Article pp.116-121 du Vol.110 n°2 (2017)

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MEDICAL ENTOMOLOGY /ENTOMOLOGIE MÉDICALE

Morphological Studies on Adult Mosquitoes (Diptera: Culicidae) and First Report of the Potential Zika Virus Vector

Aedes (Stegomyia) unilineatus (Theobald, 1906) in Iran

Études morphologiques des moustiques adultes (Diptères : Culicidés) et première évaluation du potentiel vectoriel d’Aedes(Stegomyia)unilineatus(Theobald, 1906) pour le virus Zika en Iran

M.R. Yaghoobi-Ershadi · S. Doosti · F. Schaffner · S.H. Moosa-Kazemi · K. Akbarzadeh · N. Yaghoobi-Ershadi

Received: 27 July 2016; Accepted: 2 August 2016

AbstractBeside numerous extensive studies on Anopheli- nae mosquitoes of Iran, little is known onAedesspecies in the country and existing reports are dispersed. The objective of this study was to identify adults of Culicinae species occurring in the Sistan and Baluchestan Province, southeast of Iran, during 2012–2014. Mosquito collections were carried out three times (May–June, September, October– November) in four counties by Centers for Diseases Control (CDC) light traps and human landing catches outdoors and under bed nets baited with carbon dioxide. These trapping were carried out two consecutive nights during the field studies. Several mosquito collections were also conducted with aspirator and pyrethrum spray space catches during the day. A total of 1885 mosquitoes were collected, belonging to 10 species of genusCulexincluding Cx. pipiens Com- plex, Cx. laticinctus, Cx. sinaiticus, and Cx. tritaeniorhynchus, one species of the genus Culiseta,Cs. longiareolata, and five species of the genus Aedes, Ae. caspius, Ae. vexans,Ae. detritus,Ae. albopictus, andAe. unilineatus.

Ae. vexanswas the dominant species in the area (77.7%).

During the study, sevenAe. unilineatus were collected in two villages near the city of Chabahar located in a coastal

area; this is the first record for Iran and identification was confirmed by Cytochrome oxidase (COI) sequences analysis. Confirmation of the presence ofAe. unilineatusin the country raises the number of species of the genusAedesto 12. The detection of this species reveals its probable establishment in the southeast of the country, which has implications for public health such as dengue and Zika infections and requires active entomological surveillance and implementation of adapted vector control measures in the area.

Keywords Culicinae ·Culex quinquefasciatus ·Culex laticinctus·Culex sinaiticus·Culex tritaeniorhynchus· Aedes caspius·Aedes vexans·Aedes detritus·Aedes albopictus·Aedes unilineatus· Paroomi · Voshnamdorr · Chabahar · Konarak · Saravan · Zabol · Dengue virus · Zika virus · Sistan and Baluchestan Province · Iran · Middle South Asia

Résumé Bien qu’il existe de nombreuses études très complètes sur les Anophélinés d’Iran, on connaît mal la diversité de leurs espèces, et les rapports qui existent sont difficiles à trouver. Le but de cette étude est d’identifier les imagos de Culicinés trouvés dans la Province du Sistan- et-Balouchistan, au sud-est de l’Iran, durant les années 2012 à 2014. Les collectes de moustiques ont été réalisées sur trois périodes (mai/juin, septembre, octobre/novembre) dans trois départements à l’aide de lampes trappes CDC, par captures humaines à l’extérieur des maisons et avec des moustiquaires imprégnées de dioxyde de carbone. Ces collectes ont été menées deux nuits consécutives au cours de chaque étude de terrain. Plusieurs collectes ont également été réalisées de jour à l’aide d’aspirateurs et par des pulvér- isations de pyrèthre. Au total, 1 885 moustiques ont été collectés appartenant à dix espèces du genre Culex faisant partie du complexe.Culex quinquefasciatus, Culex laticinctus, Culex sinaiticus et Culex tritaeniorhynchus, quelques

M.R. Yaghoobi-Ershadi (*) · S. Doosti · S.H. Moosa-Kazemi · K. Akbarzadeh

Department of Medical Entomology & Vector Control,

School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

e-mail : [email protected] ; [email protected] F. Schaffner

Francis Schaffner Consultancy, Riehen, Switzerland

National Center for Vector Entomology, Institute of Parasitology, University of Zürich, Switzerland

N. Yaghoobi-Ershadi

Polytechnic University of Madrid, Madrid, Spain DOI 10.1007/s13149-016-0530-1

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espèces du genre Culiseta,Cs. longiareolata, et cinq espèces du genre Aedes,Aedes caspius, Aedes vexans, Aedes detritus, Aedes albopictus et Aedes unilineatus. Ae. vexans était l’espèce la plus représentée dans cette zone (77,7 %).

