Article pp.31-37 du Vol.111 n°1 (2018)

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ENTOMOLOGIE MÉDICALE /MEDICAL ENTOMOLOGY

Genetic Population Diversity of Aedes caspius in Southern Provinces of Iran

Diversité génétique des populationsd’Aedes caspius dans les provinces sud de l’Iran

S. Doosti · M.R. Yaghoobi-Ershadi · M.M. Sedaghat · S.H. Moosa-Kazemi · K. Akbarzadeh · S.S. Hashemi-Aghdam

Reçu le 6 juin 2017 ; accepté le 26 janvier 2018

AbstractAedes caspiushas a wide distribution throughout the world and can transmit Chikungunya virus, West Nile Virus (WNV), Tahyna virus and the bacterium Franci- sella tularensis. Sequences of the mitochondrial cytochrome C-oxidase subunit 1 (COI) and cytochrome C-oxidase subunit 2 (COII) genes have been widely used to estimate phylogenetic relationships at different taxonomic levels among this species. Adult collections were carried out by human bait, Center for Deseases Control Light Traps (CDC-LT) and aspirator during February/April, June and October/

December 2013–2015 from different southern provinces of Iran and then identified morphologically with reliable keys.

A total of 3,570 adult mosquitoes were collected and identified as belonging to three genera, including five species of Culex, six species of Aedes and one species of Culiseta. In this study, 1,796 specimens ofAedes caspiuswere identified from four provinces. Based on the COI and COII sequences obtained forAe. caspius population, 12 and 11 haplotypes were identified, respectively. The present study evidenced a high degree of intraspecific variation among these populations ofAe. caspius.

Mots clésCulex perexiguus·Culex pipiensComplex · Culex laticinctus·Culex sinaiticus·

Culex tritaeniorhynchus·Aedes albopictus· Aedes unilineatus·Aedes vittatus·Aedes detritus· Aedes vexans ·Culiseta longiareolata·Aedes caspius· diversité génétique des populations · COI · COII · Sistan-et-Baluchestan · Hormozgan · Bushehr · Khuzestan · Iran · Asie du Sud-Est.

RésuméAedes caspiuslargement distribué dans le monde est le vecteur du virus du chikungunya, du virus West Nile, du virus Tahyna et de la bactérieFrancisella tularensis. Les séquences des gènes mitochondriaux COI et COII sont généralement utilisées pour estimer les relations phylogéné- tiques entre les différents niveaux taxonomiques de cette espèce. Des moustiques adultes ont été collectés sur appâts humains, à l’aide de pièges lumineux de type « CDC/light- trap » et d’aspirateurs en février/avril, juin et octobre/

décembre 2013–2015 dans les provinces au sud de l’Iran.

Les moustiques ont ensuite été identifiés d’un point de vue morphologique. Trois mille cinq cent soixante-dix moustiques adultes ont été collectés et identifiés comme apparte- nant à trois genres, dont cinq espèces de Culex, six espèces d’Aedes et une espèce de Culiseta. Dans cette étude, 1 796 spécimens d’Aedes caspius ont été identifiés dans quatre provinces. Les séquences COI et COII des populations d’Ae. caspius ont permis d’identifier respectivement 12 et 11 haplotypes. Les résultats de cette étude ont démon- tré qu’il existe une forte variation intraspécifique dans ces populations d’Ae. caspius.

Keywords Culex perexiguus·Culex pipienscomplex · Culex laticinctus ·Culex sinaiticus ·

Culex tritaeniorhynchus·Aedes albopictus · Aedes unilineatus ·Aedes vittatus·Aedes detritus· Aedes vexans·Culiseta longiareolata·Aedes caspius· Population diversity · COI · COII · Sistan-and-Baluchestan · Hormozgan · Bushehr · Khuzestan · Iran · Middle South West Asia

Introduction

Among mosquitoes, Aedes is one of the best-known genera, the species of which are important vectors of arboviruses and pathogens such as dengue fever, yellow fever, Chikungunya fever, encephalitis, filariasis, for instance [3,12,14,16,18]. It is

S. Doosti · M.R. Yaghoobi-Ershadi (*) · M.M. Sedaghat (*) · S.H. Moosa-Kazemi · K. Akbarzadeh · S.S. Hashemi-Aghdam Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

e-mail : yaghoobia@sina.tums.ac.ir, sedaghmm@tums.ac.ir DOI 10.3166/bspe-2018-0011

