J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 135 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Molecular Characterization of Anopheles sacharovi Based on Sequences of ITS2-rDNA Region and COI Gene in North of Iran Sahereh Gholami1; Hasan Bakhshi1,2; Seyyed Hassan Moosa-Kazemi1; Alireza Zahraei- Ramazani1; Alireza Chavshin3; *Mohammad Mehdi Sedaghat1 1Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 3Department of Medical Entomology, School of Public Health, Urmia University of Medical Sciences, Urmia, Iran (Received 31 Aug 2018; accepted 10 Apr 2019) Abstract Background: Malaria is an important mosquito-borne disease considered as one of the public health concerns across many countries. Anopheles mosquitoes are the main vectors of Plasmodium parasites, which cause malaria. Some of these vectors such as Anopheles maculipennis s.l. and Anopheles sacharovi are considered as complex of sibling species distributed in north of Iran. Methods: This study was conducted in north and northwest of Iran including East Azerbaijan, West Azerbaijan, Ar- dabil, Golestan and North Khorasan provinces with emphasis on the northern borders of the country during 2015– 2016. Adult specimens were collected and subjected to morphological identification as well as molecular analysis. Results: Overall, 10405 mosquitoes were collected comprising 21 species. Culex pipiens and Cx. theileri were found as the most frequent species in whole study area. Morphological identification showed that out of 1455 female Anoph- eles specimens, 77% belonged to An. maculipennis Group. Out of the identified species, ITS2 region and COI gene sequences of 8 An. maculipennis s.s. and 31 An. sacharovi representing all provinces were obtained and submitted to GenBank. The COI sequences for An. sacharovi revealed the presence of 9 haplotypes with similarity of 98.17–100%. Conclusion: Some investigations have reported An. martinius as a member of sibling species of An. sacharovi among Iranian Anopheles genus; while based on our study, there was no evidence of the presence of this species in north and northwest of Iran. Keywords: Anopheles sacharovi; Anopheles martinius; COI; ITS2-rDNA, Iran Introduction Anopheles mosquitoes are responsible for transmission of malaria parasites in humans. There are 30 definitive reported species, 3–4 biological forms and geographical races of Anopheles in Iran. There are seven primary malaria vectors recognized in Iran including An. stephensi, An. culicifacies s.l., An. fluviatilis s.l., An. superpictus s.l., An. dthali, An. macu- lipennis s.l. and An. sacharovi (1). Some of the most important species are in the Maculipennis group comprised as a primary or secondary vector of malaria parasites in the Palaearctic Region (2, 3). There is a report on the bionomics of An. maculipennis and An. sacharovi from Iran and Iraq and the distribution of the two spe- cies in central and northern areas of Iran (4). Anopheles maculipennis s.l. was reported in central and northern areas of the country (5). Twenty-two species of Anopheles were listed in Iran based on literature records. The list included An. martinius among Iranian Anoph- eles species (6); although, there is no evidence of An. martinius occurrence in Iran so far. As a result of recent molecular genetic studies, DNA sequence data are available for *Corresponding author: Dr Mohammad Mehdi Sedaghat, E-mail: sedaghmm@tums.ac.ir J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 136 http://jad.tums.ac.ir Published Online: June 24, 2019 identification of the members of the complex. Anopheles persiensis was described as a new species of the An. maculipennis complex in north of Iran (2). Two members of An. maculi- pennis complex including An. maculipennis and