Oswaldo Cruz, 100(6): 539-544.
de Patarro TF, Guirado MM, Ravazzi LM, de Bicudo H (2013) Genetic structure of Aedes aegypti populations deter- mined using pairwise comparisons. Genet Mol Res. 12(3): 3775-3787.
De Sousa GB, Blanco A, Gardenal CN (2001) Genetic relationships among Aedes aegypti (Diptera: Culicidae) popula- tions from Argentina using random amplified polymorphic DNA poly- merase chain reaction markers. J Med Entomol. 38(3): 371-375.
Failloux A-B, Darius H, Pasteur N (1995) Ge- netic differentiation of Aedes aegypti, the vector of dengue virus in French Polynesia. J Am Mosq Contr Assoc.11: 457–462.
Fatima Z, Idrees M, Bajwa MA, Tahir Z, Ullah O, Zia MQ, Hussain A, Akram M, Khubaib B, Afzal S, Munir S, Saleem S, Rauff B, Badar S, Naudhani M, Butt S, Aftab M, Ali L, Ali M (2011) Serotype and genotype analysis of dengue virus by sequenc- ing followed by phylogenetic analy- sis using samples from three mini out- breaks 2007–2009 in Pakistan. BMC Microbiol. 11: 200.
Florencio M, Díaz-Paniagua C, Gómez- Rodríguez C, Serrano L (2014) Bio- diversity patterns in a macroinverte- brate community of a temporary pond network. Insect Conserv. Diversity.7: 4–21.
Fradin MS, Day JF (2002) Comparative effi- cacy of insect repellents against mos- quito bite. N Eng J Med. 347(1): 13–18.
Fraga EC, Santos JMM, Maia JF (2003) En- zymatic variability in Aedes aegypti (Diptera: Culicidae) populations from Manaus- AM, Brazil. Genet Mol Bi- ol. 26: 181–187.
Franco FG, Munoz MDL, Fuentes SL, Salas LF, Rejon JG, Beaty BJ, Black WC (2002) Large genetic distances among Aedes aegypti populations along the south pacific coast of Mexico. AM J Trop Med Hyg. 6(5): 594–598.
Fulton, RE, Salasek ML, DuTeau NM, Black WC (2001) SSCP analysis of cDNA markers provides a dense link- age map of the Aedes aegypti ge- nome. Genetics. 158: 715–726.
Gibbons RV, Vaughn DW (2002) Dengue:an escalating problem. BMJ. 324:1563–1566.
Gilani I (2012) 2,340 malaria cases reported.Dawn News, June 04. Available at: http://dawn.com/news/407357/malaria- plagues-faisalabad.
Gorrochotegui-Escalant N, Munoz ML, Fer- nandez-Salas I, Beaty BJ and Black BJ (2000) Genetic isolation by dis- tance among Aedes aegypti popula- tions along the northeastern coast of Mexico. Am J Trop Med Hyg. 62:200–209.
Gorrochotegui-Escalante N, Gomez-Machorro C, Lozano- Fuentes S, Fernandez- Salas I, Munoz ML, Farlan-Ale JA, Garcia-Rejon J, Beaty BJ, Black WC (2002) Breeding structure of Aedes aegypti populations in Mexico varies by region. Am J Trop Med Hyg. 66:213–222.
Herrel N, Amerasinghe FP, Ensink J, Mukhtar M, Van Der Hoek W, Kon- radsen F (2001) Breeding of Anoph- eles mosquitoes in irrigated areas of South Punjab, Pakistan. Med Vet En- tomol. 15: 236–248.
Herrera F, Urdaneta L, Rivero J, Zoghbi N, Ruiz J, Carrasquel G, Martínez JA, Pernalete M, Villegas P, Montoya A, Rubio-Pais Y, Rojas E (2006) Popu- lation genetic structure of the dengue mosquito Aedes aegypti in Venezuela. Mem Inst Oswaldo Cruz. 101: 625–633.
Hiragi C, Simoes K, Martins E, Queiroz P, Lima L, Monnerat R (2009) Genetic variability in Aedes aegypti (L.) (Dip- tera: Culicidae) populations using RAPD markers. Neotrop Entomol.38: 542–547.
