A Pictorial Key for Culex pipiens Complex (Diptera: Culicidae) In Iran.

BACKGROUND: The aim of this study was to design pictorial key and taxonomic literature of Culex pipiens complex in Iran.
METHODS: Larvae were collected using standard dipping methods in 13 randomly selected areas of Bushehr, Hamedan, Kerman, Khorasan-e-Razavi, Khuzistan, Mazandaran, Tehran, Sistan and Baluchistan and Yazd Provinces from April 2009 to October 2010. The data were analyzed using SPSS Ver. 11.5.
RESULTS: Culex pipiens larvae were identified based on the Seta 1 of the abdominal segments III-IV in north and central parts of Iran. This diagnostic character had some variation among the Cx. quinquefasciatus collected from south of the country. The identification value of intersection of costa, subcosta and bifurcation of R2+3 of female veins, was calculated as 90-100 % for Cx. pipiens. This diagnostic character was varied among the Cx. quinquefasciatus specimens. The male genitalia found as the main characters to distinguish of Cx. quinquefasciatus from Cx. pipiens.
CONCLUSION: It is necessary more studies on the behavior and genetic variations of Cx. pipiens complex in Iran.

Introduction

Culex pipiens complex species have been known as important vectors of medical and veterinary arthropod-borne diseases (Kasai et al. 2008). Some vector borne diseases such as filariasis, West Nile fever, Western and Eastern Equine encephalitis, Japanese encephalitis and St Louis encephalitis are transmitted by these species complex (Smith 1973, Vinogradova 2000, Kasai et al. 2008). Cx. pipiens transmits West Nile virus among wild birds and plays an important role in enzootic cycles (Hayes et al. 2005).

Some pathogens transmitted by culicine mosquitoes such as West Nile and Sindbis viruses, Dirofilaria immitis (dog heartworm) and D. repens (dirofilariasis), and have been reported in Iran (Naficy and Saidi 1970, Saidi et al. 1976, Azari-Hamidian et al. 2007). Moreover, potential outbreaks of some mosquito-borne arboviral diseases such as Japanese encephalitis (JE) and Rift Valley fever reported in the Eastern Mediterranean Region (WHO 2004).

The morphological and ecophysiological variations of Cx. pipiens complex have been an important topic in extensive researches (Harbach 1985, 1988, Vinogradova 2000, 2003, Azari-Hamidian and Harbach 2009, Dehghan et al. 2010, 2011, 2014). Because of the complicities, more than 75 synonyms have been proposed for this complex species (Knight and Stone 1977). Barr (1982) reported a wide geographical distribution, and morphological variations among the Cx. pipiens and Cx. quinquefasciatus populations in the world. There are variations among the diagnostic characters of these species complex that attributed to some degree of expression such as species, subspecies, variety or forms (Ishii 1991).

Culex quinquifasciatus is distributed in the tropical areas with various hosts, whereas, Cx. pipiens found in the moderate areas with host preferences of nest maker birds (Vinogradova 2000).

Shahgudian (1960) and Lotfi (1976) provided identification keys for Iranian Anopheles and Culex species, respectively. The study on physiology and behavior of Cx. pipiens, the nominotypical and molestus form, was described in Iran (Lotfi 1970, 1973, 1976, Amirkhanian 1974, Zaim and Cranston 1986). Zaim (1984) reported six genera and 55 species of Iranian mosquitoes. Some years later a checklist and systematic key was provided for Iranian Culicinae by Zaim and Cranston (1986). Although, in the previous study had not been mentioned about diagnostic characters of Cx. pipiens and Cx. quinquefasciatus in the systematic keys (Zaim and Cranston 1986), but Azari-Hamidian and Harbach (2009) addressed these characters.

The present article, reviews some morphological characters of Cx. pipiens complex species, collected in some parts of Iran to facilitate conducting comprehensive research about systematics, ecology, medical and veterinary importance of the complex. Until now, seven genera, 64 species, and three subspecies of Iranian mosquitoes was reported (Azari-Hamidian 2007a).

Culex pipiens complex belongs to the Pipiens group, and was divided to several subgroups and subtypes (Harbach 2011, 2013).