An. sacharovi have been reported as vectors of malaria parasites in central and northern parts of the country. They are also considered as vectors of Plasmodium parasites from Arme- nia, Azerbaijan and Turkey (7). Members of the An. maculipennis complex are distributed mostly in northern and central areas of the country (8). The An. maculipennis complex comprises several sibling species including major vec- tors of malaria parasites of historic Europe (9). Currently, there are 10 members of the An. mac- ulipennis complex in the Palearctic Region in- cluding An. melanon, An. messeae, An. per- siensis, An. sacharovi, An. martinius, An. atro- parvus, An. daciae, An. labranchiae and An. artemievi (10). Out of the complex, six species including An. maculipennis s.s., An. maculi- pennis, An. persiensis, An. messeae, An. atropar- vus, An. labranchiae and An. sacharovi have been identified in Iran based on molecular methods (3). Anopheles melanoon and An. messeae are listed as Iranian species based on the egg morphology studies as well as mo- lecular studies (8). This study was carried out based on the molecular and morphological characters of An. maculipennis s.l. collected from north and northwest of Iran with emphasis on border lines including East Azerbaijan, West Azerbaijan, Ardabil, Golestan and North Khorasan prov- inces where are considered as important bioge- ographic regions, being the corridor between Europe and Asia. These provinces have share borders with five countries including Turkmen- istan, Armenia, Azerbaijan, Iraq and Turkey where the members of the An. maculipennis complex play an important role in malaria trans- mission. The potential occurrence of An. mar- tinius, a close species of An. sacharovi, was also considered in this study. Materials and Methods Adult mosquitoes and larvae were collected from five provinces located in north of Iran during 2015–2016 using standard methods (Fig. 1, Table 1). Animal bite traps and shelter pit methods were used for collection of adults. The collection of larvae was carried out by dip- ping method. All samples were identified to the species level by using morphological keys (11). These identifications were used to target specimens for molecular identification using ribosomal internal transcribed spacer II (ITS2-rDNA) re- gion and cytochrome oxydase I (COI) gene to differentiate cryptic species within the An. mac- ulipennis complex. Genomic DNA of the mos- quitoes was extracted using (G-spine ™) DNA Extraction Kit, according to the manufactur- er's instructions. Reactions were carried out in a total volume of 20µl using the PCR kit. The desired ITS2 fragments were amplified by using universal 5.8S (5´-TGTGAACTGC AGGACACATGAA-3´) as the forward and 28S (5´-ATGCTTAAATTAGGGGGTAGTC- 3´) as the reverse primers (12). The PCR con- ditions were as follows: 94 °C for 2min, fol- lowed by 25 cycles of 94 °C for 20sec, 50 °C for 15sec, and 70 °C for 25sec and terminat- ing with a 72 °C for 5min. The desired COI fragments were amplified using LCO1490 (5´- GGTCAACAAATC ATAAAGATATTGG-3´) as the forward and HCO2198 (5´-TAAACTT CAGGGTGACC AAAAAATCA-3´) as the re- verse primers (13). The PCR conditions were as follows: 94 °C for 2min, followed by 5 cy- cles of 94 °C for 30sec, 45 °C for 40sec, and 72 °C for 1min, followed by 35 cycles of 94 °C for 30sec, 55 °C for 30sec, and 72 °C for 1min, respectively, terminating with a 72 ºC for 5min. Accuracy and quality of the amplicon were examined using a 1% agarose gel and visualized by Gel Doc after staining with Sina- colon® (Tehran, Iran) safe stain. DNA chroma- tograms were inspected using Chromas software (Version 2.23) and the sequences were submit- ted to GenBank. Similarity with other sequenc- J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 