Open Access Open Access  Restricted Access Subscription or Fee Access

Molecular operational taxonomic units of mosquitoes (Diptera: Culicidae) collected in high Andean mountain ecosystems of Antioquia, Colombia



Accurate taxonomic identification of highland mosquito species may be complicated because of the lack of comprehensive regional morphological keys and taxonomic specialists, particularly for mosquitoes of medical or ecological importance. We applied a multi-locus approach to explore the diversity of genera/species collected, to define the Molecular Operational Taxonomic Units (MOTUs) and to perform phylogenetic clustering. Twenty MOTUs and three single sequences were revealed from 78 concatenated cox1 + ITS2 sequences, and the species name was allocated for five of these. This study provides molecular taxonomic information of culicid fauna present in high Andean mountain ecosystems in Antioquia, Colombia. However, future morphological and integrative taxonomic studies should be conducted to achieve the specific identity of all detected MOTUs.



Diptera, molecular operational taxonomic units, mosquitoes, cox1, highlands, ITS2, Colombia

Full Text:



Ahumada, M., Orjuela, L., Pareja, P., Conde, M., Cabarcas, D., Cubillos, E., López, J., Beier, J., Herrera, S. & Quiñones, M. (2016) Spatial distributions of Anopheles species in relation to malaria incidence at 70 localities in the highly endemic Northwest and South Pacific coast regions of Colombia. Malaria Journal, 15, 407.

Arnell, J.H. (1973) Mosquito studies (Diptera, Culicidae) XXXII. A revision of the genus Haemagogus. Contributions of the American Entomological Institute, 10 (2), 1–176.

Arnell, J.H. (1976) Mosquito studies (Diptera, Culicidae) XXXIII. A revision of the scapularis group of Aedes (Ochlerotatus). Contributions of the American Entomological Institute, 13 (3), 1–143.

Arregui, G., Enriquez, S., Benítez-Ortiz, W. & Navarro, J. (2015) Taxonomía molecular de Anopheles del Ecuador mediante ADN mitocondrial (Citocromo c Oxidasa I) y optimización por Parsimonia Máxima. Boletin de Malariologia y Salud Ambiental, 55, 132–154.

Ashfaq, M., Hebert, P.D., Mirza, J.H., Khan, A.M., Zafar, Y. & Mirza, M.S. (2014) Analyzing mosquito (Diptera: Culicidae) diversity in Pakistan by DNA barcoding. PLoS ONE, 9, e97268.

Barajas, J., Suaza, J.D., Torres, C., Rua-Uribe, G., Uribe, S. & Porter, C.H. (2013) Mosquitos (Diptera: Culicidae) asociados a guadua en los municipios de Anserma, Hispania y Jardín, Colombia. Revista Colombiana de Entomología, 39, 132–140.

Bargues, M., Morchón, R., Latorre, J., Cancrini, G., Mas-Coma, S. & Simón, F. (2006) Ribosomal DNA second internal transcribed spacer sequence studies of culicid vectors from an endemic area of Dirofilaria immitis in Spain. Parasitology Research, 99, 205–213.

Barratt, B., Derraik, J., Rufaut, C., Goodman, A. & Dickinson, J. (2003) Morphospecies as a substitute for Coleoptera species identification, and the value of experience in improving accuracy. Journal of the Royal Society of New Zealand, 33, 583–590.

Batovska, J., Blacket, M., Brown, K. & Lynch, S. (2016) Molecular identification of mosquitoes (Diptera: Culicidae) in southeastern Australia. Ecology and Evolution, 6, 3001–3011.

Belkin, J.N., Schick, R.X., Galindo, P. & Aitken, T.H. (1969) Mosquito studies (Diptera: Culicidae) I. a project for a systematic study of the mosquitoes of Middle America. Contributions of the American Entomological Institute, 1 (2), 1–17.

Berlin, O. (1969) Mosquito Studies (Diptera, Culicidae) XII. A revision of the Neotropical subgenus Howardina [sic] of Aedes [sic]. Contributions of the American Entomological Institute, 4 (2), 1–190.

Bickford, D., Lohman, D., Sodhi, N., Ng, P., Meier, R., Winker, K., Ingram, K. & Das, I. (2007) Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution, 22, 148–155.

Blaxter, M.L. (2004) The promise of a DNA taxonomy. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences, 359, 669–679.

