Utilizing molecular techniques to distinguish between mosquito species and populations

Marly B. Katz


Mosquitoes belong to a family of insects, called Culicidae, in the true fly order Diptera. There are over 3,000 species described worldwide, 180 of which are found in North America (Harbach & Kitching 1998, Willott & Ramberg 2007). Female mosquitoes must consume blood in order to obtain the necessary nutrients for laying eggs and readily feed on humans and other animals (Tanaka et al. 1979). In addition to being considered pests, mosquitoes are important vectors for a number of infectious diseases including malaria, West Nile virus, yellow fever, and LaCrosse encephalitis (CDC 2007). Worldwide, hundreds of millions of people suffer from the effects of mosquito borne illnesses every year and hundreds of thousands of them die (WHO 2013). The expansion of a number of species northward increases the risk for more disease outbreaks in the future (Rochlin et al. 2013).
Mitochondrial DNA (mtDNA) has been utilized in mosquito phylogenetic studies for analyzing both inter- and intraspecific variation (Birungi & Munstermann 2002). The NADH dehydrogenase 5 subunit (ND5) is an especially variable protein-coding gene, and researchers have had success in categorizing the genetic structures of mosquitoes (Besansky et al. 1997, Birungi & Munstermann 2002). For this study, five mosquito species with distributions in the eastern United States were selected for analyzing including: Aedes albopictus, Ochlerotatus japonicus, Ochlereotatus triseriatus, Anopheles punctipennis, and Anopheles quadrimaculatus. The objectives are to distinguish among species and between populations of mosquitoes by using a primer pair that amplifies a region of mtDNA in the ND5 gene.

Materials and methods

Eight adult mosquito samples representing five species were obtained from Westchester, Rockland and Sullivan counties in New York State, and Weakley county Tennessee. DNA was extracted from each specimen and amplified through PCR using an ND5 primer pair selected from the literature (Birungi & Munstermann 2002). In order to visualize DNA, the PCR products were ran on an 1% agarose gel before being purified and bands were confirmed by sequencing.

Sequences were edited using A plasmid Editor (ApE), aligned with Clustal Omega and blasted on the National Center for Biotechnology Information (NCBI) site in order to make pairwise comparisons.


The target region of mtDNA was successfully amplified in the specimens of the five species used in this study (Figure 1). Pairwise comparisons of the sequences revealed base pair differences between each of the individual species (Figure 2). The greatest homology between sequences was seen in the two Anopheles species, while the greatest differences were seen between An. quadrimaculatus and Ae.albopictus and between An. quadrimaculatus and Oc. japonicus.

The target region was also amplified in two specimens from each of three mosquito species (Figure 3). The sequences for the two Ae. albopictus individuals demonstrated 100% similarity, as did the sequences for the two Oc. triseriatus specimens Between the two An. punctipennis individuals, however, the sequences showed 99% similarity, with two base pair differences (Figure 4).


The ND5 primer pair was able to successfully amplify DNA sequences that were distinguishable among all five species. While the sequences of the two Ae. albopictus and the two Oc. triseriatus individuals were 100% homologous, there were two base pair differences between the An. punctipennis specimens from NY and from TN.   These results support the findings of previous studies indicating the potential usefulness of ND5 primer pairs in distinguishing among both species and populations of mosquitoes.

The greater distance between the NY and TN mosquitoes compared to the relatively short distances between the other two pairs in NY, may have increased the likelihood of genetic differences. A sample size of one mosquito from each location, however, is not sufficient for more concrete analysis.

In addition to determining variation among populations, interspecific analyses of invasive species can provide clues in regards to location of origin and details surrounding the introduction. Understanding trends and being able to distinguish among populations may have implications for the monitoring of future introductions and for disease transmission (Fonseca et al. 2010).

Future studies with more samples and species will need to be conducted in order to confirm the usefulness of this technique for developing mosquito species barcodes, distinguishing among mosquito populations, and for analyzing the population genetics of invasive species.


Besansky, N. J., T. Lehmann, G. T. Fahey, D. Fontenille, L. E. Hawley, W. A. Hawley, & F. H. Collins. 1997. Patterns of mitochondrial variation within and between African malaria vectors, Anopheles gambiae and An. Arabiensis, suggest extensive gene flow. Genetics 147:1817-1828.

Birungi, J., & L. E. Munstermann. 2002. Genetic structure of Aedes albopictus (Diptera: Culicidae) populations based on mitochondrial ND5 sequences: evidence for an independent invasion into Brazil and United States. Annals of the Entomological Society of America 95:125-132.

Centers for Disease Control and Prevention (CDC). 2007. Infectious Disease Information Mosquito-Borne Diseases. http://www.cdc.gov/ncidod/diseases/list_mosquitoborne.htm Accessed May 4, 2014.

Fonseca, D. M., A. K. Widdel, M. Hutchinson, S. E. Spichiger, and L. D. Kramer. 2010. Fine-scale spatial and temporal population genetics of Aedes japonicus, a new US mosquito, reveal multiple introductions. Molecular Ecology 19:1559-1572.

Harbach, R., & I. J. Kitching. 1998. Phylogeny and classification of the Culicidae (Diptera). Systematic Entomology 23:327-370.

Rochlin, I., D. V. Ninivaggi, M. L. Hutchinson, A. Farajollahi. 2013. Climate change and range expansion of the Asian tiger mosquito (Aedes albopictus) in Northeastern USA: implications for public health practitioners. PloS one 8:e60874

Tanaka, K., K. Mizusawa, and E. S. Saugstad. 1979. A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). Contributions of the American Entomological Institute 16:1-987.

Willott, E., & Ramberg, F. 2007. Identification and geographical distribution of the mosquitoes of North America, North of Mexico. Journal of Wildlife Diseases 43:806-807.

World Health Organization (WHO). 2013. World Malaria Report 2013. http://www.who.int/malaria/publications/world_malaria_report_2013/en/ Accessed May 4, 2014.


Figure 1-PCR product from 5 ng of DNA of five mosquito species on a 1% agarose gel.

Figure 2-Pairwise comparisons between the five mosquito species (% similarity).

Figure 3-PCR product from 5 ng of DNA of six individual mosquitoes constituting three species. Samples include an Ae. albopictus from Westchester, an Ae. albopictus from Rockland, an An. punctipennis from NY, an An. punctipennis from TN, an Oc. triseriatus from Sullivan, and an Oc. triseriatus from Westchester.

Figure 4-Sequence comparisons within species.


There are 180 species of mosquitoes established in the United States, many of which are important vectors for a number of diseases affecting human health. The purpose of this project was to distinguish among mosquito species and between mosquito populations using genetic analysis of mitochondrial DNA. Eight individual mosquitoes, encompassing five species, from varying geographical regions were studied for this purpose. Using a primer pair that amplified a region of the NADH dehydrogenase 5 subunit gene, it was possible to distinguish among all five species. On the population level, pairs of three different species were analyzed from differing locations. Two species pairs could not be distinguished, however the third pair resulted in 99% similarity with two base pair differences. These results indicate there is potential to utilize this technique to distinguish between both species and populations of mosquitoes.

Full Paper


I would like to thank Catharina Grubaugh and Katherine Reid for their tremendously helpful assistance and unwavering patience. Thank you to Dr. Thomas Daniels, Dr. Richard Falco and the rest of Fordham University’s Vector Ecology Lab and Dr. Jack Grubaugh for supplying mosquito specimens, and to Dr. Hekkala’s lab for providing other samples for future studies. Finally, thank you to Dr. Berish Rubin whose guidance and support were critical in the completion of this project.

This document was last modified 05/16/2014.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home