Molecular identification of Palearctic members of Anopheles maculipennis in northern Iran

Abstract

Background: Members of Anopheles maculipennis complex are effective malaria vectors in Europe and the Caspian Sea region in northern Iran, where malaria has been re-introduced since 1994. The current study has been designed in order to provide further evidence on the status of species composition and to identify more accurately the members of the maculipennis complex in northern Iran.

Methods: The second internal transcribed spacer of ribosomal DNA (rDNA-ITS2) was sequenced in 28 out of 235 specimens that were collected in the five provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan in Iran.

Results: The length of the ITS2 ranged from 283 to 302 bp with a GC content of 49.33-54.76%. No intra-specific variations were observed. Construction of phylogenetic tree based on the ITS2 sequence revealed that the six Iranian members of the maculipennis complex could be easily clustered into three groups: the An. atroparvus-Anopheles labranchiae group; the paraphyletic group of An. maculipennis, An. messeae, An. persiensis; and An. sacharovi as the third group.

Conclusion: Detection of three species of the An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as shown as new records in northern Iran, is somehow alarming. A better understanding of the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to the correct identification of species complexes, to the detection of Plasmodium composition in Anopheles vectors and to the status of insecticide resistance by looking to related genes.

Figure 1

Collection sites of Anopheles maculipennis complex specimens in Iran. Numbers quoted in the map (1–5) corresponds to the study areas; East Azerbaijan (1), Ardebil (2), Guilan (3), Mazandran (4) and Khorassan (5) provinces, accordingly.

Figure 2

Alignment showing the inter-specific variability in ITS2 sequence for six Palearctic members of An. maculipennis complex from northern Iran. The first 26 bp belongs to 5.8 s region and ITS2 starts and ends with bold nucleotides. The remainder 27 bp is 28 s region. Selected sequences used for this alignment could be reached through GenBank accession numbers; AY730269 (An. persiensis), AY050639 (An. messaea), AY050640 (An. atroparvus), AY842516 (An. labranchiae), AY730264 (An. maculipennis), AY533852 and AY842515 (An. sacharovi).

Figure 3

Phylogenetic relationship between Iranian and Nearctic members of An. maculipennis complex; Anopheles hermsi [GenBank: M64483], Anopheles freeborni [GenBank: M64484], Anopheles occidentalis [GenBank: M64482], constructed based on rDNA-ITS2 sequence. Bootstrap values under 50% have not shown.

Figure 4

rDNA-ITS2 sequence generated the phylogenetic tree, showing the relationship between members of Maculipennis complex from old and new world. An. persiensis [GenBank: AY730269, AY137847, Iran], An. maculipennis [GenBank: AY730264, AY137798, Iran; Z50104, Italy], An. sacharovi [GenBank: AY842517, AY114204, Iran; Z83198, Italy], An. labranchiae [GenBank: AY253849, Morocco; Z50102, Italy; AY842516, Iran], An. messeae [GenBank: AY050639, Iran; Z50105, Italy], An. atroparvus [GenBank: AY050640, Iran; Z50103, Italy], An. melanoon [GenBank: AJ224330, Italy], An. daciae [GenBank: AY634502], An. artemievi [GenBank: AJ849886], An. beklemishevi [GenBank: AY593958], An. hermsi [GenBank: M64483], An. freeborni [GenBank: M64484] and An. occidentalis [GenBank: M64482]. Bootstrap values under 50% have not shown.