Bourke, B., Oliveira, T., Suesdek, L., Bergo, E. & Sallum, M. (2013) A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae). Parasites & Vectors, 6, 111.

Bram, R.A. (1967) Classification of Culex subgenus Culex in the New World (Diptera, Culicidae). Proceedings of the United States National Museum Archives, 120, 1–122.

Buhay, J.E. (2009) “COI-like” sequences are becoming problematic in molecular systematic and DNA barcoding studies. Journal of Crustacean Biology, 29, 96–110.

Castrillón, J., Carlos, J. & Urcuqui, S. (2015) Dengue en Colombia, diez años de evoluciòn. Revista Chilena de Infectología, 32, 142–149.

Ceretti-Junior, W., Christe, R., Rizzo, M., Strobel, R., Junior, M., Mello, M., Fernandes, A., Medeiros-Sousa, A., Carvalho, G. & Marrelli, M. (2016) Species composition and ecological aspects of immature mosquitoes (Diptera: Culicidae) in bromeliads in urban parks in the city of São Paulo, Brazil. Journal of Arthropod-Borne Diseases, 10, 102–112.

Cienfuegos, A.V., Rosero, D.A., Naranjo, N., Luckhart, S., Conn, J.E. & Correa, M.M. (2011) Evaluation of a PCR-RFLP-ITS2 assay for discrimination of Anopheles species in northern and western Colombia. Acta Tropica, 118, 128–135.

Collins, F.H. & Paskewitz, S.M. (1996) A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species. Insect Molecular Biology, 5, 1–9.

Conn, J., Moreno, M., Saavedra, M., Bickersmith, S., Knoll, E., Fernandez, R., Vera, H., Burrus, R., Lescano, A., Sanchez, J., Rivera, E. & Vinetz, J. (2013) Molecular Taxonomy of Anopheles (Nyssorhynchus) benarrochi (Diptera: Culicidae) and Malaria Epidemiology in Southern Amazonian Peru. American Journal of Tropical Medicine and Hygiene, 88, 319–324.

Darriba, D., Taboada, G., Doallo, R. & Posada, D. (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9, 772–772.

Demari-Silva, B., Vesgueiro, F., Sallum, M. & Marrelli, M. (2011) Taxonomic and phylogenetic relationships between species of the genus Culex (Diptera: Culicidae) from Brazil inferred from the cytochrome c oxidase I mitochondrial gene. Journal of Medical Entomology, 48, 272–279.

Dupuis, J., Roe, A. & Sperling, F. (2012) Multi-locus species delimitation in closely related animals and fungi: one marker is not enough. Molecular Ecology, 21, 4422–4436.

Edgar, R.C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 5, 113.

Feo, O., Solano, E., Beingolea, L., Aparicio, M. & Villagra, M. (2009) Cambio climático y salud en la Región Andina. Revista Peruana de Medicina Experimental y Salud Pública, 26, 83–93.

Floyd, R., Abebe, E., Papert, A. & Blaxter, M. (2002) Molecular barcodes for soil nematode identification. Molecular Ecology, 11, 839–850.

Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.

Forattini, O. (2002) Culicidologia médica: identificação, biologia, epidemologia. Vol.2. Editora Universidade de São Paulo, São Paulo, 860 pp.

Friedrich, M. & Tautz, D. (1997) Evolution and phylogeny of the Diptera: a molecular phylogenetic analysis using 28S rDNA sequences. Systematic Biology, 46, 674–698.

Galvis, A.G. (1943) Biologia y distribución geográfica de los anofelinos en Colombia. Reimpreso de la Revista de la Facultad de Medicina. Editorial Cromos, Bogotá, 12, 5–55.

Githeko, A., Lindsay, S., Confalonieri, U & Patz, J. (2009) El cambio climático y las enfermedades transmitidas por vectores: un análisis regional. Revista Virtual REDESMA, 3, 21–38.

Gómez, G., Jaramillo, L. & Correa, M.M. (2013) Wing geometric morphometrics and molecular assessment of members in the Albitarsis Complex from Colombia. Molecular Ecology Resources, 13, 1082–1092.

Gómez, G.F., Bickersmith, S.A., González, R., Conn, J.E. & Correa, M.M. (2015) Molecular taxonomy provides new insights into Anopheles species of the Neotropical Arribalzagia Series. PLoS ONE, 10, e0119488.

