Identification of two cryptic species in the Anopheles (Cellia) annularis complex using ribosomal DNA PCR-RFLP

Abstract

Anopheles (Cellia) annularis Van der Wulp is a complex of two sibling species provisionally designated as species A and B and can only be differentiated on the basis of the paracentric inversion in the ovarian polytene chromosomes. To analyze the distribution of these two species and to develop a molecular method for the identification of these two cryptic species, we sequenced the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain 3 (D3) of A. annularis specimens collected from Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi (Jharkhand), and Ghaziabad (Uttar Pradesh). We did not find any sequence variation among the specimens collected from Assam, Madhya Pradesh, and Jharkhand states, whereas two types of sequences were obtained from the specimens collected from the state of Uttar Pradesh, which correspond to species A and B of the A. annularis complex. Species A was more prevalent among the all four regions studied. The ITS2 sequence of species A showed unique restriction sites for MvaI and Eco24I, while species B displayed HinfI and NruI sites. Similarly, the D3 sequence of species A showed unique restriction site for Alw26I, while species B showed a unique KpnI site. In this study, we report for the first time the development of ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism methods for identifying these two cryptic species of the Annularis complex.

The type and mysorensis forms of the Anopheles stephensi (Diptera: Culicidae) in India exhibit identical ribosomal DNA ITS2 and domain-3 sequences

“…Specimens were stored in 80% ethanol until used for DNA extraction. PCR amplification and DNA sequencing DNA from individual mosquito was isolated using AccuPrep® Genomic DNA extraction kit (Bioneer, Daejeon, Korea) with some modification as described earlier (Alam et al 2007). The ITS2 locus was amplified using ITS2 forward and ITS2 reverse universal primers as described earlier (Alam et al 2007;Collins and Paskewitz 1996;Proft et al 1999).…”

“…PCR amplification and DNA sequencing DNA from individual mosquito was isolated using AccuPrep® Genomic DNA extraction kit (Bioneer, Daejeon, Korea) with some modification as described earlier (Alam et al 2007). The ITS2 locus was amplified using ITS2 forward and ITS2 reverse universal primers as described earlier (Alam et al 2007;Collins and Paskewitz 1996;Proft et al 1999). The D3 locus of the 28S ribosomal DNA was amplified using D3a and D3b primers as described earlier (Alam et al 2007;Litvaitis et al 1994).…”

“…The ITS2 locus was amplified using ITS2 forward and ITS2 reverse universal primers as described earlier (Alam et al 2007;Collins and Paskewitz 1996;Proft et al 1999). The D3 locus of the 28S ribosomal DNA was amplified using D3a and D3b primers as described earlier (Alam et al 2007;Litvaitis et al 1994). The amplified products were resolved on a 1.2% agarose gel and the expected sized band were excised from the gel and kept in sterile tubes.…”

“…Sequencing of the polymerase chain reaction (PCR) products was done on both strands (for both ITS2 and D3) using the same primers those used for their respective PCR amplifications. The sequencing PCR conditions and other downstream sequencing protocols were the same as those described earlier (Alam et al 2007). The nucleotide sequences reported in this paper are available in the GenBank database under the accession numbers DQ981411-DQ981455, EF195701-EF195702, EF216875-EF216894 (for D3 locus), and EU359661-EU359681 (for ITS2 locus).…”

“…Ribosomal (internal transcribed spacer 2 (ITS2) and domain-3 (D3)) and mitochondrial (cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), and cytochrome b) DNA markers have widely been used for molecular taxonomy studies on Anopheles (Alam et al 2006(Alam et al , 2007Dusfour et al 2007;Hackett et al 2000;Norris 2002;Proft et al 1999). There is limited information on the population genetic structure of A. stephensi.…”