MATERIALS AND METHODS

Study area: North Khorasan Province is located in northeastern Iran between 36°37′-38°17′ N latitudes and 55°53′-58°20′ E longitudes with an area of more than 28,400 km². The province is bordered by Turkmenistan in the North, Razavi Khorasan Province in the east and southeast, Semnan Province in the southwest and Golestan Province in the west (Fig. 1). It includes seven counties, Bojnord, Esfarayen, Faruj, Garmeh, Jajarm, Maneh and Samalghan and Shirvan, with a population of more than 811,000 inhabitants (according to the 2006 census). The province has desert and mountainous areas and receives about 250 mm of rainfall annually.

Fig. 1:	Map of Iran (highlighting the position of North Khorasan Province) and Middle Asia (Lambert Conformal Conic Projection map); maps are not the same scale

Specimen and data collection: Mosquito sampling was carried out during 2005 (July), 2006 (July and August), 2007 (September), 2008 and 2009 (July, August, September). Mosquito larvae were collected by means of pipette and dipper using the standard dipping technique (350 mL dipper); adults were collected from resting sites and while landing on human and dog bait by means of aspirator. The specimens were collected in different climatic areas of five of the seven counties: Bojnord, Esfarayen, Faruj, Maneh and Samalghan and Shirvan. They were transferred to the Medical Arthropod Museum, Department of Medical Entomology and Vector Control (DMEVC), School of Public Health (SPH), Tehran University of Medical Sciences (TUMS) where the project was conducted. Larvae were preserved in lactophenol and microscope slides were prepared using de Faure’s medium. Adult mosquitoes were pinned. The adults and third- and fourth-instar larvae were identified using the keys of Gutsevich et al. (1974), Harbach (1988) and Azari-Hamidian and Harbach (2009). The mosquito generic abbreviations follow Reinert (2009), which reflects the current formal classification of the family Culicidae (http://mosquito-taxonomic-inventory.info/). The specimens collected during the study were deposited in the Medical Arthropod Museum, DMEVC, SPH, TUMS.

Molecular identification of species: The specific identity of five adult mosquitoes, morphologically identified as Anopheles maculipennis s.l. [collected in Babaaman (37°28′ N 57°26′ E), Bojnord County, North Khorasan Province] was determined by sequencing the ITS2 region of ribosomal DNA (GenBank accession FJ210892). PCR reaction mixes and thermocycler parameters were those described previously by Linton et al. (2001). The ITS2 region was amplified using the universal 5.8S forward (5′-ATC ACT CGG CTC GTG GAT CG-3′) and 28S reverse (5′-ATG CTT AAA TTT AGG GGG TAG TC-3′) primers (Collins and Paskewitz, 1996). Sequences were edited and aligned using Sequencher^TM version 4.8 (Genes Codes Corporation, Ann Arbor, Michigan, USA) and similarities with sequences in GenBank were assessed using FASTA search (http:/www.ebi.ac.uk/fasta33/). Template DNA from this study is retained in the Molecular Systematics Laboratory of the Natural History Museum, London, for future reference.

RESULTS

In total, 682 adult mosquitoes representing 10 species of four genera were collected and identified based on morphology: Anopheles maculipennis s.l., An. pulcherrimus, An. superpictus, Culex modestus Ficalbi, Cx. perexiguus Theobald, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus, Ochlerotatus caspius s.l. and Uranotaenia unguiculata (Table 1, 2). The ITS2 sequences obtained from the five mosquitoes identified morphologically as An. maculipennis s.l. (GenBank accession FJ210892) shared 100% identity with An. maculipennis from Iran (Djadid et al., 2007; GenBank accessions AY730267-AY730268), Greece (Patsoula et al., 2007; DQ118166) and Romania (Nicolescu et al., 2004; AY634548 and AY634550).

