ITS2 characterization and Anopheles species identification of the subgenus Cellia
Graphical abstract
ITS2-based PCR protocol was developed to identify Anopheles species under the subgenera Cellia.
Highlights
► We study the ITS2 sequence divergence, phylogeny and secondary structure of Anophelines. ► ITS2 length was variable and no intraspecific variations were found. ► Significance was found between repeats, minimum free energy and RNA secondary structures. ► Four domain types in secondary structure and unique microsatellites were identified. ► ITS2 region is more species-specific and useful for Anopheles species identification.
Introduction
Malaria is one of the major public health problems in the world with 3.3 billion people at risk, 216 million cases and 655,000 deaths recorded in 2010 (WHO, 2011). Malaria is prevalent in 11 countries of the South East Asian region, 85.7% are exposed to the risk of malaria, where India is one of the highest number of cases and deaths reported (World Malaria Report, 2011). Malaria is a life-threatening disease in the North East India (12% infection cases) with 174,000 cases and 290 deaths recorded during 2010 (NVBDCP, 2010). The high risk populations are ethnic tribes living in the forested pockets of the North Eastern states and other parts of India (Dash et al., 2008). In Mizoram, out of 10.32 lakhs, 29 deaths were reported during 2011, about 94.47% being due to Plasmodium falciparam (Mizoram Health and family welfare Department, 2011). Among the 25 species of anophelines identified in Mizoram by taxonomic characters (Rita et al., 2009), An. dirus and An. minimus have been implicated as the primary malaria vectors in Northeast India (Prakash et al., 2006), while an increasing number of other Anopheles species (NRHM, 2009, Bhattacharyya et al., 2010, Sarma et al., 2012a, Sarma et al., 2012b) have been reported as incriminated vectors.
According to Harbach and Kitching (2005), the genus Anopheles includes 484 recognized species and only 30–40 commonly transmit Plasmodium parasites that cause malaria. The genus has been subdivided into seven subgenera in which Cellia (239 species) is the largest type and all the species are found in Old World (Krzywinski and Besansky, 2003, Rattanarithikul et al., 2006). It has been divided into six series wherein Myzomyia (69 species), Neocellia (33 species), and Pyretophorus (22 species) are of medical importance carrying vectors of malarial protozoa and microfilariae (Garros et al., 2005) especially in Southeast Asia (Harbach, 2004). Among the three series, Funestus (An. jeyporiensis, An. aconitus, An. varuna, An. minimus), Annularis (An. annularis, An. nivipes), Jamesii (An. jamesii), Maculatus (An. maculates), Vagus (An. vagus) and Subpictus (An. subpictus) groups are considered important in the epidemiology of malaria in Bangladesh (Maheswary et al., 1993, Maheswary et al., 1994, Alam et al., 2010), India (Prakash et al., 2004, Tikar et al., 2011), China (Ma et al., 2006), Hongkong (Greenwood et al., 2005) and Vietnam (Van Bortel et al., 2001). Therefore, it is necessary to develop accurate and reliable methods for the identification of malaria vectors. The genus Anopheles includes many complexes of isomorphic or similar sibling species and this may result in imprecise morphological identification of mosquitoes. Further, the problem of vector identification has been solved by the application of DNA based methods. Ribosomal DNA has transcribed and non-transcribed spacer regions that have high and low intraspecific variabilities, making them useful for the study of relationships of closely related species, also allow unambiguous identification of species in a range of closely related mosquitoes and to solve taxonomic problems (Li and Wilkerson, 2007, Alam et al., 2007). In mosquitoes, each transcriptional unit is made up of an external transcribed spacer, an 18S subunit, an internal transcribed spacer one (ITS1), a 5.8S subunit, an internal transcribed spacer two (ITS2), and a 28S subunit (Hwang, 2007, Kampen, 2005). The repetitive DNA sequences in the ITS2 region contribute to polymorphism aiding in molecular taxonomic studies regulate key functions and are responsible for genomic instability (Zhang and Hewitt, 2003, Li and Wilkerson, 2007).
In the present study, PCR based ribosomal DNA marker (ITS2 region) of the 10 Anopheline species prevalent in Mizoram was compared and addresses the following questions: (1) Are there conserved regions of the secondary structures that can be identified among the Anopheles species? (2) Is there any relationship between ITS2 repeats and secondary structure? (3) Does the ITS2 secondary structure in Anopheles fit the canonical 3–6 domain model as seen in other eukaryotes? (4) And, what is the resolution of ITS2 DNA sequences in inferring the phylogeny of six groups belonging to three series under Cellia subgenera?
Section snippets
Mosquito collection
The list of anopheline species collected for the present study and their origin is given in Table 1. Adult mosquitoes were collected in cattle shed and human dwellings during June–October 2009–2011 from five districts of Mizoram state (Neipui: Lawngtlai; Lengpui: Mamit; Thenzawl: Serchhip; Chawnpui and buhchang: Kolasib; Turial: Aizawl). Whole night collections (18:00 h–06:00 h) were done using Centers for Disease Control (CDC) miniature light traps and aspirators (18:00–22:00 h and 02:00–06:00 h).
Results and discussion
Mizoram is one of the malaria prone areas in Northeast India and the forests of Mizoram have remained an area of intense malaria transmission providing a focus for reinfection for the border regions of Myanmar and Bangladesh. Several Anopheles species apart from the primary vectors involved in transmitting malaria in the north-east India had been suspected for a long time (Malhotra, 1998, Prakash et al., 2004). Understanding of malaria epidemiology in this area demands studies on the whole
Conclusion
Eukaryotic ribosomal DNA (rDNA) has several properties that make it useful for studying genetic variability and divergence within and between species: tandemly repeated genes, secondary structure of transcribed regions, differential rates of evolution between spacers and coding regions, and concerted evolution (Arnheim, 1983, Gerbi, 1985, Tautz et al., 1987). Unique species-specific conserved regions of secondary structures of the ten Anopheles species were identified; significant fixed
Acknowledgements
The authors thank CSIR, New Delhi (No. 37(1362)/09/EMR-II dt 3 March 2009) and Directorate of Health Services, Government of Mizoram, Mizoram D 12030/1/2003 – DHS (M)/21 dt. 06 March 2009 for supporting research projects related to mosquito. We are also thankful to the Department of Biotechnology, New Delhi, India for the financial assistance in the form of Bioinformatics Infrastructure Facility and State Biotech Hub. We thank Dr. Praveen Karanth, Indian Institute of Science, Bangalore for
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