Volume 76, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


A multiplex PCR assay has been developed for detection of cryptic species, their human host preference, and presence in the mosquito. PCR conditions were optimized using primer sets specific for cryptic species, and and evaluated with field-collected mosquitoes. A unique mosquito processing method was used for screening carrying capacity and human host preference of mosquitoes in first-round multiplex PCR. The vectorial status of the mosquito for parasite was confirmed in second-round PCR. Of the 121 collected mosquitoes, 92 were of S type, 26 of T type, and 3 were of other types. Human host preference was dominant in S type, of which 4% were sporozoite positive. This assay and processing method can also be used to evaluate vector competence of other anophelines.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. World Health Organization, 2003. The World Health Report 2003: Shaping the Future. Geneva: World Health Organization.
  2. World Health Organization andUnited Nations Children’s Fund, 2005. World Malaria Report 2005. Geneva: World Health Organization and UNICEF.
  3. Fontenille D, Louchouarn L, 1999. The complexity of the malaria vectorial system in Africa. Parassitologia 41 : 267–271. [Google Scholar]
  4. Subbarao SK, 1998. Anopheles Species Complexes in South East Asia Region. Technical Publication No. 18. New Delhi: World Health Organization, South East Asia Regional Office.
  5. Beebe NW, Saul A, 1995. Discrimination of all members of the Anopheles punctulatus complex by polymerase chain reaction-restriction fragment length polymorphism analysis. Am J Trop Med Hyg 53 : 478–481. [Google Scholar]
  6. Cornel AJ, Porter CH, Collins FH, 1996. PCR diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences. J Med Entomol 33 : 109–116. [Google Scholar]
  7. Kampen H, Sternberg A, Proft J, Bastian S, Schaffner F, Maier WA, Seitz HM, 2003. Polymerase chain reaction-based differentiation of the mosquito sibling species Anopheles claviger s.s. and Anopheles petragnani (Diptera: Culicidae). Am J Trop Med Hyg 69 : 195–199. [Google Scholar]
  8. Proft J, Maier WA, Kampen H, 1999. Identification of six sibling species of the Anopheles maculipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay. Parasitol Res 85 : 837–843. [Google Scholar]
  9. Sharpe RG, Hims MM, Harbach RE, Butlin RK, 1999. PCR based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism. Med Vet Entomol 13 : 265–273. [Google Scholar]
  10. Reimer LJ, Tripet F, Slotman M, Spielman A, Fondjo E, Lanzaro GC, 2005. An unusual distribution of the kdr gene among populations of Anopheles gambiae on the island of Bioko, Equatorial Guinea. Insect Mol Biol 14 : 683–688. [Google Scholar]
  11. Surendran SN, Ramasamy MS, De Silva BG, Ramasamy R, 2006. Anopheles culicifacies cryptic species B and E in Sri Lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides. Med Vet Entomol 20 : 153–156. [Google Scholar]