The adult specimen of Ur. unguiculata was collected only from resting sites and those of An. pulcherrimus and Cx. perexiguus during night landing catches on human and on human and dog bait, respectively. The remaining species were collected during both resting and night landing catches (Table 2).

Six hundred and fifty-four larvae representing 11 species of five genera were collected: An. claviger, An. maculipennis, An. superpictus, Cx. hortensis, Cx. mimeticus, Cx. modestus, Cx. pipiens, Cx. theileri, Cs. longiareolata, Oc. caspius s.l. and Ur. unguiculata (Table 1).

Table 1:	Relative abundance of adult and larval mosquitoes collected in North Khorasan Province, Iran, 2005-2009

Table 2:	The adult mosquitoes collected by different methods in North Khorasan Province, Iran, 2005-2007

Culex mimeticus, Cx. modestus, Cx. perexiguus, Cx. pipiens and Oc. caspius s.l. are recorded from North Khorasan Province for the first time. Anopheles pulcherrimus, Cx. perexiguus and Cx. tritaeniorhynchus were collected only as adults and An. claviger, Cx. hortensis, Cx. mimeticus and Cs. longiareolata only as larvae (Table 1). Fourteen species representing five genera were collected overall (Table 1). Places where specimens of each species were collected are summarized below and the species collected in each of the five counties of North Khorasan Province are listed in Table 3.

Table 3:	Distribution of mosquitoes collected in five counties of North Khorasan Province, Iran, 2005-2009
Anopheles multicolor in Bojnord County and An. maculipennis in Shirvan County are based on Saebi (1987) and An. marteri in Bojnord County is based on records of the Institute of Malariology and Parasitology (1953), *: Present, -: Absent

Anopheles claviger-Shirvan County: Eslamabad (2 larvae); Bojnord County: Firoozeh (1 larva).

Anopheles maculipennis-Maneh and Samalghan County: Shoorak (2♀), Kashkabad (1♀, 11 larvae), Hassegah (4♀); Bojnord County: Babaaman (20♂, 2 larvae), Bojnord (5 larvae), Sangsaar (1 larva).

Anopheles pulcherrimus-Maneh and Samalghan County: Hassegah (1♀).

Anopheles superpictus-Maneh and Samalghan County: Kashkabad (62♀), Hassegah (1♀), Mehmanak (1♀), Borj (2♀), Keykanloo (2♀), Mohammadabad (1 larva), Kalimani (4 larvae); Esfarayen County: Rooeen (18 larvae); Bojnord County: Bachehdareh (19 larvae), Ayerghayeh (6 larvae), Firoozeh (4 larvae), Babaaman (1 larva), Armodli (6 larvae); Shirvan County: Eslamabad (1 larva).

Culex hortensis-Esfarayen County: Rooeen (32 larvae); Maneh and Samalghan County: Kashkabad (7 larvae), Mohammadabad (3 larvae) Kalimani (4 larvae); Shirvan County: Glian (2 larvae); Bojnord County: Firoozeh (4 larvae), Ayoob (3 larvae), Oshtoot (1 larva), Ayerghayeh (2 larvae).

Culex mimeticus-Maneh and Samalghan County: Borj (5 larvae); Bojnord County: Firoozeh (4 larvae).

Culex modestus-Maneh and Samalghan County: Hassegah (11♀), Kashkabad (4♀); Bojnord County: Babaaman (2♀, 1 larva), Sangsaar (1 larva).

Culex perexiguus-Maneh and Samalghan County: Kashkabad (14♀).

Culex pipiens-Maneh and Samalghan County: Kashkabad (3♀), Shoorak (2♀), Hassegah (1♀); Bojnord County: Bojnord (29♀), Babaaman (18 larvae) Yengeghale (27 larvae); Shirvan County: Glian (18 larvae), Eslamabad (3 larvae).

Culex theileri-Maneh and Samalghan County: Kashkabad (210♀), Hassegah (75♀), Keykanloo (1♀); Bojnord County: Bojnord (1♀, 7 larvae), Babaaman (4♀, 7 larvae); Faruj County: Segonbad (1 larva).