À l’occasion de cette étude, septAe. unilineatusont été col- lectées dans deux villages près de la ville de Chabahar située sur la côte. C’est la première identification de cette espèce en Iran et elle a été confirmée par l’analyse des séquences COI.

La présence d’Ae. unilineatus dans le pays y fait passer à 12 le nombre d’espèces du genre Aedes. La découverte de ces espèces montre leur probable installation au sud-est du pays avec des implications possibles en santé publique sur la dengue et l’infection par le virus Zika, et elle nécessite une surveillance entomologique active ainsi que la mise en place de mesures adaptées de contrôle vectoriel dans cette zone.

Mots clésCulicinae ·Culex quinquefasciatus · Culex laticinctus·Culex sinaiticus·

Culex tritaeniorhynchus·Aedes caspius·Aedes vexans· Aedes detritus·Aedes albopictu·Aedes unilineatu· Dengue · Zika · Paroomi · Voshnamdorri · Chabahar · Konarak · Saravan · Zabol · Province du Sistan- et-Balouchistan · Iran · Moyen-Orient

Introduction

Among all insect pest and vector species, mosquitoes are of paramount importance, being a threat to human, livestock, and wildlife health. Evolution and diversity of the mosquito fauna have allowed these insects to colonise almost all envir- onments. A large number of the world mosquito fauna belong to the subfamily Culicinae, which includes 11 tribes. Genus Aedes(sensu Wilkerson et al. [19]), represents one-third of the members of the subfamily Culicinae, and some of them are vectors of arboviruses such as dengue, Yellow fever, chikungunya, Rift Valley fever, Ross River, and Zika [19].

Sixty-two mosquito species including 21 and 41 from the subfamilies Anophelinae and Culicinae, respectively, are cur- rently known to be occurring in Iran (School of Public Health, Tehran University of Medical Sciences, unpublished data).

Among these are several potential vectors of pathogens and parasites such as malaria plasmodia, dengue, West Nile, and Sindbis viruses, dirofilarial and setarial worm. Moreover, occurrences of epidemics of mosquito-borne viral infections such as dengue, chikungunya, West Nile, and Rift Valley fevers in neighboring countries and risk of introduction of exotic vectors into Iran has raised the country at a significant risk level for these severe human diseases [4].

Although there are a number of informative reports on Anophelinae mosquito fauna of Iran, little is known about Aedesspecies in the local context as well as in southwestern

Asia in general. Therefore, studies targeting these species are of crucial importance in a public health perspective.

Aedesspecies of Iran have been mainly studied by Dow in 1953, Gutsevich in 1974, Minar in 1974, and finally mosquito catalogue of the world by Knight and Stone in 1977 who reported six species of the genusAedes: Ae. vexans,Ae.

geniculatus,Ae. cabalus,Ae. caspius,Ae. pulchritarsis, and Ae. aegypti [5,7,10,11]. Zaim et al. in 1984 identified five other species of the genusAedes,Ae. vittatus,Ae. echinus, Ae. detritus,Ae. flavescens, andAe. leucomelas[22]. Doosti et al. in 2016 reportedAe. albopictusfor the first time in the southeast of the country [4].

Materials and methods Study area

The study was carried out during 2012–2014 in four counties:

Chabahar, Konarak, Saravan, and Zabol in the Sistan and Baluchestan Province (25°3′–31°27′N, 58°50′–63°21′E).

The province is the largest in the country with an area of 187,502 km². It borders over about 1260 km with Pakistan and Afghanistan and 300 km with Oman Sea. Date, banana, citrus fruit, mango, papaya, pistachio, and vine are grown in the area, as well as wheat, barely, Indian corn, potato, tomato, alfalfa, rice, onion, garlic, fig, vegetables, olive, and cumin.