Culex pipiens including form molestus and Cx. quinquefasciatus have been reported in Iran (Azari-Hamidian 2007a, Azari-Hamidian and Harbach 2009). Furthermore, the larval habitat of Cx. pipiens form molestus was reported by Golestani (1967) in Tehran City. The polygene chromosomal pattern of the autogenous Tehran strain of Culex pipiens molestus was described by Amirkhanian in 1974. Further studies about distribution of Cx. pipiens have been reported in Iran (Zaim 1987, Azari-Hamidian et al. 2005, 2011, Azeri-Hamidian 2007b, Moosa-Kazemi et al. 2009, 2010, Dehghan et al. 2013). Distribution of Cx. quinquefasciatus has been reported in Yazd and Kermanshah Provinces (Ghaffary 1955). More studies reported distribution of this species in southern parts of Iran (Zaim 1987, Mousa-kazemi 2000, Azari-Hamidian et al. 2005, Moosa-Kazemi et al. 2009, Azari-Hamidian et al. 2010, Moosa-Kazemi et al. 2010, Khoobdel et al. 2012). The distribution of this species has been reported in tropical areas in south of Iran based on molecular identification (Azari-Hamidian et al. 2010, Dehghan et al 2013).

Morphological studies on Cx. pipiens complex, revealed that, male genitalia and DV/D ratio are the main morphological characters for distinguishing adults of Cx. quinquefasciatus and Cx. pipiens (Sundararaman 1949). The main reliable character for identification in larval stage has been known as the number of branches of seta 1 on the abdominal III–IV segments (Harbach 1988, Dehghan et al. 2013).

Since there are scatter studies about the morphological variations and distribution of Cx. pipiens complex in Iran, (Zaim 1987, Azari-Hamidian and Harbach 2009, Azari-Hamidian et al. 2010) it is necessary to obtain more accurate data on the variability of the species. On the other hand, the final decision about taxonomic status needs more complete data that will be obtained from further studies in different geographical areas. The results of this study can be useful to identify Cx. pipiens complex species, as well as designing plans for vector control programs in the future.

Materials and Methods

Study area

Chabahar (25°17′N, 60°37′E) and Nikshahr (26°04′N, 60°37′E) counties were selected from Sistan and Baluchistan Province with tropical warm and humid climate. In southern Iran, Jiroft (28.5°N, 57.8°E) from Kerman, Borazjan (29°15′N, 51°12′E) from Bushehr, Ahvaz (31°19′N 48°41′E) and Bostan (31°27′N 48°04′E) counties from Khuzistan Province with subtropical warm and humid climate were selected. Yazd, Zarch (54°04′N 31°59′E) from Yazd and Kerman (30°17′N 57°04′E) from Kerman Province with hot and dry desert climate were selected. For cool and moist Mediterranean climate Neka (36°42′N 53°33′E) county from Mazandaran and Mashhad (36°18′N 59°36′E) from Khorasan-e-Razavi Province were chosen. Hamedan (34°48′N 48°31′E) from Hamedan, and Tehran (35°45′N 51°35′E) from Tehran Province were selected from cold and dry climate (Fig. 1).

Fig. 1.

The study areas and distribution of Culex pipiens and Culex quinquefasciatus in different stratum of Iran, 2010

Mosquito sampling and morphological studies

This cross sectional study was conducted in 13 randomly selected areas in Iran. Larval stages of the mosquitoes were collected, using standard dipping technique from April 2009 to October 2010.

The larvae were collected from different regions of the country using WHO standard dipping method (WHO 1992). The samples were transferred to the Entomology Laboratory, Department of Entomology and Parasitology, School of Medical Sciences, Tarbiat Modares University. The mosquitoes’ larvae maintained in specific cage for rearing in insectarium condition (22–25 °C, 70–75% RH). Microscopic slides were used to mounting of some parts of the adult body such as wings and maxillary palps using Canada balsam diluted with xylen. The caudal abdominal segment of males were removed, and placed in KOH 10% for 20 to 30 minutes, then washed with distilled water and placed in ethanol 96% for dehydration (Barr 1957, Jakob et al. 1979). Microscope slides of the samples were prepared using Puri’s medium. The taxonomic figures were drawn using light Zeiss microscope with a Nikon drawing tube accessory long arm (9.1 inches) (22.5 cm). The morphological features were used to identify the Cx. pipiens complex includes:

Seta 1 of abdominal segments III–IV (Fig. 2) (Azari-Hamidian and Harbach 2009).