Culex tritaeniorhynchus-Maneh and Samalghan County: Kashkabad (3♀), Hassegah (17♀).

Culiseta longiareolata-Maneh and Samalghan County: Kashkabad (1 larva); Shirvan County: Glian (25 larvae); Bojnord County: Ayoob (33 larvae), southern Ayoob (94 larvae), Cheheldokhtar Ayerghgayeh (5 larvae), Yooghan Borj (22 larvae), Firoozeh (4 larvae), Oshtoot (61 larvae), Ayerghayeh (6 larvae); Faruj County: Segonbad (32 larvae).

Ochlerotatus caspius s.l.-Maneh and Samalghan County: Kashkabad (202♀), Hassegah (5♀), Keykanloo (1♀); Bojnord County: Babaaman (3 larvae), Ghazi (129 larvae).

Uranotaenia unguiculata-Maneh and Samalghan County: Hassegah (1♀); Bojnord County: Babaaman (5 larvae); Shirvan County: Eslamabad (2 larvae).

DISCUSSION

In total, 1,336 mosquito specimens (682 adults and 654 third- and fourth-instar larvae) representing 14 species and five genera were collected in North Khorasan Province during the surveys and identified based on morphology and ITS2 sequence. Four of the six species of Anopheles previously recorded in the province, including An. claviger, An. maculipennis, An. pulcherrimus and An. superpictus, were collected. Anopheles marteri and An. multicolor, which were recorded in unpublished documents (Institute of Malariology and Parasitology, 1953; School of Public Health, 1970; Saebi, 1987), were not found. Gaffary (1954) mentioned the possible occurrence of An. fluviatilis s.l. and An. moghulensis in northeastern Iran, but their occurrence in this area of the country has never been verified-they have only been found in southern and southeastern Iran, respectively (Saebi, 1987).

Of the species of the Maculipennis Group, only An. maculipennis has been recorded in northeastern Iran, based on molecular data (GenBank accession AY730264; Djadid et al., 2007). The ITS2 sequences of the An. maculipennis specimens analyzed in the present study (GenBank accession FJ210892 from Bojnord) have a 90% overlap with the sequence obtained by Djadid et al. (2007) and this region shares 99% identity; however, they share 100% similarity with many other sequences for this species from Iran (AY730267-AY730268; Djadid et al., 2007), Greece (DQ118166; Patsoula et al., 2007) and Romania (AY634548 and AY634550; Nicolescu et al., 2004). Seven species of the Maculipennis Group are listed in the most recent checklist of Iranian mosquitoes (Azari-Hamidian, 2007). There are no records for An. martinius (Shingarev) in the country; but its occurrence in northeastern Iran seems possible in view of its presence in Kazakhstan, Turkmenistan and Uzbekistan (Ejov, 2008). Anopheles maculipennis is a rare species in Middle Asia. It has been recorded only from Kopet Dag in southern Turkmenistan, adjoining Iran. Anopheles messeae Falleroni has been recorded in Kazakhstan, Kyrgyzstan and Turkmenistan, An. artemievi Gordeyev, Zvantsov, Goryacheva, Shaikevich and Yezhov in Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan and An. beklemishevi Stegnii and Kabanova in Kazakhstan (Ejov, 2008; Bezzhonova, Personal Communication). Also, Tarbinsky (1996) previously reported An. beklemishevi in Kyrgyzstan. The type localities of An. artemievi and An. martinius are in Kyrgyzstan (Ejov, 2008) and Uzbekistan (Knight and Stone, 1977), respectively. Anopheles artemievi, An. beklemishevi and An. martinius have not been recorded in Iran (Azari-Hamidian, 2007). Country records for other anopheline species are as follow: Anopheles algeriensis Theobald, An. barianensis James, An. claviger, An. hyrcanus, An. multicolor, An. plumbeus Stephens, An. pulcherrimus and An. superpictus have been recorded in Turkmenistan (Mamednyasov, 1995) and the recent report of An. sergentii sergentii in southern Turkmenistan is noteworthy (Mamednyazov and Yerokhin, 2005). Mamednyasov (1995) mentioned An. barianensis, a species similar to An. plumbeus, in Turkmenistan, but Gutsevich et al. (1974) had explained previously that the specimens from Tajikistan are typical An. plumbeus. Gutsevich et al. (1974) and Gutsevich and Dubitskiy (1987) did not mention An. barianensis in the checklist of the mosquitoes of the former U.S.S.R. In addition to its distribution in the Palaearctic Region, this species also occurs in India and Pakistan (Knight and Stone, 1977) of the Oriental Region. It seems that the presence of this species in Middle Asia is doubtful and needs to be verified.