The area is arid with annual precipitation varying between 70 and 200mm and annual temperature between 22°C and 33°C. The maximum temperature is higher than 40°C in all counties. The mean humidity ranges between 70% and 80%

around the coastal area of Oman Sea.

Mosquito collections

Field studies were carried out three times (May–June, Sep- tember, October–November) during 2012–2014, coincident with mosquito activity in different parts of selected rural and urban areas of four counties: Chabahar, Konarak, Saravan, and Zabol in the Sistan and Baluchestan Province. Human landing catches were conducted outdoors (in gardens, stables, and animal shelters) under bed nets baited with carbon dioxide cylinder and with one or more sides tacked up about 30 cm, from 14:00 to 20:00h (during the period of maximum biting activity forAedes species, with two local men hired as baits). Four CDC miniature light traps baited with carbon dioxide cylinders were suspended in animal shelters or on trees near breeding sites or in the middle of the village. Traps were operated from sunset to sunrise. Ani- mal baits included a calf, a young goat, or a camel placed near traps from 14:00 to 20:00h. All mosquito collections were carried out for two consecutive nights.

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In addition, several mosquito collections were conducted outdoors with aspirators during the day. Adult mosquitoes were also collected indoors with aspirators and pyrethrum spray space catches. Adult mosquitoes were pinned in the field and kept in collection boxes. A collection form was used to record all pertinent data for adult collections in the field. All specimens were transferred to the laboratory of Medical Entomology at the School of Public Health, Tehran University of Medical Sciences, for identification.

Morphological identification

Morphological identification of females and some male mosquitoes were carried out using the keys of Becker et al.

[3], Huang [8], and the interactive CD of Schaffner et al.

[16]. Specimens tentatively identified as Ae. unilineatus were sent to the third author’s laboratory for morphological and molecular confirmation. Some specimens ofAe. unili- neatuswere deposited at the museum of Medical Entomol- ogy, Department of Medical Entomology & Vector Control, Tehran University of Medical Sciences.

Genetic identification

DNA isolation and amplification of part (460 bp) of the mitochondrial cytochrome oxidase subunit I gene (mt COI) using primers C1-J-1718_mod (5′-GWGGRTTTGGWAA YTGAYTAG-3′) and C1-N-2191_mod (5′-GTAAAAT- TAAAATATAAACTTCTGG-3′) at a concentration of 1 μM each were done as described [15]. Amplifications were performed in 50μl volumes containing master mix of the multiplex PCR kit (Qiagen, Hildesheim, Germany) with the following reaction conditions: Initial DNA polymerase activation at 95°C for 15min, followed by 40 cycles at 95°C

for 30s, annealing at 50°C for 30s, and extension at 72°C for 60 s. A final elongation step at 72°C for 10min was included. Sequencing of the purified amplicons (minelute PCR kit, Qiagen) was done by a private company (Syner- gene, Switzerland).

Results

In total, 1885 mosquitoes were collected belonging to 10 Culicinae species (Table 1). They consisted of four species of the genusCulex,Cx. pipiensComplex, 5.1%,Cx. laticinctus 1.5%, Cx. sinaiticus 0.2%, and Cx. tritaeniorhynchus 2.4%; one species of genus Culiseta, Cs. longiareolata 0.2%, and five species of genusAedes,Ae. caspius10.5%, Ae. vexans77.7%,Ae. detritus1.6%,Ae. albopictus 0.4%, andAe. unilineatus 0.4%. Table 2 shows the places where species were collected in each of the four counties of the Sistan and Baluchestan province. Species richness is consid- erable in Chabahar where 9 out of 10 species have been identified. During the study seven Ae. unilineatus (0.4%) were detected among the specimens (6♀and 1♂) from the villages of Paroomi (25.45011 N, 60.93016 E) and Vosh- namdorri (25.41010 N, 60.75012 E) located in a coastal locality and situated 25 km from the city of Chabahar for the first time in November 2013 (Fig. 1). All of these specimens were collected on human bait between 12:00 (noon) and 19:00 PM under the bed net and shade of trees baited with slow releasing carbon dioxide cylinder. CO1 sequences (462 bp) of two specimens (one male and one female) were 99.53% identical toAe. unilineatusGenBank and Barcode of Life Data Systemes (BOLD) entries. This sequence is deposited on GenBank under the accession num- bers KU351084 and KU351085.