Fig. 2.

Identification of Culex pipiens and Culex quinquefasciatus larvae based on abdominal segments of III–IV

Seta 1a–S and 1b–S in the siphon (Fig. 3) (Harbach 1988).

Fig. 3.

Identification Culex pipiens and Culex quinquefasciatus larvae based on siphon characters

Shape of the siphon (Fig. 3) (Harbach 1988, Azari-Hamidian and Harbach 2009).

DV/D: The ratio used for adult identification, also used as a confirmation for molecular studies. DV was described as the distance between two tips of dorsal and ventral arms and D was defined the distance between two tips of dorsal arms (Fig. 4) (Mattingly et al. 1951, Barr 1957, Kamura 1959, Vinogradova 2003, Smith and Fonseca 2004).

Fig. 4.

Identification of adult male in Culex pipiens and Culex quinquefasciatus by male genitalia and different of dorsal and ventral arms

Costa and subcosta intersection with R2+3 furcation (Fig. 5) (Harbach 1985, Azari-Hamidian and Harbach 2009).

Fig. 5.

Identification of adult female in Culex pipiens and Culex quinquefasciatus based on vegetation of wings

Female Characters (Wing venation)

Data were analyzed using SPSS Ver. 11.5.

Results

Overall, 304 larvae and 419 adults (177 males and 242 females) were randomly selected. The branch number of Seta 1 on abdominal segments III–IV, 1a–S tuft, 1b–S tuft and siphon shape are showed in Table 1. Culex pipiens larvae were identified in the samples of Mashhad, Tehran, Neka, Yazd and Zarch1 areas whereas, Cx. quinquefasciatus was found in Zarch 2 and Kerman based on the Seta 1 abdominal segments III–IV. The findings showed variations at the mentioned character among the samples collected from Borazjan, Chabahar, Jiroft, and Nikshahr.

Table 1.

The variations of some morphological characters of Culex pipiens and Cx. quinquefasiatus larvae, Islamic Republic of Iran, 2009–2010

Areas	No	Seta 1 abdominal segment III – IV		Seta 1a-S			Seta 1b-S			Siphon shape
		Single	Double	Mean	Max	Min	Mean	Max	Min	Gradually narrowing	Widen in middle
Mashhad▲	21	0 (0%)	21(100%)	3.2	7	2	3.2	5	2	21 (100%)	0 (0%)
Neka▲	29	0 (0)	29 (100)	2.7	5	2	2.8	4	2	29 (100)	0 (0)
Tehran▲	50	0 (0)	50 (100)	4.3	7	2	4.4	7	2	45 (100)	5 (0)
Yazd▲	19	0 (0)	19 (100)	4.7	7	3	4.4	6	3	16 (84.2)	3 (15.8)
Zarch 1▲	46	0 (0)	46 (100)	4.5	6	3	4.2	7	2	46 (100)	0 (0)
Borazjan△	21	4 (19)	17 (81)	6.3	8	4	6.2	10	3	1 (5)	20 (95)
Chabahar△	40	20 (50)	20 (50)	6.9	10	4	6.9	10	4	4 (10)	36 (90)
Jiroft△	23	4 (17.4)	19 (82.6)	7.9	9	6	7.8	13	6	17 (74)	6 (26)
Kerman△	16	0 (0)	15 (100)	6.7	10	4	6.3	8	4	15 (93.7)	1 (6.3)
Nikshahr△	24	8 (33.3)	16 (76.7)	6.2	9	4	6	10	4	4 (16.7)	20 (83.3)
Zarch 2△	15	0 (0)	15 (100)	6.3	8	2	6.5	8	5	0 (0)	15 (100)
Total	304	–	–	–	–	–	–	–	–	–	–

All of the species identified as Culex pipiens

All of the species identified as Culex quinquefasciatus

The mean average of 1a–S tuft branches were calculated 2.7 to 4.7 and range of 2 to7 and 6.2 to 7.9, range 2 to 10 for Cx. pipiens and Cx. quinquefasciatus respectively. The mean average of 1b–S tuft branches were counted 2.8 to 4.4, rage 2 to7 and 6 to 7.8, range 3 to 13 for Cx. pipiens and Cx. quinquefasciatus respectively.