Tarbinsky (1996) mentioned the occurrence of An. algeriensis, An. claviger, An. hyrcanus, An. pulcherrimus and An. superpictus in Kyrgyzstan. The type locality of An. chodukini Martini of the Hyrcanus Group is Tashkent, Uzbekistan and An. algeriensis, An. claviger, An. hyrcanus, An. pulcherrimus and An. superpictus have been found in the country (Gutsevich et al., 1974; Ejov, 2005, 2008). Within Middle Asia, An. marteri sogdianus Keshishian and An. lindesayi Giles are only recorded from Tajikistan (the type locality of An. marteri sogdianus is Stalinabad, Tajikistan), however Gutsevich and Dubitskiy (1987) doubted the occurrence of An. lindesayi and a subspecies of An. marteri in Middle Asia. Other anophelines recorded from Tajikistan include An. algeriensis, An. claviger, An. hyrcanus, An. plumbeus, An. pulcherrimus and An. superpictus (Gutsevich et al., 1974; Ejov, 2005, 2008). Finally, Gutsevich et al. (1974) recorded An. hyrcanus, An. pulcherrimus and An. superpictus in Kazakhstan. In total, 17 anopheline species (13 in the subgenus Anopheles and four in the subgenus Cellia) have been recorded in Middle Asia. Six of these species, An. artemievi, An. beklemishevi and An. martinius of the Maculipennis Group and An. barianensis, An. chodukini and An. lindesayi, have not been found in Iran; hence, 65% of the anopheline fauna of Middle Asia occurs in the country. However, at least 15 Anopheles species (and some unnamed sibling species) are known to occur in Iran, especially southern areas, which are not recorded in Middle Asia (Azari-Hamidian et al., 2006; Azari-Hamidian, 2007). Except for An. barianensis and An. lindesayi, the species of the subgenus Anopheles recorded in Middle Asia are exclusively Palaearctic (Gutsevich et al., 1974; Knight and Stone, 1977). Anopheles lindesayi (including four of its nominal subspecies) is mainly Oriental, but it is also recorded in Afghanistan and the eastern Palaearctic (China, Japan and Korea) (Gutsevich et al., 1974; Knight and Stone, 1977). Two species of the subgenus Cellia, An. multicolor and An. pulcherrimus, are found in the Palaearctic and western Oriental (India and Pakistan) Regions. The distribution of An. multicolor also extends into the Afrotropical Region (Yemen). Anopheles sergentii and An. superpictus are Palaearctic and Oriental (Pakistan) species, but An. sergentii macmahoni Evans is Afrotropical (Gutsevich et al., 1974; Knight and Stone, 1977). Species of the Hyrcanus Group in the western Palaearctic Region include An. chodukini, An. pseudopictus Grassi and An. hyrcanus (and its different forms and/or synonyms), but the taxonomic status of these nominal forms needs to be studied in detail (Azari-Hamidian et al., 2006). There are no records of An. pseudopictus in Middle Asia. Gutsevich et al. (1974) and Gutsevich and Dubitskiy (1987) considered the three forms to be a single polytypic species, i.e. An. hyrcanus. Recently, Poncon et al. (2008) suggested that An. hyrcanus and An. pseudopictus may belong to a single species in southeastern France and Djadid et al. (2009) proposed a new species of the group in southwestern Iran based on molecular evidence. Anopheles maculipennis and An. superpictus are known malaria vectors in Iran and An. pulcherrimus and An. hyrcanus are considered to be probable vectors in the southeastern and northern areas of the country, respectively (Edrissian, 2006; Djadid et al., 2009). Anopheles martinius, An. pulcherrimus and An. superpictus are assumed to be malaria vectors in Turkmenistan (Amangeldiev, 2001). This is important in view of the autochthonous cases of malaria in the area of Turkmenistan that borders Iran (Ejov, 2005). Anopheles messeae is a known malaria vector in Kazakhstan and Kyrgyzstan and An. pulcherrimus and An. superpictus are probable vectors in Tajikistan and Uzbekistan (Ejov, 2008). Anopheles maculipennis is a vector of Setaria nematodes in northwestern Iran (Azari-Hamidian et al., 2009).