Table 1 Relative abundance of adult Culicinae mosquitoes collected by different methods in Sistan and Baluchestan Province, southeastern Iran, 2012–2014 / Abondance relative des Culicinés adultes collectés par diverses méthodes dans la Province du Sistan- et-Balouchistan, sud de l’Iran, 2012-2014.

Counties

Species Chabahar Konarak Saravan Zabol N %

Cx. pipiens complex 29 0 67 0 96 5.1

Cx. laticinctus 19 0 10 0 29 1.5

Cx. sinaiticus 1 0 3 0 4 0.2

Cx. tritaeniorhyncus 5 38 2 0 45 2.4

Cs. longiareolata 0 0 3 0 3 0.2

Ae. caspius 150 3 1 45 199 10.5

Ae. vexans 1390 75 0 0 1465 77.7

Ae. albopictus 7 0 0 0 7 0.4

Ae. detritus 18 12 0 0 30 1.6

Ae. unilineatus 7 0 0 0 7 0.4

Total 1626 128 86 45 1885 100

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Aedes (Stegomyia) unilineatus(Theobald) Diagnosis

Maxillary palpus with white scales, scutum with dorsocentral setae, scutum without a distinct patch of broader cress-shaped white scales on fossal area, scutum with a median longitudinal white stripe of narrow scales extending from anterior margin to about the level of wing root, subspiracular and postspira-

cular areas with broad white scales, hypostigmal and meta- meron areas with broad white scales, paratergite with broad white scales, scutellum with broad white scales on all lobes, white knee-spot present on all femora, all tibiae anteriorly dark, without any white lines, midfemur with a large white spot on anterior surface, hindtarsus with a basal white band on tarsomeres 1–4, hind tarsomere 5 all white, female fore- and midlegs with tarsal claws equal, all toothed, and male fore- and midlegs with tarsal claws unequal, all toothed [8].

Table 2 Contribution of adult Culicinae mosquitoes collected in four counties in Sistan and Baluchestan province, southeastern Iran, 2012–2014 /Distribution des Culicinés adultes récoltés dans 4 contés de la Province du Sistan-et-Balouchistan, sud de l’Iran, 2012-2014.

Counties

Species Chabahar Konarak Saravan Zabol

Cx. pipiens complex + +

Cx. laticinctus + +

Cx. sinaiticus + +

Cx. tritaeniorhyncus + + +

Cs. longiareolata +

Ae. caspius + + +

Ae. vexans + + +

Ae. albopictus +

Ae. detritus + +

Ae. unilineatus +

Fig. 1 Localities of collection ofAe. unilineatusin Chabahar, Sistan and Baluchestan Province, 2012–2014 /Localisation des collectes de Ae. unilineatus à Chabahar, Province du Sistan-et-Balouchistan, 2012-2014

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Discussion

The tribe Aedini contains 1261 species and is the largest among the 11 tribes within Culicinae mosquitoes (3119 species). Many species of the genusAedesare major vectors of human pathogens and arboviruses such as West Nile, Rift Valley fever, dengue, chikungunya, and Zika viruses, as well asWuchereria bancroftiworms and so forth [19].

During our entomological survey in southeastern Iran in 2012–2014, the presence ofAe. unilineatuswas detected for the first time along withAe. albopictuson human bait near an area covered with bush in November 2013 in the villages of Paroomi and Voshnamdorri, 25 km from the coastal city of Chabahar. It is located in an area which has warm and humid summers and moderate winters. The highest average temperature is 31.1°C in July and the lowest average temperature is 18.8°C in January. Precipitation is the lowest in May with average of 0mm but most precipitation falls in January with an average of 34 mm (Meteorological station, Sistan and Baluchestan Province, personal communication).

The adults ofAe. unilineatusresembles very much toAe.

albopictus but the existence of a single median line with white scales on the scutum and a small white scale patch on the anterior face of the mid-femur allows differentiation fromAe. albopictus. The specimen were examined by the third author morphologically and also by DNA barcoding to confirm identification. Molecular studies with COI gene revealed that the genetic structure of the species is very sim- ilar to specimen from Pakistan, registered in GenBank [1]. It is possible thatAe. unilineatuswas introduced into Iran from Pakistan by population or goods movement. Confirmation of the presence ofAe. unilineatusin the country increases the number of species of genusAedesto 12 in Iran.