137 http://jad.tums.ac.ir Published Online: June 24, 2019 es in GenBank was assessed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) online tool. Phylogenetic tree was constructed by MEGA7 (ver. 7.0.21) software (Molecular Evo- lutionary Genetic Analysis) using Maximum Likelihood method with 1000 replicates of bootstrapping. Phylogenetic tree of fifteen COI sequences obtained from this study (An. sacharovi: 11 and An. maculipennis: 4) and four COI Genbank sequences as outgroup (KU950429: An. martinius, KM224658: An. melanoon, KM258220: An. messeae and KU 380466: An. gambiae) were created (Fig. 2). Results Morphological investigations Out of 10405 specimens, 6556 adult sam- ples and 3849 larvae were collected. Morpho- logical identifications revealed that the spec- imens represented five genera and 21 species including An. maculipennis s.l., An. sacharovi, An. claviger, An. hyrcanus, An. superpictus s. l., An. psudopictus, Culex hortensis, Cx. pipiens, Cx. theileri, Cx. modestus, Cx. mimeticus, Cx. perexiguus, Cx. tritaeniorhynchus, Aedes cas- pius, Ae. geniculatus, Ae. vexans, Ae. flavescens, Ae. echinus, Culiseta longiareolata, Cs. sub- ochrea and Uranotaenia unguiculata. Culex pipiens (30.2%), Culiseta loniareolata (23.8%) and Cx. theileri (22.3%) were the dominant species, and accounted for 76.3% of the col- lected samples (Table 2). Culex pipiens and Cx. theileri were found as frequent species in whole study area. Uranotaenia unguiculata (n=1, 0.009%), Cs. subochrea (n=4, 0.003 %) and Ae. vexans (n=4, 0.003%) were considered as infrequent species. Uranotaenia unguicu- lata was only collected in West Azerbaijan Province. Among Aedes mosquitoes, Ae. caspius (1.2%) was found in whole study area and Ae. flavescens in sympatry with Ae. vexans in Ar- dabil Province. Among Anopheles mosquitoes, An. clavi- ger and An. hyrcanus were widespread across the whole study area. Anopheles maculipennis s.l. (5.8%) was found in five provinces and An. sacharovi (0.3%) was found in sympatry with An. maculipennis s.l. only in three provinces located in northwest. Anopheles superpictus s.l. was also found with frequency of 3.9%. The last three species are the main malaria vectors in Iran. Molecular investigations Anopheles maculipennis s.l. and An. sacha- rovi were subjected to molecular study using ITS2 and COI sequences. Eight sequences of An. maculipennis s.s. for the ITS2 region (n=4) and COI gene (n=4) were obtained and the se- quences submitted to GenBank under accession numbers KY225560, KY225561, KY225562, KY225563 and KY196448, KY196449, KY 196450, KY196462 for ITS2 and COI regions respectively. All specimens were identified as An. maculipennis s.s. by an identity of 100 %. Thirty one sequences for An. sacharovi for the ITS2 region (n=19) and COI gene (n=12) were obtained and the sequences were sub- mitted to GenBank under accession numbers KY225557-KY225559, KY263795-KY263806 and KY 196451-KY196461 for the ITS2 and COI regions respectively. All ITS2 sequences for An. sacharovi were identical with 100% similarity. The COI sequences for An. sacha- rovi showed that there were 9 haplotypes with similarity of 98.17–100%. The base composi- tion of the COI fragments showed an AT bias with all sequences being between 66.4 and 67.8% AT rich (mean= 66.8%). Seventeen single nucleotide polymorphisms among the haplotypes were observed (Table 3). Based on the COI sequences of An. sacha- rovi and An. maculipennis s.s. (about 601bp), phylogenetic tree was constructed. A tree for 15 COI sequences including 11 An. sacharovi, 4 An. maculipennis as well as 3 sequences of An. maculipennis complex deposited in Gen- Bank (AN: KU950429 (An. martinius), KM 224658 (An. melanoon), KM258220 (An. mes- seae) and