Hlaing T, Tun-Lin W, Somboon P, Socheat D, Setha T, Min S, Thaung S, Anyaele O, Silva BD, Chang MS, Prakash A, Linton Y, Walton C (2010) Spatial genetic structure of Aedes aegypti mos- quitoes in mainland Southeast Asia. Evol Applic. 3(4): 319–339.
Huber K, Le Loan L, Hoang TH, Ravel S, Rodhain F, Failloux AB (2002) Ge- netic differentiation of the dengue vector, Aedes aegypti (Ho Chi Minh City, Vietnam) using microsatellite markers. Mol Ecol. 11: 1629–1635.
Idrees S, Ashfaq UA (2102) A brief review on dengue molecular virology, diag- nosis, treatment and prevalence in Pakistan. Gen Vacc Ther. 10: 6.
Itrat A, Khan A, Javaid S, Kamal M, Khan H, Javed S, Kalia S, Khan AH, Sethi MI, Jehan I (2008) Knowledge, aware- ness and practices regarding dengue fever among the adult population of dengue hit cosmopolitan. PLoS ONE.3(7): 1–6.
Julio NB, Chiappero MB, Rossi HJ, Rondan Duenas JCR, Gardenal CN (2009) Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a re- cently reinfested area. Mem Inst Os- waldo Cruz. Rio de Janeiro. 104(4):626–631.
Khan E, Kisat M, Khan N, Nasir A, Ayub S, Hasan R (2010) Demographic and clinical features of dengue fever in Pakistan from 2003–2007: A retro- spective cross-sectional study. PLoS ONE. 5(9): 1–6.
Lerdthusnee K, Chareonviriyaphap T (2002) Genetic differentiation of Aedes ae- gypti mainland and island popula- tions from Southern Thailand. J Am Mosq Contr Assoc. 18: 173–177.
Morlais I, Severson DW (2003) Intraspecific DNA variation in nuclear genes of the mosquito Aedes aegypti. Insect Mol Biol. 12: 631–639.
Naeem-Ullah U, Akram W, Suhail A, Rana SA (2010) Grouping of different mosquito species on the bases of lar- val habitats. Pak J Agri Sci. 47(2):124–131.
Nei M (1978) Estimation of average hetero- zygosity and genetic distance from a small number of individuals. Genet- ics. 89: 583–590.
Nikookar SH, Moosa-Kazemi SH, Oshaghi MA, Vatandoost H, Kianinasab A (2010) Species composition and di- versity of mosquitoes in Neka County, Mazandaran Province, northern Iran. J Arthropod-Borne Dis. 4(2): 26–34.
Ocampo CB, Wesson DM (2004) Population dynamics of Aedes aegypti from a dengue hyperendemic urban setting in Colombia. Am J Trop Med Hyg.71(4): 506-513.
Paduan KS, Araújo-Júnior JP, Ribolla PEM (2006) Genetic variability in geograph- ical populations of Aedes aegypti (Dip- tera, Culicidae) in Brazil elucidated by molecular markers. Genet Mol Bi- ol. 29: 391–395.
Paupy C, Vazeille-Falcoz M, Mousson L, Rodhain F, Failloux AB (2000) Aedes aegypti in Tahiti and Moorea (French Polynesia): isozyme differentiation in the mosquito population according to human population density. Am J Trop Med Hyg. 62: 217–224.
Paupy C, Chantha N, Reynes JM, Failloux AB (2005) Factors influencing the population structure of Aedes aegypti from the main cities in Cambodia. Heredity. 95: 144–147.
Paupy C, Le Goff G, Brengues C, Guerra M, Revollo J, Simon ZB, Herve JP, Fon- tenille D (2012) Genetic structure and phylogeography of Aedes aegypti, the dengue and yellow-fever mosquito vec- tor in Bolivia. Infect Genet Evol.12(6): 1260–1269.
Pinheiro FP, Corber SJ (1997) Global situa- tion of dengue and dengue haemor- rhagic fever, and its emergence in the Americas. WHO Stat Q. 50: 161–169.
Raheel U, Faheem M, Riaz MN, Kanwal N, Javed F, Zaidi NS, Qadri I (2011) Dengue fever in the Indian subconti- nent: an overview. J Infect Dev Ctries.5(4): 239–247.