González, R. & Carrejo, N. (2009) Introducción al estudio taxonómico de Anopheles de Colombia: claves y notas de distribución. Segunda Edición. Programa Editorial Universidad de Valle, Cali, 209 pp.

González, R., Carrejo, N., Wilkerson, R.C., Alarcon, J., Alarcon-Ormasa, J., Ruiz, F., Bhatia, R., Loaiza, J. & Linton, Y.-M. (2010) Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material. Memorias do Instituto Oswaldo Cruz, 105, 1001–1009.

Gutiérrez, L.A., González, J.J., Gómez, G.F., Castro, M.I., Rosero, D.A., Luckhart, S., Conn, J.E. & Correa, M.M. (2009) Species composition and natural infectivity of anthropophilic Anopheles (Diptera: Culicidae) in the states of Cordoba and Antioquia, Northwestern Colombia. Memorias do Instituto Oswaldo Cruz, 104, 1117–1124.

Hajibabaei, M., Janzen, D., Burns, J., Hallwachs, W. & Hebert, P. (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences of the United States of America, 103, 968–971.

Harbach, R. (2017) Culicidae classification [Internet]. Mosquito taxonomic inventory. Accessed March 11, 2017. Available from: (accessed 17 May 2017)

Harrison, B., Ruiz-lopez, F., Falero, G., Savage, H., Pecor, J. & Wilkerson, R. (2012) Anopheles (Kerteszia) lepidotus (Diptera: Culicidae), not the malaria vector we thought it was: Revised male and female morphology; larva, pupa, and male genitalia characters; and molecular verification. Zootaxa, 3218, 1–17.

Hebert, P., Cywinska, A., Ball, S. & deWaard, J. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270, 313–321.

Hlaing, T., Tun-Lin, W., Somboon, P., Socheat, D., Setha, T., Min, S., Chang, M.S. & Walton, C. (2009) Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti mosquitoes: implications for past and future population genetic studies. BMC Genetics, 10, 11.

Hoyos-López, R., Uribe-Soto, S. & Gallego-Gómez, J. (2015) Evolutionary relationships of West Nile virus detected in mosquitoes from a migratory bird zone of Colombian Caribbean. Virology Journal, 12, 80.

Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755.

Jones, M., Ghoorah, A. & Blaxter, M. (2011) JMOTU and taxonerator: turning DNA barcode sequences into annotated operational taxonomic units. PLoS ONE, 6, e19259.

Judd, D.D. (1998) Review of a bromeliad-ovipositing lineage in Wyeomyia and the resurrection of Hystatomyia (Diptera: Culicidae). Annals of the Entomological Society of America, 91, 572–589.

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P. & Drummond, A. (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649.

Keller, A., Schleicher, T., Schultz, J., Müller, T., Dandekar, T. & Wolf, M. (2009) 5.8S-28S rRNA interaction and HMM-based ITS2 annotation. Gene, 430, 50–57.

Khoshdel-Nezamiha, F., Vatandoost, H., Oshaghi, M., Azari-Hamidian, S., Mianroodi, R., Dabiri, F., Bagheri, M., Terenius, O. & Chavshin, A. (2016) Molecular characterization of mosquitoes (Diptera: Culicidae) of northwestern Iran using rDNA-ITS2. Japanese Journal of Infectious Diseases, 69, 319–322.

Krishna-Krishnamurthy, P. & Francis, R. (2012) A critical review on the utility of DNA barcoding in biodiversity conservation. Biodiversity and Conservation, 21, 1901–1919.

Lane, J. (1953) Neotropical Culicidae. Vols I and II. Universidade de São Paulo, São Paulo, 548 pp.

Levi-Castillo, R. (1954) The Haemagogus of South America (Diptera: Culicidae). Revista Ecuatoriana de Entomologia y Parasitología, 2, 269–302.

López-Rubio, A., Suaza-Vasco, J., Marcet, P., Ruíz-Molina, N., Cáceres, L., Porter, C. & Uribe, S. (2016) Use of DNA barcoding to distinguish the malaria vector Anopheles neivai in Colombia. Zootaxa, 4175 (4), 377–389.