The siphon shape of Cx. pipiens larvae were found gradually narrowing toward the end of siphon in all samples that were collected from Mashhad, Tehran, Neka, and Zarch1 whereas, this character included 84.2 % of Cx. pipiens samples collected from Yazd. The variation was found among the siphon shape of Cx. quinquifasciatus larvae in Borazjan, Chabahar, Jiroft, Kerman, and Nikshahr samples (Table 1).

The species of Cx. pipiens were identified based on the morphological characters of female wings vegetation in Hamedan, Yazd and Bostan, while it was found varied among the samples collected from Neka, Zarch, Ahvaz, Chabahar, Nikshahr and Jiroft. Culex quinquefasciatus was identified in Chabahar (97%), Ahvaz (88.9%), and Nikshahr (73.7 %) (Table 2).

Table 2.

The variations of some morphological characters of wings in the females of Culex pipiens and Cx. quinquefasciatus, Islamic Republic of Iran, 2009–2010

Areas	No	Costa and Subcosta Intersection
		At or beyond level of Furcation of R2+3 (Cx. pipiens)		Before level of Furcation of R2+3 (Cx. quinquefasciatus)
		Prevalence	%	Prevalence	%
Hamedan▲	16	16	100	0	0
Neka▲	34	33	97.06	1	2.94
Yazd▲	5	5	100	0	0
Zarch▲	84	75	90	9	10
Ahvaz△	27	3	11.1	24	88.9
Bostan▲	9	9	100	0	0
Chabahar△	33	1	3	32	97
Jiroft△	15	14	93.33	1	6.67
Nikshahr△	19	5	26.3	14	73.7
Total	242	–	–	–	–

All of the species identified as Culex pipiens

All of the species identified as Culex quinquefasciatus

Table 3 shows the DV/D and D/V ratios. The DV/D ratio calculated −1.23–0.12 in mean average and range −0.21–0.2 in Cx. pipiens. This ratio was calculated in mean average of 0.5–1.09 and range 0.33–2.37 for Cx. quinquefasciatus. The D/V ratio of Cx. pipiens samples were calculated in mean average of 0.95–1.35, and range of 0.7–1.75. In addition, mean average of this ratio was calculated 0.35–0.64 and the range was 0.17–0.78 in Cx. quinquefasciatus.

Table 3.

The ratio variations of male genitalia in Culex pipiens and Cx. quinquefasciatus, Islamic Republic of Iran, 2009–2010

Areas	No	DV/D			D/V
		Mean	Max	Min	Mean	Max	Min
Hamedan▲	20	0.09	0.05	−0.17	1.27	1.51	0.92
Neka▲	25	0.07	0.14	− 0.21	1.21	1.75	0.78
Tehran▲	9	−1.23	0.00	− 0.19	1.35	1.61	1.00
Yazd▲	30	0.12	0.20	− 0.19	0.95	1.63	0.70
Ahvaz△	24	0.90	1.54	0.47	0.35	0.55	0.25
Chabahar △	22	0.50	1.55	0.41	0.64	0.78	0.24
Jiroft△	19	1.09	2.29	0.33	0.36	0. 60	0.18
Kerman△	7	0.90	1.68	0.40	0.38	0.56	0.23
Nikshahr△	21	0.79	2.37	0.38	0.46	0.62	0.17
Total	177	–	–	–	–	–	–

All of the species identified as Culex pipiens

All of the species identified as Culex quinquefasciatus

Discussion

In present research, the seta 1 on the abdominal segments III–IV was found the main reliable characters for identification of Cx. pipiens and Cx. quinquefasciatus in north and central of the Country. This character was not found a reliable character for Cx. quinquefasciatus in southern parts of the country. Moosa-Kazemi et al. (2010) was reported the occurrence of Cx. pipiens in Kurdistan Province, northeast of Iran. This study showed that seta 1 of abdominal segments III–IV of Cx. pipiens had two branches. Harbach (1988) and Azari-Hamidian and Harbach (2009) described this character in Cx. pipiens as well.

Our finding showed, siphonal seta 1a-S and 1b-S of Cx. pipiens had range of 2–7 branches. These results are supported by previous study, Knight and Malek (1951) reported an average 4 and a range 2–9 branches for Cx. pipiens.