KU380466 (An. gambiae) was con- structed (Fig. 2). The phylogenetic tree revealed the inter-population differences of the studied https://blast.ncbi.nlm.nih.gov/Blast.cgi J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 138 http://jad.tums.ac.ir Published Online: June 24, 2019 species. The smallest genetic distance was shown between the An. sacharovi populations from three provinces including East Azerbai- jan, West Azerbaijan and Ardabil provinces ra- ther than Golestan and North Khorasan prov- inces. The three mentioned provinces are close- ly located in northwest of the country (Fig. 1). Moreover, An. melanoon, An. messeae and An. martinius were recognized in separate clades from all An. sacharovi and An. maculipennis s.s. populations. Fig. 1. The study area: 1. North Khorasan, 2. Golestan, 3. Ardabil, 4. East Azerbaijan, 5. West Azerbaijan Table 1. Geographical coordinates of the study areas No. Province County Latitude Longitude 1 West-Azerbaijan Poldasht 39° 20' 49.69'' N 45° 3' 59.61'' E Shahindej 36° 40' 26.74'' N 46° 34' 12.48'' E Oshnavieh 37° 2' 11.17'' N 45° 5' 49.69'' E Makoo 39° 17' 44.24'' N 44° 30' 51.07'' E 2 Ardabil Parsabad-Moghan 39° 37' 14.80'' N 47° 54' 18.22'' E Aslan-Duz 39° 26' 29.57'' N 47° 24' 40.25'' E Meshgin Shahr 38° 23' 41.38'' N 47° 39' 53.46'' E 3 East-Azerbaijan Kaleybar 38° 51' 51.27'' N 47° 2' 25.94'' E Azarshahr 37° 44' 39.54'' N 45° 59' 13.95'' E 4 North-Khorasan Bojnord 37° 28' 12.74'' N 57° 18' 51.61'' E Shirvan 37° 24' 33.25'' N 57° 55' 39.42'' E 5 Golestan Gorgan 36° 50' 44.31'' N 54° 26' 21.61'' E J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 139 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 2. Phylogenetic tree constructed by 15 COI sequences (about 601bp) obtained from Anopheles sacharovi=11 and Anopheles maculipennis=4. The sequences KU950429: Anopheles martinius, KM224658: Anopheles melanoon, KM258220: Anopheles messeae and KU380466: Anopheles gambiae (as the out-group) were derived from GenBank J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 140 http://jad.tums.ac.ir Published Online: June 24, 2019 Table 2. Species composition of mosquitoes collected from East-Azarbaijan: EA, West-Azarbaijan: WA, Ardabil: A, Golestan: G, North-Khorasan: NKh No. Species Adults Species Species Larvae Adults (%) Larvae (%) Total (Adults and Larvae) (%) EA WA A G NKh EA WA A G NKh 1 An. maculipennis 88 97 33 19 24 - 155 118 - 72 261 (3.98) 345 (8.96) 606 (5.8) 2 An. sacharovi 26 3 5 - - - - - - - 34 (0.51) 34 (32) 3 An. claviger 64 43 99 14 2 - 99 50 - 104 222 (3.38) 253 (6.57) 475 (4.56) 4 An. hyrcanus 12 - 23 20 9 - - - - - 64 (0.97) - 64 (0.61) 5 An. superpictus - 3 - 13 116 - 15 - - 266 132 (2.01) 281 (7.3) 413 (3.96) 6 An. psudopictus - - - 15 - - - - - 15 (0.22) - 15 (0.14) 7 Cx. hortensis 120 9 - 20 3 - 180 106 - 28 152 (2.31) 314 (8.15) 466 (4.48) 8 Cx. pipiens 1326 9 63 109 46 - 336 1254 - 8 1553 (17.58) 1598 (41.51) 3151 (30.28) 9 Cx. theileri 1703 14 - 39 13 - 281 243 - 35 1769 (26.98) 559 (14.525) 2328 (22.38) 10 Cx. modestus - 3 - - - - 47 50 - - 3 (0.04) 97 (2.52) 100 (0.96) 11 Cx. mimeticus - - - - 17 - 7 6 - 13 17 (0.25) 26 (0.67) 43 (0.41) 12 Cx. perexiggus - - 3 17 3 - - 18 - 3 23 (0.35) 21 (0.54) 44 (0.42) 13 Cx. tritarincus - - - - - - - 15 - - - 15 (0.38) 15 (0.14) 14 Ae. caspius 1 6 32 31 8 - - 33 - 15 78 (1.18) 48 (1.24) 126 (1.21) 15 Ae. geniculatus - 4 - 6 - - - - - - 10 (0.15) - 10 (0.096) 16 Ae. vexans - - - - - - - 4 - - 4 (0.1) 4 (0.038) 17 Ae. flavecence - - - - - - - 11 - - 11 (0.28) 11 (0.1) 18 Ae. echinus - - - 15 - - - - - - 15 (0.22) 15 (0.14) 19 Cs. loniareolata 2080 42 - 71 11 - 215 55 - 6 2204 (33.61) 276 (7.17) 2480 (23.83) 20 Cs. subochrea - - - 4 - - - - - - 4 (0.06) 4 (0.038) 21 Ur. ungiuiculata - - - - - - 