Integration of Anopheles beklemishevi (Diptera: Culicidae) in a PCR assay diagnostic for palaearctic Anopheles maculipennis sibling species

Abstract

A few years ago a PCR-based assay for a quick and reliable identification of six palaearctic sibling species of the Anopheles maculipennis complex was presented making use of differences in the nucleotide sequence of the ITS2 ribosomal mosquito DNA. An. beklemishevi, which is distributed in Scandinavia and Russia only, has now been integrated into this test after analysis of its ITS2 region which turned out to be much longer than those of the other sibling species. Three oligonucleotides putatively specific for An. beklemishevi were constructed and tested in combination with a universal genus-specific primer for the amplification of an An. beklemishevi-specific ITS2 DNA-fragment. Two of the three oligos generated accurate and specific PCR products, even when used in a multiplex PCR together with the specific primers for the other six sibling species. Cross-hybridization of the primers to heterologous culicid DNA was never observed. The amplicons that identify An. beklemishevi consist of 554 and 735 bp, respectively, and are easily distinguished from those specific for the other sibling species after gel electrophoresis.

Molecular identification of Palearctic members of Anopheles maculipennis in northern Iran

“…maculipennis species complex generated in this study and by others [7,26,29,30,45,46] and also three Nearctic taxa [42] were examined for phylogenetic analysis.…”

Occurrence and host preferences of Anopheles maculipennis group mosquitoes in England and Wales

“…maculipennis group, except An. beklemishevi (853 bp) (Kampen, ) and the close genetic relationship between the species, other closely related species may produce similarly sized fragments following the restriction with enzyme BstU I (CG↓CG). Thus, to avoid such misidentification, An.…”

Identifying Canadian mosquito species through DNA barcodes

“…Methods based on PCR, such as satellite DNA (Krzywinski et al , 2005), restriction fragment length analysis, single‐strand conformation shifts, or heteroduplex analysis, have been applied to detect diagnostic differences among PCR products in mosquito species (Moriais & Severson, 2003; Santomalazza et al , 2004; Weeto et al , 2004; Garros et al , 2005; Goswami et al , 2005). Most PCR assays have examined sequence diversity in specific nuclear loci (Scott et al , 1993; Beebe & Saul, 1995; Singh et al , 1997; Koekemoer et al , 1999; Proft et al , 1999; Walton et al , 1999; Hackett et al , 2000; Favia et al , 2001; Manonmani et al , 2001; Cohuet et al , 2003; Kent et al , 2004; Smith & Fonseca, 2004; Kampen, 2005; Marrelli et al , 2005). Other researchers have examined the taxonomic insights that can be gained by combining information from two or more genes (Nguyen et al , 2000; Krzywinski et al , 2001; Linton et al , 2001; Mitchell et al , 2002; Linton et al , 2003; Dusfour et al , 2004; Shaikevitch & Vinogradova, 2004; Cook et al , 2005).…”