Louton, J., Gelhaus, J. & Bouchard, R. (1996) The aquatic macrofauna of water-filled bamboo (Poaceae: Bambusoideae: Guadua) internodes in a Peruvian lowland tropical forest. Biotropica, 28, 228–242.

Masters, B., Fan, V. & Ross, H. (2011) Species delimitation-a geneious plugin for the exploration of species boundaries. Molecular Ecology Resources, 11, 154–157.

Mattingly, P. (1971) Contributions to the mosquito fauna of Southeast Asia. ‒ XII. Illustrated keys to the genera of mosquitoes. Contributions of the American Entomological Institute, 7 (4), 1–84.

Monaghan, M.T., Balke, M., Gregory, T.R. & Vogler, A.P. (2005) DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences, 360, 1925–1933.

Motta, M. & Lourenço-de-Oliveira, R. (2000) The Subgenus Dendromyia Theobald: a review with redescriptions of four species (Diptera: Culicidae). Memorias do Instituto Oswaldo Cruz, 95, 649–683.

Müller, G. & Marcondes, C. (2006) Bromeliad-associated mosquitoes from Atlantic forest in Santa Catarina Island, southern Brazil (Diptera, Culicidae), with new records for the State of Santa Catarina. Iheringia Série Zoologia, 96, 315–319.

Naranjo-Díaz, N., Altamiranda, M., Luckhart, S., Conn, J. & Correa, M. (2014) Malaria vectors in ecologically heterogeneous localities of the Colombian Pacific region. PLoS ONE, 9, e103769.

Navarro, J., Liria, J., Piñango, H. & Barrera, R. (2007) Biogeographic area relationships in Venezuela: A parsimony analysis of Culicidae-phytotelmata relationships distributions in national parks. Zootaxa, 1547, 1–19.

Navarro, J., Del Ventura, F., Zorrilla, A. & Liria, J. (2010) Registros de mayor altitud para mosquitos (Diptera: Culicidae) en Venezuela. Revista de Biologia Tropical, 58, 245–254.

Ng’endo, R.N., Osiemo, Z.B. & Brandl, R. (2013) DNA barcodes for species identification in the hyperdiverse ant genus Pheidole (Formicidae: Myrmicinae). Journal of Insect Science, 13, 27. [online]

Ortega-Morales, A., Zavortink, T., Huerta-Jimenez, H., Sanchez-Ramos, F., Teresa Valdes-Perezgasga, M., Reyes-Villanueva, F., Siller-Rodriguez, Q. & Fernandez-Salas, I. (2015) Mosquito records from Mexico: the mosquitoes (Diptera: Culicidae) of Tamaulipas State. Journal of Medical Entomology, 52, 171–184.

Pecor, J. & Gaffigan, T. (1997) Collecting, rearing, preserving, mounting and shipping techniques for mosquitoes [Internet]. Walter Reed Biosystematics Unit, Washington, DC. Accessed February 17, 2014. Available from: (accessed 17 May 2017)

Pemola Devi, N. & Jauhari, R. (2004) Altitudinal distribution of mosquitoes in mountainous area of Garhwal region: Part-I. Journal of Vector Borne Diseases, 41, 17–26.

Pinault, L. & Hunter, F. (2011) New highland distribution records of multiple Anopheles species in the Ecuadorian Andes. Malaria Journal, 10, 236.

Prado, M. (2003) Caracterización morfológica y molecular de tres especies del subgénero Kerteszia de Anopheles (Diptera: Culicidae) en la región andina venezolana. Trabajo Especial de Grado, Escuela de Biología, Universidad Central de Venezuela, Caracas, 50 pp.

Puillandre, N., Lambert, A., Brouillet, S. & Achaz, G. (2012) ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21, 1864–1877.

Quintero, J., Brochero, H., Manrique-Saide, P., Barrera-Pérez, M., Basso, C., Romero, S., Caprara, A., De Lima Cunha, J., Beltrán-Ayala, E., Mitchell-Foster, K., Kroeger, A., Sommerfeld, J. & Petzold, M. (2014) Ecological, biological and social dimensions of dengue vector breeding in five urban settings of Latin America: a multi-country study. BMC Infectious Diseases, 14, 38.

Ratnasingham, S. & Hebert, P. (2013) A DNA-based registry for all animal species: the barcode index number (BIN) system. PLoS ONE, 8, e66213.