This species has been reported from Angola, Burkina Faso, Gabon, Ghana, Kenya, India, Malawi, Nepal, Nigeria, Nyasaland, Senegal, Sudan, Pakistan, Saudi-Arabia, South Africa, and Zambia [12,20]. Data on the ecology ofAe. uni- lineatusare scarce. Most researchers have collected its larvae in tree holes and rock holes [2,9,14]. In 2014, Rohi Naz collected larvae from Bamboo glasses and discarded con- tainers in Pakistan [13].

Although the main vector of Zika virus isAe. aegypti, Ae. unilineatus, and Ae. albopictus are in the list of 20 other species which have been tested positive to this virus [17]. Natural infection of Ae. unilineatus to Zika virus has been reported from southeastern Senegal by Diallo et al. in 2014 [6]. The minimum infection rate (estimated number of positive mosquitoes per 1000 mosquitoes tested has been calculated to be 250.00 and entomological inocula- tion rate (number of infected mosquito bites per person per evening) was 0.01 in June, 2011 in Senegal.

The local transmission of Zika virus has been reported in almost 60 countries and territories, of which 46 have experi- enced a first outbreak since 2015. The situation was declared as a public health emergency of international concern by the World Health Organization in 2016 [21]. Finding ofAe. unilineatusin southeast of Iran is of importance and it can be suspected to be distributed all along the southern coastal areas of the country. This mosquito species has been reported as a probable vector of dengue in Karachi, Pakistan, and it should be pointed out that serological evidence of Zika virus has also been recorded from this territory neighboring Iran [18]. BothAe. unilineatusandAe. albopictusmay have been introduced in the southeast of Iran by ships coming from Karachi. Their presence is a threat for dengue, Zika, chikungunya, and Rift Valley fever viruses in the south of Iran and national public health authorities might increase preparedness and remain vigilant forever.

Chabahar is officially designated as a Free Trade and Industrial zone by the government of Iran in 1992. The area reached its importance from its geographical location as the shortest route connecting central Asian independent states and Afghanistan to warm waters and its proximity to one of the largest oil, gas, and mineral resources of the world and as the only ocean port of the country. Recently India, Iran, and Afghanistan have extended their relations with India being supposed to finance the development of Chaba- har port, within a project creating regional trade, investment and transportation hub with links from the Indian Ocean to central Asia. The establishment of Ae. unilineatusandAe.

albopictus in Chabahar port could cause significant health problems in the coming years and it may be impossible to eradicate them but continuous entomological surveillance and active application of control measures are challenged.

It is worth mentioning that common sources of information such as newspapers and television should be used to inform about vector control and in particular community participa- tion for reduction of larval habitats and personal preventive measures on Zika and dengue infections on a large scale.

Conclusion

The purpose of the current study was to determine adults of Culicinae species occurring in the Sistan and Baluchestan Province because of existence of epidemics of some mosquito-borne viral infections in neighboring countries and risk of introduction of exotic vectors into Iran. The study showed that 10 species of the genusCulex, one species of the genus Culisetaand five species of the genusAedes are present in the province. Ae. (Stegomyia) unilineatus the potential Zika virus vector is reported for the first time that was confirmed by CO1 sequence analysis. Finding of this mosquito species discloses its establishment in the country,

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which has indications for dengue and Zika infections and requires continuous entomological surveillance and active implementation of control measures in the area.

AcknowledgmentsWe thank Dr. M.M. Gooya, Head of the Communicable Diseases Management Center and Dr. M.R.

Shirzadi, Chair of zoonosis section of Iranian Ministry of Health and Medical Education for their collaboration and support. We also thank Dr. Akbari, Dean of School of Public Health, Tehran University of Medical Sciences for his kind support and encouragement. Thanks are also due to the staff at the Sistan and Baluchestan Province Health Center for their assistance in the fieldwork. This research was finan- cially supported by Iran National Science Foundation, Proj- ect no. 86052/18 and partly by Tehran University of Medical Sciences: grant no: 88-03-27-9200.

Conflict of interest:The authors do not have any conflict of interest.

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