The five species of the subfamily Culicinae previously recorded in North Khorasan Province, Cx. hortensis, Cx. theileri, Cx, tritaeniorhynchus, Cs. longiareolata and Ur. unguiculata (Zaim et al., 1985, 1986), were found in the present study. Also, Cx. mimeticus, Cx. pipiens and Oc. caspius s.l., which have wide distributions in Iran and were recorded in the former Khorasan Province (Zaim, 1987) and Culex modestus and Cx. perexiguus, which were not recorded previously, were collected for the first time in the province. Culex arbieeni, Cx. laticinctus, Cx. territans and Culiseta subochrea recorded in the former Khorasan Province by Zaim (1987) were not found in the present study.

Ochlerotatus caspius s.l., which is widely distributed in Iran, was found in this study, but none of the other aedine species are known to occur in northeastern Iran (Zaim, 1987). There is, in general, less information for aedine mosquitoes in the country than for other culicine mosquitoes. A species of Aedimorphus, i.e. Am. vexans (Meigen), a species of Fredwardsius Reinert, i.e., Fr. vittatus (Bigot), six species of Ochlerotatus and two species of Dahliana are documented in Iran. Ochlerotatus berlandi (Seguy), Oc. chelli (Edwards), Oc. dorsalis (Meigen) and Stegomyia aegypti (Linnaeus) are listed in old Iranian records, but they are not currently known to occur in the country (Azari-Hamidian, 2007). Aedimorphus vexans, Ae. cinereus Meigen, Ae. esoensis Yamada, Da. geniculata (Olivier) and 21 species of Ochlerotatus, Oc. campestris (Dyar and Knab), Oc. caspius s.l., Oc. cataphylla (Dyar), Oc. communis (De Geer), Oc. cyprius (Ludlow), Oc. detritus (Haliday), Oc. dorsalis, Oc. euedes (Howard, Dyar and Knab), Oc. excrucians (Walker), Oc. flavescens (Mueller), Oc. gutzevichi (Dubitsky and Deshevykh), Oc. kasachstanicus (Gutsevich), Oc. leucomelas (Meigen), Oc. montchadskyi (Dubitsky), Oc. pulcritarsis (Rondani), Oc. pullatus (Coquillett), Oc. punctor (Kirby), Oc. rempeli (Vockeroth), Oc. simanini (Gutsevich), Oc. stramineus (Dubitzky) and Oc. subdiversus (Martini), are recorded in Middle Asia (Gutsevich et al., 1974; Gutsevich and Dubitskiy, 1987; Mamednyasov, 1995; Tarbinsky, 1996). Mamednyasov (1995) listed Ae. cinereus, Am. vexans (as Ae. vexans), Da. geniculata (as Ae. geniculatus) and eight species of Ochlerotatus, including Oc. simanini [as Oc. niphadopsis (Dyar and Knab)], in Turkmenistan. Tarbinskyi (1996) mentioned the presence of Am. vexans, two species of Aedes, Da. geniculata (as Ae. geniculatus) and nine species of Ochlerotatus in Kyrgyzstan. Gutsevich et al. (1974) recorded Oc. caspius s.l. and Oc. pulcritarsis in Tajikistan and Oc. pulcritarsis and Oc. simanini