Ratnasingham, S. & Hebert, P.D.N. (2007) The Barcode of Life Data System BOLD. Molecular Ecology Notes, 7, 355–364.

Rosenberg, N. (2007) Statistical tests for taxonomic distinctiveness from observations of monophyly. Evolution, 61, 317–323.

Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X. & Rozas, R. (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19, 2496–2497.

Rozo-Lopez, P. & Mengual, X. (2015) Mosquito species (Diptera, Culicidae) in three ecosystems from the Colombian Andes: identification through DNA barcoding and adult morphology. ZooKeys, 513, 39–64.

Rubinoff, D. (2006) Utility of mitochondrial DNA barcodes in species conservation. Conservation Biology, 20, 1026–1033.

Ruiz-López, F., González-Mazo, A., Vélez-Mira, A., Gómez, G., Zuleta, L., Uribe, S. & Vélez-Bernal, I.D. (2016) Presencia de Aedes (Stegomyia) aegypti (Linnaeus, 1762) y su infección natural con el virus dengue en alturas no registradas para Colombia. Biomédica, 36, 303–308.

Rutar, T., Baldomar Salgueiro, E.J. & Maguire, J.H. (2004) Introduced Plasmodium vivax malaria in a Bolivian community at an elevation of 2,300 meters. American Journal of Tropical Medicine and Hygiene, 70, 15–19.

Ryberg, M. (2015) Molecular operational taxonomic units as approximations of species in the light of evolutionary models and empirical data from Fungi. Molecular Ecology, 24, 5770–5777.

Santacoloma-Varón, L., Chaves-Córdoba, B. & Brochero, H. (2010) Susceptibilidad de Aedes aegypti a DDT, deltametrina y lambdacialotrina en Colombia. Revista Panamericaca de Salud Publica, 27, 66–73.

Song, H., Buhay, J.E., Whiting, M.F. & Crandall, K.A. (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proceedings of the National Academy of Sciences, 105, 13486–13491.

Stone, A. (1944) Notes on the genus Trichoprosopon (Diptera, Culicidae). Revista de Entomologia, 15, 335–341.

Suaza-Vasco, J., López-Rubio, A., Galeano, J., Uribe, S., Vélez, I. & Porter, C. (2015) The sabethines of northern Andean coffee-growing regions of Colombia. Journal of the American Mosquito Control Association, 31, 125–134.

Suaza-Vasco, J., Ruiz-Molina, N., Galeano, E., Lopez, A., Uribe, S. & Porter, C. (2013a) Evidencia molecular de presencia de Anopheles (Kerteszia) grupo boliviensis (Diptera: Culicidae) en reserva ecológica Alto Romeral departamento de Antioquia, Colombia. Entomotropica, 28, 160.

Suaza-Vasco, J., Ruiz-Molina, N., Uribe, S. & Porter, C. (2013b) Contribución al conocimiento de Anopheles (Kerteszia) grupo “boliviensis” (Diptera: Culicidae) en Colombia: Taxonomía y variabilidad genética. Entomotropica, 28, 160.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.

Wesson, D., Porter, C. & Collins, F. (1992) Sequence and secondary structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae). Molecular Phylogenetics and Evolution, 1, 253–269.

Wilkerson, R., Linton, Y., Fonseca, D., Schultz, T., Price, D. & Strickman, D. (2015) Making mosquito taxonomy useful: a stable classification of Tribe Aedini that balances utility with current knowledge of evolutionary relationships. PLoS ONE, 10, e0133602.

Wolff, M., Niehi, S. & de Carvalho, C. (2016) Catalogue of Diptera of Colombia: an introduction. Zootaxa, 4122 (1), 8–14.

WRBU (2017) Systematic catalog of Culicidae [Internet]. Walter Reed Biosystematics Unit [accessed March 13, 2017]. Available from: (accessed 17 May 2017)

Zavortink, T. (1979) Mosquito studies (Diptera, Culicidae) XXXV. The new sabethine genus Johnbelkinia and a preliminary reclassification of the composite genus Trichoprosopon. Contributions of the American Entomological Institute, 17 (1), 1–61.



  • There are currently no refbacks.

ISSN 1175-5326 (Print Edition) & ISSN 1175-5334 (Online Edition)
Published by Magnolia Press, Auckland, New Zealand