Results of this study showed the branch number of seta 1a-S and 1b-S had a mean average of 6–7.9 and range of 2–13 for Cx. quinquefasciatus. Similarly, Harbach (1988) reported more branches in Cx. quinquefasciatus than Cx. pipiens.

In this study, shape of the siphon was studied among 165 larvae samples and confirmed vast most of the Cx. pipiens species. As mentioned above, there are character variations among the samples which were collected from south of Iran. Generally, the length of siphon in Cx. pipiens was reported longer than in Cx. quinquefasciatus (Harbach 1988, Azari-Hamidian and Harbach 2009).

According to our observations, the seta 1 branches of abdominal segments III–IV and shape of the siphon in larval samples were found as valuable characters, which can easily used to distinguish of Cx. pipiens from Cx. quinquefasciatus. In our study, some characters were overlapped between Cx. pipiens and Cx. quinquefasciatus species, therefore, it is recommended the whole characters should be evaluated to accurate identification.

Culex pipiens complex is considered cosmopolitan species. Although, the distribution patterns of the complex species have been reported in Iran (Zaim 1987, Harbach 1988, Azari-Hamidian et al. 2010) results of this study indicated the distribution of Cx. pipiens in Bostan, Yazd, Neka, Mashhad, Hamedan and Tehran. Nevertheless, the distribution of Cx. quinquefasciatus was limited in Chabahar, Nikshahr, Jiroft, Kerman, Borazjan, Ahvaz and Zarch.

The occurrence of Cx. quinquefasciatus in the Iranian Persian Gulf islands had been reported previously (Azari-Hamidian et al. 2010, Khoobdel et al. 2012). In addition this species was reported in Kermanshah, western Iran (Ghaffary 1954), and in Bandar-e Anzali Northern Iran (Harbach 1988). There are scattered data about the species composition of Cx pipiens and Cx. quinquefasciatus in southern Iran. As mentioned, the male genitalia reported as the main morphological characters to identification of the Cx. pipiens complex species. In our research DV/D ratio of Cx. quinquefasciatus was more than 0.4 (0.56–1.89, mean 1.03), But in Cx. pipiens this ratio found ranges −0.14 to zero (Harbach 1988). The DV/D ratio of Cx. pipiens, calculated at mean average of −1.23–0.12 and range of 0.21–0.2 while, calculated at mean average of 0.5–1.09 and ranges 0.33–2.37 for Cx. quinquefasciatus.

The D/V ratio was calculated at mean average of 0.95–1.35, and range of 0.7–1.75 for Cx. pipiens samples collected in south of the country, whereas it was calculated at mean average of 0.35–0.64 and range of 0.17–0.78 for Cx. quinquefasciatus. Further supports for these results also came from some previous studies, Sasa (1967) express the D/V ratio in range of 0.4–0.9 in Cx. pipiens pallens and less than 0.3 in Cx. quinquefasciatus and average of 1.2 in Cx. pipiens form molestus. Moreover, Choochote (1987) reported the D/V ratio at average of 0.35 for Cx. quinquefasciatus. In parallel to the present research, Mohsen et al. (1995) reported D/V ratio as 1.43 for Cx. pipiens form molestus and 0.337 for Cx. quinquefasciatus.

In recent research, dorsal arms of male genitalia of Cx. quinquefasciatus described as narrow, sharp apex and parallel as the base toward the end. In addition, the ventral arms were flat and leaf shape. Whereas, in Cx. pipiens dorsal arms were described quite broad, truncate at the apex, and diverges in the base toward the end of arms, which, indicated the occurrence of Cx. pipiens in north to central parts of Iran. Further support to these results also came from a previous study, Harbach (1988) described the dorsal arms of phalosoma as divergent, broad and nearly truncate at the apex for Cx. pipiens.

Conclusion

Identification of the Cx. pipiens complex species using morphological key have some difficulties because of occurring variations among closely related complex species. In present pictorial key, the colors of the siphon and male’s genitalia are not referring to the color of original samples. However, the described taxonomical characters in this article should be included in other characters, which were reported in the previous literatures. The most complex species as well as Cx. pipiens complex should be identified by morphological characters in the first step, although some researchers prefer molecular studies such as Ace. 2 gene, microsatellite loci and COI gene for solving the morphological taxonomic problems.

Comprehensive studies such as phylogenic and molecular are necessary to obtain new information for identification of Cx. pipiens complex species in the future.