  12. Sharma VP, 1998. Fighting malaria in India. Curr Sci 75 : 1127–1140. [Google Scholar]
  13. Rao TR, 1984. The Anophelines of India. New Delhi: Malaria Research Centre, Indian Council of Medical Research.
  14. Subbarao SK, Nanda N, Vasantha K, Dua VK, Malhotra MS, Yadav RS, Sharma VP, 1994. Cytogenetic evidence for three sibling species in Anopheles fluviatilis (Diptera: Culicidae). Ann Entomol Soc Am 87 : 116–121. [Google Scholar]
  15. Nanda N, Joshi H, Subbarao SK, Yadav RS, Shukla RP, Dua VK, Sharma VP, 1996. Anopheles fluviatilis complex: host feeding pattern of species S, T and U. J Am Mosq Control Assoc 12 : 147–149. [Google Scholar]
  16. Nanda N, Yadav RS, Subbarao SK, Joshi H, Sharma VP, 2000. Studies on Anopheles fluviatilis and Anopheles culicifacies sibling species in relation with malaria in forested hilly and deforested riverine eco-systems in northern Orissa in India. J Am Mosq Control Assoc 16 : 199–205. [Google Scholar]
  17. Sharma SK, Nanda N, Dua VK, Joshi H, Subbarao SK, Sharma VP, 1995. Studies on the bionomics of Anopheles fluviatilis sensu lato and sibling species composition in the foothills of Shivalik range (Uttar Pradesh), India. Southeast Asian J Trop Med Public Health 26 : 566–572. [Google Scholar]
  18. Manonmani A, Townson H, Adeniran T, Jambulingam P, Sahu S, Vijayakumar T, 2001. rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis. Acta Trop 78 : 3–9. [Google Scholar]
  19. Singh OP, Chandra D, Nanda N, Raghavendra K, Sunil S, Sharma SK, Dua VK, Subbarao SK, 2004. Differentiation of members of the Anopheles fluviatilis species Complex by an allele-specific polymerase chain reaction based on 28s ribosomal dna sequences. Am J Trop Med Hyg 70 : 27–32. [Google Scholar]
  20. Edrissian GH, Manouchehry AV, Hafizi A, 1985. Application of an enzyme-linked immunosorbent assay (ELISA) for determination of the human blood index in anopheline mosquitoes collected in Iran. J Am Mosq Control Assoc 1 : 349–352. [Google Scholar]
  21. Savage HM, Duncan JF, Roberts DR, Sholdt LL, 1991. A dipstick ELISA for rapid detection of human blood meals in mosquitoes. J Am Mosq Control Assoc 7 : 16–23. [Google Scholar]
  22. Thapar BR, Sharma SN, Dasgupta RK, Kaul SM, Bali A, Chhabra K, Lal S, 1998. Blood meal identification by using Microdot ELISA in vector mosquitoes. J Commun Dis 30 : 283–287. [Google Scholar]
  23. Collins RT, Narasimham MV, Dhal KB, Mukherjee BP, 1991. Gel diffusion analysis of Anopheles bloodmeals from 12 malarious study villages of Orissa State, India. J Am Mosq Control Assoc 7 : 595–603. [Google Scholar]
  24. Kent RJ, Norris DE, 2005. Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome B. Am J Trop Med Hyg 73 : 336–342. [Google Scholar]
  25. Oshaghi MA, Chavshin AR, Vatandoost H, Yaaghoobi F, Mohtarami F, Noorjah N, 2006. Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes. Exp Parasitol 12 : 232–236. [Google Scholar]
  26. Appawu MA, Bosompem KM, Dadzie S, McKakpo US, Anim-Baidoo I, Dykstra E, Szumlas DE, Rogers WO, Koram K, Fryauff DJ, 2003. Detection of malaria sporozoites by standard ELISA and VecTest™ dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana. Trop Med Int Health 8 : 1012–1017. [Google Scholar]
  27. Arez AP, Lopes D, Pinto J, Franco AS, Snounou G, do Rosario VE, 2000. Plasmodium sp.: optimal protocols for PCR detection of low parasite numbers from mosquito (Anopheles sp.) samples. Exp Parasitol 94 : 269–272. [Google Scholar]
  28. Moreno M, Cano J, Nzambo S, Bobuakasi L, Buatiche JN, Ondo M, Micha F, Benito A, 2004. Malaria Panel Assay versus PCR: detection of naturally infected Anopheles melas in a coastal village of Equatorial Guinea. Malar J 3 : 20. [Google Scholar]
  29. Tassanakajon A, Boonsaeng V, Wilairat P, Panyim S, 1993. Polymerase chain reaction detection of Plasmodium falciparum in mosquitoes. Trans R Soc Trop Med Hyg 87 : 273–275. [Google Scholar]
  30. Wilson MD, Ofosu-Okyere A, Okoli AU, McCall PJ, Snounou G, 1998. Direct comparison of microscopy and polymerase chain reaction for the detection of Plasmodium sporozoites in salivary glands of mosquitoes. Trans R Soc Trop Med Hyg 92 : 482–483. [Google Scholar]
  31. Orlandi PA, Lampel KA, 2000. Extraction-free, filter-based template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. J Clin Microbiol 38 : 2271–2277. [Google Scholar]
  32. Christophers SR, 1933. The Fauna of British India. Dipter, Vol IV. Family-Culicidae. Tribe-Anophelini. New Delhi: Today and Tomorrow’s Printers and Publishers.