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a large tribe of mosquitoes with 1,255 species classified in 10 genera [34]. There is relatively little information about this genus in Iran. Twelve species ofAedeshave been reported in Iran till now, including Aedes vexans, Aedes geniculatus, Aedes caballus, Aedes caspius, Aedes pulchritarsis, Aedes detritus,Aedes flavescens,Aedes leucomelas,Aedes vittatus, Aedes echinus[36],Aedes albopictusandAedes unilineatus[11].Aedes caspiushas a wide distribution throughout the world from Europe to Mongolia, north and west China, North Africa, West and Middle Asia. It is a polycyclic and halophylic species. Females are strongly exophagic but can enter inhabited areas, houses and cattle sheds [15]. They often bite during the day as well as at night, but usually search for a blood meal actively at dusk. They may migrate for long dis- tances, up to 10 km [4].Aedes caspiuscan transmit West Nile virus (WNV), Tahyna virus and the bacterium Franci- sella tularensis[10]. This species has a wide distribution in Iran and has been reported from many provinces of the coun- try, including Guilan, West Azarbaijan, Zanjan, Lorestan, Khorassan, Esfahan, Yazd, Kerman, Fars, Bushehr, Hormoz- gan, Khuzestan and Sistan & Baluchestan [41].

Aedes caspiushas been reported as a complex of species in Europe [8] and Lebanon [19]. In Italy, based on the iso- morphic analysis of populations ofAe. caspius, two forms of species were detected, A and B [10], and similar findings were reported in France [27].

Effective control of mosquitoes requires correct identification. Different molecular studies, such as DNA-based approaches, showed that they were very practical to rapid description of species and biodiversity [5,17,22].

Use of DNA barcode techniques based on a single gene in Culicinae species and analysis of DNA sequences of this gene may provide a useful tool for species identification and could be advantageous and practicable. Among the mitochondrial genes, cytochrome C-oxidase subunit 1 (COI) has been reported to be the most conserved gene with regard to the amino acid sequences, and therefore shows distinct advantage for taxonomical studies [20]. In addition, the sequence of the mitochondrial cytochrome C-oxidase subunit 2 (COII) gene has been widely used to estimate the

phylogenetic relationships at different taxonomic levels among the species.

Here, we present the DNA barcoding ofAe. caspius s.l., which is distributed in different parts of the southern provinces of Iran.

Materials and methods Study area

The study was carried out in four provinces (Sistan and Balu- chestan, Hormozgan, Bushehr and Khuzestan, (Fig. 1)) based on the reports of some cases of dengue fever from the province of Sistan and Baluchestan [9] and other provinces being at risk of dengue fever transmission (Table 1). The southern coastal plains of Iran have mild winters but scorching hot and exceedingly humid summer days. The temperature sometimes exceeds 49 °C during the months of July and August. The total annual precipitation varies between 90 and 600 mm, and the relative humidity varies from 30% to 90% in the studied provinces.

Table 1 Range of altitudes and biomes of studied provinces of Iran, and mean annual precipitation during the study period, 2013–2015 /Altitudes, biomes et précipitations annuelles moyennes des provinces iraniennes étudiées, pendant la période d’étude 2013–2015

Province Altitude (m a.s.l.) Biome Total annual

precipitation (mm)

Max Min

Sistan and Baluchestan 1 394 8 Subtropical desert, warm temperate desert scrub 89.3

Hormozgan 30 2.14 Subtropical desert 176.1

Bushehr 940 0 Subtropical desert 268

Khuzestan 1457 3 Subtropical desert scrub 209.2

Fig. 1 Map showing the study areas in Iran, 2013–2015 /Carte de la zone d’étude en Iran, 2013–2015

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Mosquito collection

Adult collections were carried out using human bait under bed net with carbon dioxide gas which was released slowly and also by three CDC light traps in different parts of Sistan and Baluchestan, Hormozgan, Bushehr and Khuzestan provinces during three sessions (February/April, June, October/

December) 2013–2015 (Fig. 1). Adult mosquitoes were also collected by aspirator from 14:00 to 20:00 hrs (during the period of maximum biting activity for Aedes sp.) in indoors, transferred into paper cups and stored in cold box. All the data including date, temperature, humidity, collector’s name and time of collection were noted on the collection forms in the field. All specimens were transported to the laboratory at the Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences. Specimens were identified morphologically with reliable keys of Zaim and Cranston [40,41], the an interactive Compact Disc of Schaffner et al. and Becker et al. [4].