in Uzbekistan (Kokand in the Fergana Valley of Uzbekistan is the type locality of Oc. simanini). Gutsevich et al. (1974) and Gutsevich and Dubitskiy (1987) listed Am. vexans, two species of Aedes and 18 species of Ochlerotatus in Kazakhstan. Gutsevich et al. (1974) indicated that Oc. caspius s.l. is widespread in Middle Asia. Of the species of the tribe Aedini that occur in Middle Asia, only seven (28%) are found in Iran. Three Iranian species, Fr. vittatus, Oc. caballus (Theobald) and Da. echinus (Edwards), are not found in Middle Asia (Azari-Hamidian, 2007). Aedimorphus vexans occurs principally in the Holarctic and Oriental Regions with extensions into the Australasian Region (not Australia) and Central America and populations in South Africa, Ae. cinereus is a northern Holarctic species and Ae. esoensis occurs in the Palaearctic Region, including far eastern Russia, Korea and Japan (Knight and Stone, 1977). Dahliana geniculata and nine species of Ochlerotatus, Oc. leucomelas, Oc. detritus, Oc. simanini, Oc. montchadskyi, Oc. stramineus, Oc. gutzevichi, Oc. kasachstanicus, Oc. cyprius and Oc. subdiversus, are only found in the Palaearctic Region and five species of the genus, Oc. dorsalis, Oc. communis, Oc. rempeli, Oc. cataphylla and Oc. euedes, are Holarctic. Four species, Oc. pullatus, Oc. flavescens, Oc. excrucians and Oc. punctor, occur mainly in northern areas of the Holarctic Region (Knight and Stone, 1977). Ochlerotatus pullatus also occurs in the Oriental Region (India and Pakistan) (Aslamkhan, 1971; Kaur, 2003). Ochlerotatus caspius s.l. and Oc. pulcritarsis have distributions in the Palaearctic and Oriental Regions: Oc. caspius s.l. is found in Pakistan (Aslamkhan, 1971) and Oc. pulcritarsis in Pakistan and Thailand (Knight and Stone, 1977). Of the Middle Asian aedine fauna, 10 species of Ochlerotatus (Oc. communis, Oc. montchadskyi, Oc. rempeli, Oc. gutzevichi, Oc. euedes, Oc. kasachstanicus, Oc. excrucians, Oc. subdiversus, Oc. cyprius and Oc. punctor) are only recorded from Kazakhstan (Gutsevich et al., 1974; Gutsevich and Dubitskiy, 1987) [Oc. kasachstanicus, Oc. montchadskyi, Oc. stramineus and Oc. gutzevichi were originally described from localities in Kazakhstan]. Mamednyasov (1995) and Gutsevich and Dubitskiy (1987) noted the presence of Oc. campestris, a Nearctic species, in Turkmenistan and Kazakhstan, respectively and Gornostayeva (2000) listed it among the Russian mosquito fauna. Gutsevich and Dubitskiy (1987) noted some differences in the male genitalia and larva of specimens from Kazakhstan and the Western Hemisphere; hence, the occurrence of Oc. campestris in Middle Asia and Russia requires further investigation.