  33. Nagpal BN, Srivastava A, Saxena R, Ansari MA, Dash AP, Das SC, 2005. Pictorial Identification Key for Indian Anophelines. New Delhi: Malaria Research Centre (ICMR).
  34. Casimiro S, Coleman M, Mohloai P, Hemingway J, Sharp B, 2006. Insecticide resistance in Anopheles funestus (Diptera: Culicidae) from Mozambique. J Med Entomol 43 : 267–275. [Google Scholar]
  35. Casimiro S, Coleman M, Hemingway J, Sharp B, 2006. Insecticide resistance in Anopheles arabiensis and Anopheles gambiae from Mozambique. J Med Entomol 43 : 276–282. [Google Scholar]
  36. Kelly-Hope LA, Yapabandara AM, Wickramasinghe MB, Perera MD, Karunaratne SH, Fernando WP, Abeyasinghe RR, Siyambalagoda RR, Herath PR, Galappaththy GN, Hemingway J, 2005. Spatiotemporal distribution of insecticide resistance in Anopheles culicifacies and Anopheles subpictus in Sri Lanka. Trans R Soc Trop Med Hyg 99 : 751–761. [Google Scholar]
  37. Overgaard HJ, Sandve SR, Suwonkerd W, 2005. Evidence of anopheline mosquito resistance to agrochemicals in northern Thailand. Southeast Asian J Trop Med Public Health 36 (Suppl 4): 152–157. [Google Scholar]
  38. Collins FH, Paskewitz SM, 1996. A review of the use of ribosomal DNA to differentiate among cryptic Anopheles species. Insect Mol Biol 5 : 1–9. [Google Scholar]
  39. World Health Organization, 1975. Manual on practical entomology in malaria. Part II. Offset Publication 13. Geneva: World Health Organization (WHO).
  40. Wirtz RA, Burkot TR, Andre RG, Rosenberg R, Collins WE, Roberts DR, 1985. Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay. Am J Trop Med Hyg 34 : 1048–1054. [Google Scholar]
  41. Wirtz RA, Sattabongkot J, Hall T, Burkot TR, Rosenberg R, 1992. Development and evaluation of an ELISA for Plasmodium vivax-VK247 sporozoites. J Med Entomol 29 : 854–857. [Google Scholar]
  42. Fucharoen D, Tirawanchai N, Wilairat P, Panyim S, Thaithong S, 1988. Differentiation of Plasmodium falciparum clones by means of a repetitive DNA probe. Trans R Soc Trop Med Hyg 82 : 209–211. [Google Scholar]
  43. Ryan JR, Dave K, Collins KM, Hochberg L, Sattabongkot J, Coleman RE, Dunton RF, Bangs MJ, Mbogo CM, Cooper RD, Schoeler GB, Rubio-Palis Y, Magris M, Romero LI, Padilla N, Quakyi IA, Bigoga J, Leke RG, Akinpelu O, Evans B, Walsey M, Patterson P, Wirtz RA, Chan AST, 2002. Extensive multiple test centre evaluation of the VecTest™ malaria antigen assay. Med Vet Entomol 16 : 321–327. [Google Scholar]
  44. Kabiru EW, Mbogo CM, Muiruri SK, Ouma JH, Githure JI, Beier JC, 1997. Sporozoite loads of naturally infected Anopheles in Kilifi District, Kenya. J Am Mosq Control Assoc 13 : 259–262. [Google Scholar]
  45. Beier JC, Davis JR, Vaughan JA, Noden BH, Beier MS, 1991. Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi. Am J Trop Med Hyg 44 : 564–570. [Google Scholar]
  46. Cano J, Berzosa PJ, Roche J, Rubio JM, Moyano E, Guerra-Neira A, Brochero H, Mico M, Edu M, Benito A, 2004. Malaria vectors in Bioko Island (Equatorial Guinea): estimation of vector dynamics and transmission intensities. J Med Entomol 41 : 158–162. [Google Scholar]
  47. Kain KC, Wirtz RA, Fernandez I, Franke ED, Rodriguez MH, Lanar DE, 1992. Serological and genetic characterization of Plasmodium vivax from whole blood-impregnated filter paper discs. Am J Trop Med Hyg 46 : 473–479. [Google Scholar]

Data & Media loading...

  • Received : 07 Aug 2006
  • Accepted : 13 Dec 2006

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error