Isolation of genomic DNA

Mosquito DNA was extracted using the G-spine™Total kit with Cat No 17045. Genomic DNA extraction was carried out according to the manufacturer instructions by grinding of individual mosquito in an Eppendorf micro tube and then tubes were sealed with parafilm and stored at–4 °C.

PCR amplification

The primers used for COI in the present study (barcode) were : the LCO 1490 (5′-GGTCAACAAATCATAAAGA- TATTGG-3′) with 710 bp and HCO 2198 (5′-TAAACTT- CAGGGTGACCAAAAAATCA-3′) [15] and for COII, A-tLEU (5′-ATGGCAGATTAGTGCAATGG-3′) and HCO 2198 (5′-GTTTAAGAGACCAGTACTTG-3′) with 730 bp [22]. The total polymerase chain reaction (PCR) mixture of 25μl was made by mixing 20.5μl (ddH2O), 2μl of premix 10X (Intron, Korea), 1μl each of forward and reverse primer and 0.5μl of template DNA in each tube and then the tubes were placed on a thermal cycler.

The PCR protocol for COI (barcode) includes two cycling stages. For the initial 5 cycles, the steps are as follows: initial denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 45 °C for 40 s and extension at 72 °C for 1 min. For the next 35 cycles: denaturation at 94 °C for 30 s, annealing at 1 °C for 40 s and extension at 72 °C for 1 min.

PCR protocol for COII includes 40 cycles: initial denaturation at 95 °C for 5 min, denaturation at 93 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 90 s.

A final extension was carried out at 72 °C for 10 min and the temperature was held at 4 °C.

COI gene (barcode) and COII sequencing

The COI (barcode) and COII genes were submitted for sequencing to Fazapajhoh Bio tech. Ltd, Tehran, Iran. Deter- mination of sequence done in GenBank by BlastN and aligned with ClustalW was checked using basic local alignment search tool (BLAST) analysis software (www.ncbi.

nlm.nih.gov/BLAST).

Results

A total of 3,570 adult mosquitoes were collected and identified. They belong to three genera, including five species of Culex(Culex perexiguus,Culex pipiensComplex,Culex laticinctus,Culex sinaiticus,Culex tritaeniorhynchus), six species ofAedes(Aedes albopictus, Aedes caspius, Aedes unilineatus, Aedes vittatus, Aedes detritus, Aedes vexans) and one species of Culiseta (Culiseta longiareolata). In this study, 1,796 specimens of Aedes caspius were identified from four provinces including Sistan and Baluchestan (n= 199), Hormozgan (n= 23), Bushehr (n= 227) and Khu- zestan (n= 1347) during the period 2013–2015.

Genomic DNA of Ae. caspiuswas extracted after morphological identification of 30 individual specimens from the study areas (Table 2). From these, 14 COI (barcode) and 16 COII sequences were derived and aligned together and with otherAe. caspiussamples obtained from Genbank.

The alignment length of the sequences was 710 bp for COI (barcode) and 730 bp for COII gene (Figs 2,3) The phylogram of these sequences revealed 12 and 11 separate haplotypes, respectively (Figs 4,5).

Based on the COI and COII sequences obtained for Ae. caspius, phylogenetic trees were constructed. The COI sequences forAe. caspiusshowed 12 haplotypes with a 99% similarity. Regarding the phylogram of Ae. caspius for COI (barcode), the specimen numbers 28 and 29 from Zabol and Chabahar counties (Sistan and Baluchestan) are very close to Ae. caspius of Turkey (GenBank accession number HM535298) and are associated with the same clade. The specimen number 16 from Zabol is located in another clade and is similar to Ae. caspius from France, Greece, Serbia, Romania and Turkey (GenBank accession numbers HM535266, HM535291, HM535287, HM535297 and HM535299) (Fig. 3). In addition, specimen 44 from Bandar Lenge (Hormozgan) was located in the same clade and similar to the Spain and Belgium samples (GenBank accession numbers HM535287 and KM258356). Other specimens were similar to Iranian specimens recorded in GenBank and were included in the same clade (GenBank accession numbers FJ210902, FJ21090, FJ210903 and J210904).