Coquillettidia richiardii (Ficalbi) is widely distributed in Middle Asia, with occurrence records in Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan (Gutsevich et al., 1974; Mamednyasov, 1995; Tarbinsky, 1996). Although this species is present in northern and western provinces of Iran (Zaim, 1987; Azari-Hamidian, 2007; Azari-Hamidian and Harbach, 2009; Azari-Hamidian et al., 2009), it was not found in the present study and is not known to occur in northeastern Iran. The genus includes two species in the western Palaearctic Region, Cq. richiardii and Cq. buxtoni (Edwards) (Gutsevich et al., 1974; Knight and Stone, 1977).

Thirteen species of the genus Culex are recorded from countries in Middle Asia, including Cx. hortensis, Cx. martinii Medschid, Cx. mimeticus, Cx. modestus, Cx. orientalis Edwards, Cx. perexiguus (as Cx. univittatus Theobald), Cx. pipiens, Cx. pusillus Macquart, Cx. territans, Cx. theileri, Cx. torrentium Martini, Cx. tritaeniorhynchus and Cx. vagans Wiedemann (Gutsevich et al., 1974; Mamednyasov, 1995; Tarbinsky, 1996). Seven of these species were collected during the present study, but three others that are known to occur in Iran (Cx. pusillus, Cx. torrentium and Cx. territans) were not encountered. Culex martinii, Cx. orientalis and Cx. vagans have never been found in Iran; thus, 77% of the Culex species that occur in Middle Asia also occur in Iran. However, nine species that occur in Iran are not found in Middle Asia (Azari-Hamidian, 2007). It should be noted that the occurrence of Cx. orientalis in Middle Asia is doubtful as it otherwise only occurs in eastern areas of the Palaearctic and Oriental Regions, including the Khabarovsk, Ussuri and Maritme (Perimori) territories of Russia (Gutsevich et al., 1974). Among other species, Cx. torrentium and Cx. martinii are exclusively Palaearctic and Cx. pipiens is cosmopolitan. Culex pusillus is mainly Palaearctic but is also found in the Afrotropical Region (Sudan). Culex territans is Holarctic. Culex modestus, Cx. hortensis and Cx. vagans are mainly Palaearctic, however they are also found in the Oriental Region: Cx. modestus in India, Pakistan and China, Cx. hortensis in India and Cx. vagans in southern China and India. Culex mimeticus is found mainly in southern areas of the Palaearctic Region but its distribution extends across southwestern areas of the Oriental Region. Culex theileri, Cx. perexiguus and Cx. tritaeniorhynchus are found in the Palaearctic, Afrotropical and Oriental Regions, however Cx. theileri and Cx. perexiguus are mainly Palaearctic and Cx. tritaeniorhynchus is mainly Oriental (Knight and Stone, 1977; Harbach, 1988). As explained by Harbach (1988), the species recorded as Cx. univittatus in areas outside of the Afrotropical Region, including Iran and Middle Asia, is Cx. perexiguus.

Five species of the g enus Culiseta, Cs. alaskaensis Ludlow, Cs. annulata (Schrank), Cs. longiareolata, Cs. morsitans (Theobald) and Cs. subochrea, are recorded from countries in Middle Asia (Gutsevich et al., 1974; Maslov, 1989; Mamednyasov, 1995; Tarbinsky, 1996). These five species also occur in Iran (Azari-Hamidian, 2007), however only the widespread Cs. longiareolata was found during the present study. Culiseta longiareolata occurs in the Afrotropical, Oriental (India and Pakistan) and southern Palaearctic Regions; Cs. alaskaensis occurs in the northern Nearctic, northern and central Palaearctic and Oriental (India and Pakistan) Regions. Both Cs. annulata and Cs. subochrea are western Palaearctic species and Cs. morsitans is Holarctic (Knight and Stone, 1977; Maslov, 1989).

Uranotaenia unguiculata, which was collected in the present study, is widespread in Iran (Zaim, 1987), but it does not seem to be an abundant species (Azari-Hamidian et al., 2009). The species occurs in Turkmenistan and Kyrgyzstan (Mamednyasov, 1995; Tarbinsky, 1996) and in Middle Asia in general (Gutsevich et al., 1974). Uranotaenia includes two species in the Palaearctic Region, Ur. unguiculata and Ur. mashonaensis Theobald. The latter, which is an Afrotropical species, was recently recorded from Israel (Harbach and Schnur, 2007).

