Firooziyan et al. Malar J (2018) 17:366 https://doi.org/10.1186/s12936-018-2523-y Malaria Journal RESEARCH Open Access Speculation on the possibility for introducing Anopheles stephensi as a species complex: preliminary evidence based on odorant binding protein 1 intron I sequence Samira Firooziyan1,2, Navid Dinparast Djadid3 and Saber Gholizadeh1,2* Abstract Background: Anopheles stephensi is considered an important malaria vector in Iran, Asia, and recently in the Horn of Africa. Recently, Ansteobp1 intron I sequence has been introduced a new molecular marker for identifcation of its biological forms including, mysorensis, intermediate and type, using insectary colony specimens. Methods: In the current study, new marker ability in molecular identifcation of biological forms has been evaluated with An. stephensi specimens collected from Iran and Afghanistan malarious provinces. Following DNA extraction and PCR amplifcation, sequence analysis and constructed phylogenetic tree revealed that type and intermediate forms are distributed in Iran. Results: The specimens collected from Afghanistan identifed as intermediate and mysorensis forms. Therefore, intermediate form is sympatric species in both countries. Based on the results of Ansteobp1 intron I sequences, An. stephensi could be suggested as new Anopheles complex species including An. stephensi sibling A (type form), An. stephensi sibling B (intermediate form) and An. stephensi sibling C (mysorensis form). This is the frst report on the pres- ence of An. stephensi biological forms in Afghanistan. Conclusions: Iran is going to eliminate malaria transmission from the country, precise species identifcation, espe- cially in complex species will be helpful in the prevention of malaria resurgence in the country, mainly because of common fauna of Anopheles species and through border malaria and population movement within Afghanistan, Pakistan, and Iran. Keywords: Anopheles stephensi complex species, An. stephensi sibling A, An. stephensi sibling B, An. stephensi sibling C Background reported as a malaria vector in the African continent in Anopheles stephensi is one of the approximately 60 Djibouti, on the horn of Africa. Te high susceptibility of Anopheles species considered in malaria transmission as this species to Plasmodium falciparum and its tolerance Asian malaria vector [1, 2]. Tis species is geographically to urban habitats may challenge the global malaria con- distributed in South Asia and the Arab Peninsula [3, 4]. trol and elimination programmes in the future [1]. Anopheles stephensi is a key vector in malaria transmis- About one century ago, in 1921, malaria was reported sion not only, in Iran and India, but also in Asia especially from Iran for the frst time [10]. Malaria control inter- in the Persian Gulf area [1, 3, 5–9]. Recently, this species vention was started in 1956 in the country [11]. Now, in the Eastern Mediterranean Region, the malaria con- trol programme is in the elimination phase in Iran and *Correspondence: [email protected]; [email protected] 2 Medical Entomology Department, School of Public Health, Urmia Saudi Arabia, however, transmission is still reported University of Medical Sciences, Urmia, Iran from southeastern parts of Iran [12]. Te number of Full list of author information is available at the end of the article malaria cases in Iran has been decreased from 1847 © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Firooziyan et al. Malar J (2018) 17:366 Page 2 of 7 (year) to 81 cases in 2016 [13]. Te total number of three forms has been reported from malarious regions malaria cases until the end of November 2017 was in Hormozgan, Sistan–Baluchistan, and Fars prov- 800 (736 imported and 63 indigenous) (A. Raeisi, pers. inces of Iran [25]. Recently, by using laboratory reared comm.). specimens of An. stephensi, AsteObp1 intron I has been So far, there are 30 Anopheles species in Iran based introduced as a new molecular marker for the identi- on morphological and molecular markers [14], of which fcation of mysorensis, intermediate and type forms of four species, including Anopheles maculipennis, Anoph- An. stephensi [4]. In the current study, AsteObp1 intron eles culicifacies, Anopheles fuviatilis, and Anopheles I was examined as a molecular marker for identifca- superpictus, belong to species complex based on ribo- tion of An. stephensi biological forms on feld-collected somal DNA internal transcribed spacer 2 (rDNA-ITS2) specimens from the Iran and Afghanistan. sequences [14–20]. However, despite various studies on An. stephensi, this species was just considered as races Methods or subspecies [4, 14]. Te available information is inad- Anopheles