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The COII sequences forAe. caspius showed that there were 11 haplotypes with 99% similarity. According to phylogenetic tree based on COII gene, specimens 29 and 33 (from Sistan and Baluchestan province), 31 and 41 (from Bushehr province) and 34 (from Khuzestan province) shared the same clade and others were placed in different clades.

Figure 5 shows that three specimens from Bushehr, four specimens from Hormozgan and one specimen from Sistan and Baluchestan are very closely related to each other and share the same clade. Two specimens from Sistan and Baluchestan

and one specimen from Hormozgan were similar to Ae. caspiusof Italy (GenBank accession number DQ300496, DQ300494, DQ300491 and DQ300483) and were placed in the same clade (Fig. 5).

The COI (barcode) haplotypes sequenced from the study areas showed that there were 25 different nucleotides in alignment and the intraspecific variation amongAe. caspiuspopu- lation was 4.2%, whereas the sequenced COII gene haplotypes showed 17 differentiations in the nucleotide alignment, and the calculated intraspecific variation was 3.2%.

Fig. 2 PCR production of COI (barcode) gene, 710 bp, inAedes cas- piuspopulation from: 1, 2: Sistan and Baluchestan; 3, 4: Khuzestan;

5: Bushehr; 6, 7: Hormozgan; N: negative control /PCR des gènes COI (code-barres), 710 bp, chez les populations d’Ae. caspius au : 1, 2 : Sistan and Baluchestan, 3, 4 : Khuzestan, 5 : Bushehr, 6, 7 : Hormozgan ; N : témoin négatif

Table 2 Number of PCR products used for sequencing Aedes caspiusbased on COI and COII genes, place and date of collection in the south of Iran, 2013–2015 /Nombre de PCR utilisées pour le séquençage d’Ae. caspius sur la base des gènes COI et COII, lieux et dates des collectes au sud de l’Iran, 2013–2015

Provinces County COI (barcode) code COII code Collection date

Sistan and Baluchestan Zabol 16 16, 24 June 2014

Chabahar 29 29 April 2013

Zabol 28 28 June 2014

Konarak 33 33 December 2013

Hormozgan Doran 53 53 December 2014

Bandar Lenge 44 44 October 2014

Sirik 45 45 November 2014

Roudan 36 36 December 2014

Bandar Khamir 43 43 October 2014

Bushehr Tangestan 30 30 October 2014

Bushehr 46 46 March 2014

Bushehr – 31 February 2015

Khuzestan Abadan 34 34 March 2014

Total number of sequencing reactions performed forAedes caspius

14 16 30

Fig. 3 PCR production of COII gene, 730 bp, inAedes caspius populations from: 1, 2: Sistan and Baluchestan; 3: Hormozgan; 4, 5: Khuzestan; 6, 7: Bushehr; N: negative control /PCR des gènes COII, 730 bp, chez les populations d’Ae. caspius au : 1, 2 : Sistan and Baluchestan, 3 : Hormozgan, 4, 5 : Khuzestan, 6, 7 : Bushehr ; N : témoin négatif

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Discussion

The taxonomy of some mosquito species of the genus Aedes is still questionable [30–32,35,38]. Comparative analysis of morphological characters showed insufficient effectiveness in solving the problems of taxonomy and establishing relationships in some species [7,16,31].

Genetic study of the Ae. caspius populations provides good information for a better understanding of interspeci- fic genetic divergence [21,23,34]. Particular attention was drawn on the fact thatAe. caspiuscan be a potential vector of human pathogens [26].

For more comprehensive understanding of the role of Ae. caspius as a disease vector, it is needed to review up- to-date results of the taxonomy, biology, genetic variability, genetic relationships and distribution of this mosquito species [28].