Overall, the main difference between the mosquito fauna of Iran (64 species) (Azari-Hamidian, 2007) and Middle Asia (62 species) (Gutsevich et al., 1974; Gutsevich and Dubitskiy, 1987; Mamednyasov, 1995; Tarbinsky, 1996; Mamednyazov and Yerokhin, 2005; Ejov, 2008) involves species in southern Iran that have their principal distributions in southwestern Asia and in the Afrotropical Region [such as Cx. antennatus (Becker) and Oc. caballus] or Oriental Region [such as An. peditaeniatus (Leicester), An. subpictus Grassi s.l. and Cx. pseudovishnui Colless]. The fauna of southwestern Asia includes many species that occur in two or more zoogeographical regions, such as Cx. quinquefasciatus Say (cosmotropical), Cx. bitaeniorhynchus Giles and Cx. sitiens Wiedemann (Palaearctic, Afrotropical, Oriental and Australasian Regions), An. culicifacies Giles s.l., An. dthali Patton, An. fluviatilis s.l., An. turkhudi Liston and Fr. vittatus (Palaearctic, Oriental and Afrotropical Regions), An. moghulensis and An. stephensi Liston (Palaearctic and Oriental Regions), Cx. arbieeni, Cx. deserticola Kirkpatrick, Cx. laticinctus and Cx. sinaiticus Kirkpatrick (Palaearctic and Afrotropical Regions). Among these species, An. culicifacies s.l. and An. fluviatilis s.l. are mainly Oriental, with populations in southwestern Asia that extend into the Afrotropical Region (Yemen and Ethiopia). Dahliana echinus, which occurs in Iran but not in Middle Asia, is exclusively Palaearctic (Knight and Stone, 1977). Two species originally discovered in Iran, An. apoci Marsh and An. persiensis Linton, Sedaghat and Harbach, have limited distributions. In addition to Iran, An. apoci also occurs in Iraq (Knight and Stone, 1977) and An. persiensis occurs in the Azerbaijan Republic (Ejov, 2008). Among the exclusively Palaearctic species, An. beklemishevi and 10 species of the genus Ochlerotatus have only been found in one country (Kazakhstan) of Middle Asia and three species, Oc. gutzevichi, Oc. montchadskyi and Oc. kasachstanicus, are endemic to the country.

Among the species of Culicinae that were found in the present study, Cx. perexiguus, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus and Oc. caspius s.l. are potential or proven vectors of pathogens that cause diseases in humans and domesticated animals in various areas of the world (Gutsevich et al., 1974; Harbach, 1988). Culex theileri is a known vector of canine heartworm in northwestern Iran (Azari-Hamidian et al., 2009). Many questions about the ecology of medically important species and the taxonomy of species complexes in Iran remain to be answered, especially in areas of northeastern Iran that have yet to be investigated.

ACKNOWLEDGMENTS

The authors are grateful to Dr. O. Bezzhonova (Department of Entomology, Faculty of Biology, Moscow State University, Moscow, Russian Federation) for kindly providing literature and information published in Russian. The authors also thank Dr. N. Nikparast (Health Deputy, North Khorasan University of Medical Sciences) and Mr. M. Heydarpoor (Disease Control Unit, Shirvan Health Center, North Khorasan University of Medical Sciences) for their collaboration. Dr. Y.-M. Linton (Department of Entomology, The Natural History Museum, London, UK) is appreciated for her collaboration in the Molecular Systematics Laboratory. This study was financially supported by the Institute of Public Health Research, Academic Pivot for Education and Research, Tehran University of Medical Sciences: project No. 241.83.77. Molecular work conducted at the Natural History Museum, London, was supported with funds provided by the senior author (REH).