stephensi mosquitoes equate to introduce it as a complex species. Mosquitoes were collected form Hormozgan (Bandar- A population or series of populations of organisms Abbas district), Sistan and Baluchistan (Chabahar, that are capable of interbreeding freely with each other Nikshahr and Iranshahr districts) and Fars (Kazerun but not with members of other species is referred to district) provinces in Iran and Nangarhar province (Pol- species [21]. Tere are conficting reports on crossing e-tarache and Ali Khan villages) in Afghanistan by hand experiments between biological or geographical forms catch collection method in 2015 (Fig. 1). Te details of of An. stephensi. A defnite incompatibility existed in sampling locations were presented in Table 1. Adult An. cross-mating between An. stephensi type and mysoren- stephensi specimens were identifed morphologically sis biological forms [22]. However, intra-specifc vari- using keys to the adult females and fourth-instar larvae of ation in the reproductive capacity was demonstrated the mosquitoes of Iran [26]. in this species [23]. On the other hand, any hybrid ste- rility was not found in the crossing between An. ste- DNA extraction and PCR amplifcation phensi type and mysorensis strains collected from Iran, DNA was extracted from 100 An. stephensi specimens using India, and Iraq [24]. Based on egg morphology charac- YTA Genomic DNA Extraction Mini Kit (Yekta Tajhiz ters, An. stephensi has three biological forms including Azma, Tehran, Iran). Each mosquito was homogenized in type, intermediate and mysorensis. Distribution of all the 200-μl TG1 bufer using a micropestle. Te mixture was Nangarhar Kazerun Bandar Abbas Iranshahr Nikshahr Chabahar Fig. 1 Collection sites of Anopheles stephensi specimens in Iran and Afghanistan Firooziyan et al. Malar J (2018) 17:366 Page 3 of 7 Table 1 The details of Anopheles stephensi samples collected aligned and compared using Clustal Omega [27]. Te from Iran and Afghanistan fnal sequences were aligned with three representa- Locations Latitude (oN) Longitude (oE) Sequenced tive sequences in the GenBank. Te phylogenetic tree specimens was constructed using distance Neighbor-joining and Iran maximum likelihood Methods based on the Tamura– Bandar Abbas 27°11′11″ 56°15′29″ BaU1, BaU3 Nei model’s model in Molecular Evolutionary Genet- Chabahar 25°27′39″ 60°39′28″ ChU1, ChU2, ChU3, ics Analysis version 6.0. (MEGA6) [28]. Nucleotide ChU4, ChU5 sequences are available in the GenBank, European Nikshahr 26°14′14″ 60°13′46″ NiU1 Molecular Biology Laboratory (EMBL), and DNA Iranshahr 27°12′22″ 60°40′49″ IrU1, IrU3, IrU4 Data Bank of Japan (DDBJ) databases [GenBank ID: Kazerun 29°37′26″ 51°38′57″ KaU3 MG797525-MG797537]. Afghanistan Results Nangarhar 34°26′03″ 70°26′52″ 1-H-H, 2-H-H, 3-H- H, 4-H-H, 5-H-H An 845 bp fragment was amplifed in 100 feld-collected specimens from Iran and Afghanistan using OBP1F1 and OBP1R1 primers [4]. In total, 18 specimens were applied incubated at 60 °C for 1 h, after the addition of 20-μl pro- for direct sequencing form Iran (n = 13) and Afghani- teinase K. After incubation, TG2 bufer (200-μl) was added stan (n = 5), randomly. Te length of intron I region was and re-incubated for 10 min at 70 °C. following adding 200- 115 bp and 120 bp of sequenced specimens. Te compar- μl cold ethanol, the mixture was transferred to TG mini ison of these sequences with representative mysorensis column and centrifuged for 1 min at 8000 rpm. DNA was [GenBank: KJ557449], intermediate (KJ557452) and type washed two times with 500-μl and 700-μl of W1 and wash (KJ557463) biological forms intron I sequences showed bufers with a centrifuge for 1 min at 14,000 rpm, respec- that Afghani specimens were mysorensis (n = 2) and type tively. Te DNA was eluted from the column using 100-μl (n = 3) forms, while Iranian specimens were intermediate (n 4) and type (n 9) forms. Terefore, An. stephensi elution bufer and stored at − 20 °C until use. Te Anste- = = obp1 intron I region was amplifed using OBP1F1 (CGT type form was the prevalent biological form in Iranian AGG TGG AAT ATA GGT GG) as forward and OBP1R1 specimens. (TCG GCG TAA CCA TAT TTG C) as reverse primers [4]. Te sequences obtained from An. stephensi speci- PCR reactions of the Ansteobp1 intron I region were mens collected from Afghanistan were two groups. performed in a 25-μl volume of Master Mix (Yekta Te sequence similarity within each group was 100%, Tajhiz Azma, Tehran, Iran). Te optimized reactions while it was 86.67% between both groups. Te multiple sequence alignment of KT587049 and KT587051 with contained 12.5 μl of Master Mix, 8.5 μl ddH2O, 1 μl each of specifc primers, and 2 μl of genomic DNA.