The subgenus Ochlerotatus includes many salt marshes, inland or littoral mosquitoes. About one-half of them have been registered in the Holarctic and nearly one-quarter in each of the Australian and the Neotropical regions [4]. The taxonomy of the Caspius group within and with other taxa of the family Culicidae is still a matter of disagreement. It is assumed that the ancestor of this group originated in Central Asia and later spread to Western Europe and North America [24,33]. The classification of Ochlerotatus as a genus was followed by some workers but ignored by others [6,13]. Pre- viously, Ochlerotatus was assumed to be an individual genus, but Wilkerson’s studies in 2015 changed its status to subgenus according to a new classification system [39].

Gutsevich et al. [16] showed that, this group had four species within the subgenusOchlerotatus:caspius(compri- sing caspius, mariae and pulchritarsis), cantans, rusticus and communis. Based on the morphological characters, Stacklberg (1937) classified into two subspeciesAe. caspius caspius(occurring in saline marshes of Central Russia) and Ae. caspius dorsalis(in lakes and ponds of lower alkalinity in Northern Russia), and an intermediate form (in overlap- ping area in the Volga valley) [36].

Cianchi et al. (1980) in Italy have defined two sibling species, A and B, forAe. caspiuscomplex [10]. Minar [25]

noted that the specimens from Iran showed typical characters and there was no sibling species here, but our results showed 12 haplotypes based on COI (barcode) gene, whereas Azari et al. [1] showed four haplotypes based on this gene with intraspecific variation of 0.46% in Abu-Musa Island, south of Iran. Moreover, intraspecific variation for this species was calculated to be 0.3% based on the same gene in Ardebil province, northwest of Iran [2]. In our study, the intraspecific variation was found to be 4.2% based on COI (barcode) gene and 3.2% based on COII gene. The differences in the results of these two studies are probably related to the limited area, Fig. 5 Dendrogram based on 830 bp COII gene sequence data

of the relationship of the haplotypes withinAedes caspius/Den- drogramme fondé sur le séquençage du gène COII 830 bp par rapport aux haplotypes chez Ae. caspius

Fig. 4 Phylogram based on 710 bp COI gene mt-DNA sequence data of the relationship of the haplotypes withinAedes caspius/ Phylogramme fondé sur le séquençage ADN-mt du gène COI 710 bp par rapport aux haplotypes chez Ae. caspius

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conducted only in Ardebil province, whereas the present survey was conducted in four provinces in the south of Iran that included different climates and covered a vast area.

In a study, which was carried out by Talbalaghi and Shai- kevich in northwest of Italy in 2011 [37], four unique haplotypes ofAedes caspiuswere found, based on the sequences of COI gene (the barcode fragments) and Internal Transcribed Spacer Region 2 (ITS2). These sequences were achieved from six populations. The individuals investigated differed by less than 1%. Three populations were identical in the 563-bp DNA fragment, and DNA in one population varied by only one nucleotide [37]. Porretta et al. in 2011 [29] recognized 67 haplotypes from 112 individuals considered (haplotype diversity = 0.971, nucleotide diversity = 0.0067) based on COI and COII genes in the Mediterranean region, north and central of Europe. In spite of the considerable genetic diversity, they found neither strong phylogenetic divergence among haplotypes (uncorrected mean sequence divergence of 0.8%) nor any phylogeographic arrangement throughout the study region.

Our study evidences a high degree of intraspecific variation among populations ofAe. caspiuscoming from different regions. These areas represent about one-third of surface area of Iran with different climatic conditions, which affect the population dynamics and genetic diversity. It is suggested that comprehensive studies should be carried out based on specific gene such as ITS2 to determine the population genetic diversity and the probable presence of sibling species in Ae. caspiusin the southern provinces of Iran (Table 2).

AcknowledgmentsThe authors would like to thank Dr MM Gooya, Head of the Communicable Diseases Management Center, and Dr MR Shirzadi, Zoonosis Section, Iranian Ministry of Health and Medical Education, for their collabo- ration and support. We also thank Prof Akbari, Dean of the School of Public Health, Tehran University of Medical Sciences, for his kind support and encouragement. Thanks are also due to the staff at the Center of Health Services for their assistance in the fieldwork and city residents for their cooperation during the survey. This research was financially supported by Iran National Science Foundation (INSF) (pro- ject no 86052/18) and partly by Tehran University of Medi- cal Sciences (grant no 88-03-27-9200)

Conflict of interest: Authors do not have any conflict of interest to declare.

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