1 - - - - 1 (0.02) 1 (0.009) Total (%) 5420 (52.09) 234 (2.24) 258 (2.48) 393 (3.78) 252 (2.42) 1336 (12.83) 1963 (18.86) - 550 (5.3) 6556 (63) 3849 (37) 10405 (100) 140 http://jad.tums.ac.ir Published Online: June 24, 2019 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 141 http://jad.tums.ac.ir Published Online: June 24, 2019 Table 3. DNA sequence comparison of about 601bp of COI gene of Anopheles sacharovi distributed in the study area. Totally 9 haplotypes were identified within the sequenced samples, *: non-synonymous base change, Dots show identical sequences to the top sequence Position 38 116 155 170 203 209 278 323 326 341 347 368 401 487 509 557 563 Poldasht T G A T A T C A T C A G A C G A A Azarshahr . . . . . . . . . . . . . . . . . Parsabad- Moghan . . . . . . . . . . . . . . T . . Shahindej . . . . . . . . . . . . . . T . . Makoo . . . . . . . . . . . . . . T . . Kaleybar . . . . . . . G . . . . . . T . . Azarshahr . . G . . . . . . . G . . . . . G Poldasht C A . C G C T . . . G A . . T G . Kaleybar . . . . . . . . . . . . G . T . . Parsabad- Moghan . . G . . . . . C . G A . G* . . . Oshnavieh . . . . . . . . . . G . . . T . . Aslan-Duz . . . . . . . . . T . . . . T . . Discussion The Maculipennis subgroup currently com- prises 10 members including An. artemievi, An. atroparvus, An. daciae, An. labranchiae, An. maculipennis, An. martinius, An. melanoon, An. messeae, An. persiensis and An. sacharovi. Six members of An. maculipennis complex have been identified as primary or secondary vec- tors of malaria parasites in the Palearctic Re- gion (2). These members are very close relat- ed species which are difficult to be identified by morphological characteristics. Although it is possible to distinguish An. sacharovi from other members by morphological characteris- tics, An. martinius is remained as a sibling spe- cies of An. sacharovi which makes it impos- sible to identify them by their morphological characteristics; these two species can be de- tected by cytological studies (14). Although the occurrence of An. martinius in Iran had been mentioned, there is no evidence for distribu- tion of this species in the country so far. The most ambiguity is the distribution of An. mar- tinius in east of Caspian Sea and east of Iran. Although it was reported in 1941 (15), but there is no new evidence of occurrence of this spe- cies in north of Tajikistan as well (16). This is in concordance with another study (17). Cy- togenetic study showed that only An. maculi- pennis s.s. was present in this region. Howev- er, An. artemievi described as a homosequential species with An. maculipennis, could be erro- neously identified as An. martinius (18). Anoph- eles artemievi is a new and predominate spe- cies in Kyrgyzstan, where it was identified as An. martinius (18). On the other hand, there are cytological or molecular evidences for occur- rence of An. martinius in northeast of Turkmen- istan, the Turkmen-Khorasan Mountain Range, Karakalpakstan and the Khorezm areas of Uz- bekistan (19, 20). Our molecular studies on the An. macu- lipennis complex were conducted to elucidate J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 142 http://jad.tums.ac.ir Published Online: June 24, 2019 the possible occurrence of An. martinius. Mem- bers of the An. maculipennis complex were identified by sequence analysis of the ITS2- rDNA and COI gene. Morphological charac- ter-based identification showed that out of 1455 female Anopheles specimens, 1121 (77%) belonged to An. maculipennis complex. Mo- lecular analysis of the complex indicated the presence of An. sacharovi and An. maculipennis s.s. in northwest and north of Iran. This result is in agreement with other studies (2, 3). Three genetically distinct species of the