Rasheed SB, Boots M, Frantz AC, Butlin RK (2013) Population structure of the mosquito Aedes aegypti (Stego- myia aegypti) in Pakistan. Med Vet Entomol. 27: 430–440.
Ravel S, Herve JP, Diarrassouba S, Kone A, Cuny G (2002) Microsatellite mark- ers for population genetic studies in Aedes aegypti (Diptera: Culicidae) from Côte d’Ivoire: evidence for a micro- geographic genetic differentiation of mosquitoes from Bouaké. Acta Trop.82: 39–49.
Sajid A, Ikram A, Ahmed M (2012) Dengue fever outbreak 2011: clinical profile of children presenting at Madina teach- ing hospital Faisalabad. J Univ Med Dent Coll. 3 (1): 42–47.
Santos JMM, Fraga EC, Maia JF, Tadei WP (2011) Genetic Diversity in Dengue Mosquito, Aedes aegypti (Diptera: Cu- licidae) from Amazon Region: Com
-parative Analysis with Isozymes and RAPD Loci. The Open Trop Med J.4: 11–20.
Scarpassa VM, Cardoza TB, Cardoso-Júnior RP (2008) Population genetics and phylogeography of Aedes aegypti (Dip- tera, Culicidae) from Brazil. Am J Trop Med Hyg. 78: 895–903.
Severson DW, Zaitlin D, Kassner VA (1999) Targeted identification of markers linked to malaria and filarioid nem- atode parasite resistance genes in the mosquito Aedes aegypti. Genet Res.73: 217–224.
Severson DW, Meece JK, Lovin DD, Saha G, Morlais I (2002) Linkage map or- ganization of expressed sequence tags and sequence tagged sites in the mos- quito, Aedes aegypti. Insect Mol Biol.11: 371–378.
Shortall CR, Moore A, Smith E, Hall MJ, Woiwod IP, Harrington R (2009) Long-term changes in the abundance of flying insects. Insect Conserv Di- vers. 2: 251–260.
Siddiqui FJ, Haider S, Bhutta ZA (2009) En- demic Dengue Fever: a seldom rec- ognized hazard for Pakistani children. J Infect Dev Ctrie. 3(4): 306–312.
Souza GB de, Blanco A, Gardenal CN (2001) Genetic relationships among Aedes ae- gypti (Diptera: Culicidae) populations from Argentina using random ampli- fied polymorphic DNA polymerase chain reaction markers. J Med En- tomol. 38: 371–375.
Thavara U, Tawatsin A, Chansang C, Kong- ngamsuk W, Paosriwong S, Boon- Long J, Rongsriyam Y, Komalamisra N (2001) Larval occurrence, ovipo- sition behavior and biting activity of potential mosquito vectors of dengue on Samui Island, Thailand. J Vector Ecol. 26: 172–180.
Wallis GP, Tabachnick WJ, Powell J (1984) Genetic heterogeneity among Carib-bean populations of Aedes aegypti.Am J Trop Med Hyg. 33: 492–498. WHO (1997) Dengue hemorrhagic fever,
diagnosis, treatment and control, 2nd ed. World Health Organization, Ge- neva, Switzerland. WHO (2009) Den- gue guidelines for diagnosis, treat- ment, prevention and control. New edition. Geneva, Switzerland.
WHO (2013) Dengue Fever in Pakistan.Weekly Epidem. Monit. Available at:
-emro-weekly-epidemiological-moni- tor-volume-volume-6-issue-37-dengue- fever-0.
WHO (2014) Dengue and severe dengue. Fact sheet No117. Available at: http:// www. who.int/mediacentre/factsheets/ fs117/ en/#.
Yáñez P, Mamani E, Valle J, García MP, León W, Villaseca P, Torres D, Cab- ezas C (2013) Genetic variability of Aedes Aegypti determined by mito- chondrial gene ND4 analysis in elev- en endemic areas for dengue in Peru. Rev Peru Med Exp Salud Públi- ca. 30(2): 246–250.
Zahoor MK, Suhail A, Zahoor S, Iqbal A, Awan FS (2013) Molecular charac- terization of Scarab beetles (Scarabae- idae: Coleoptera) using RAPD mark- ers. Pak J Life Soc Sci. 11: 238–243.