An. maculi- pennis complex were reported in Iran (2): An. maculipennis s.s., An. sacharovi and An. per- siensis. However, the last species was not found in this study as it was found as a dominant species of the complex in the southern Caspi- an Sea littoral provinces of Guilan and Ma- zandaran (2). Six members of the group were reported based on molecular approach, while there was no An. martinius in the study area in northern Iran (21). No species of this com- plex was found but An. maculipennis s.s. based on molecular study in Zanjan Province located in the northwest of Iran (22). Anopheles mac- ulipennis s.s. and An. sacharovi were found in nine provinces from northwest to central re- gions of Iran, it was no molecular evidence for presence of An. martinius either (23). Molecular and phylogenetic analysis of the present investigation indicates more spe- cies diversity of An. sacharovi than has been recognized up to now. Divergence among the members of mosquito complexes varies but can be fundamental. Twenty-two species of Anoph- eles were reported in Iran including An. mar- tinius (6). Apparently, there is no molecular evidence of An. martinius presence across the whole study area. The results correspond with other findings (24). The base composition of the COI frag- ments showed an AT bias with all sequences being between 66.4% and 67.8% AT rich (mean=66.8%). These levels fall within the range of AT bias in mitochondrial genomes of other members of An. maculipennis com- plex including An. maculipennis (25), An. mes- seae (26), An. sacharovi (2, 3) and An. mar- tinius (AN: KU950429). The COI sequences of An. sacharovi showed that there were 9 unique mtDNA haplotypes with similarity of 98.17–100%. The sequences were translated into amino acids to obtain the mitochondrial code. Translation of the sequences into amino acids showed all but one of the twelve speci- mens shared the same amino acid haplotype. Only one specimen (KY196454) from Parsa- bad-Moghan in Ardabil Province showed two nucleotide transversion (G C, C G) at the second and third codon position bases of 478 and 479bp (Table 3). These nonsynonymous bases change altered the codon from the con- sensus CCG (Arginine) to CGC (Proline), thus resulting in a unique amino acid haplotype. Conclusion We collected three important malaria vec- tors in north of Iran. The permanent presence of historical vectors of pathogens results in po- tential epidemiological threats. Some malaria foci have been spotted in the northwest of Iran. Significant increases in commercial activities and travel from the neighbouring countries has led to increase concern about malaria and oth- er vector-borne diseases in the Northern prov- inces. This has increased the concern of ma- laria cases occurrence in the area. A better un- derstanding of the accurate identification of sympatric sibling species and their distribu- tions remain important as the malaria control programs depend on the accurate identifica- tion of the vectors. Here, there is no evidence for occurrence of An. martinius in north of the country. Acknowledgements The authors are grateful to Prof Mohebali and Eng. Zarei for cooperation during the study. This study was financially supported by Tehran University of Medical Sciences (Grant No. 94-02-27-28914). javascript:popRef('f3') J Arthropod-Borne Dis, June 2019, 13(2): 135–144 S Gholami et al.: Molecular Characterization of … 143 http://jad.tums.ac.ir Published Online: June 24, 2019 The authors declare that there is no con- flict of interests. References 1. Salahimoghadam A, Khoshdel A, Barati M, Sedaghat M (2015) An overview and mapping of Malaria and its vectors in Iran. Hormozgan Med J. 18(5): 473–485. 2. 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