Microsoft Word - 4-Doosti_RTL.doc Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 28 Original Article Applying Morphometric Variation of Seta 2 (Antepalmate Hair) among the Larvae of the Members of the Maculipennis Subgroup (Diptera: Culicidae) in Iran S Doosti 1, H Vatandoost 1, MA Oshaghi 1, M Hosseini 2, *MM Sedaghat 1 1Dept. of Medical Entomology and Vector Control, School of Public Health and Institute of Public Health Research, Medical Sciences /University of Tehran, Iran 2Dept. of Epidemiology and Biostatistics, School of Public Health and Institute of Public Health Research, Medical Sciences /University of Tehran, Tehran, Iran (Received 20 Oct 2006; accepted 1 May 2007) ABSTRACT The members of Anopheles maculipennis subgroup (Diptera: Culicidae) are the most important vectors of malaria in the north, west, and central plateau of Iran. This study was carried out to differentiate the species composition of this subgroup based on morphometric variation seta 2-IV and V (antepalmate hair) among 149 larval specimens that were deposited at the Medical Arthropods Museum, the School of Public Health, Tehran University of Medical Sciences by using the light micro- scope. The mean numbers of larval seta 2-IV and V of the specimens belong to different locations of Iran, were calculated by SPSS (11.5) software package, followed by cluster analysis, and four different groups (clusters) were identified. The means were compared with the similar and available published data. After analyzing, four clusters recognized. The first cluster was fitted in ten localities in Esfahan, East Azarbaijan, West Azarbaijan, Khorassan, Kurdistan, and Mazandaran Provinces with its mean and standard deviation (SD) of 14.89±1.13 (n= 79); the second group with one location in Gilan Province (11±1.58, no= 5); the third one with two locations in Fars and Western Azarbaijan Provinces (27.43±0.31, n=20), and the final group with four locations in Khuzestan, West Azarbaijan, and Qazvin Provinces (36.84±1.91, n= 45) were identified and corresponded to Anopheles messeae, An. atroparvus, An. melanoon, and An. sacharovi respectively. This work provides comparative information on the Maculipennis Subgroup based on morphometric examination at the larval stage in Iran. Keywords: Maculipennis group, Malaria, Mosquito, Numerical taxonomy, Iran INTRODUCTION The anopheline fauna in Iran is diverse and at least seven species have been incriminated as malaria vectors in the country. Among them the members of Anopheles maculipennis subgroup (Diptera: Culicidae) have involved with malaria transmission mostly in northern and central area of Iran (Sedaghat et al. 2003a). According to the last classification, the ge- nus Anopheles (Diptera: Culicidae) includes six subgenera and at least 484 species in the world (Harbach 2004). The members of Anopheles maculipennis subgroup are the most important vectors of the western Palaearctic region (Se- daghat et al. 2003a). An. maculipennis was de- scribed and named for the first time by Migen in 1818 and for many years, all scientists ex- pected that it was one species with single mor- phological type and biological characters (White 1978). In fact the Maculipennis Group was the first sibling species complex, to be discovered among mosquitoes (Falleroni 1926, van Thiel 1927). Since this discovery, extensive efforts *Corresponding author: MM Sedaghat, Tel: +98 21 88951393, E-mail: sedaghat@doctor.com Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 29 have been made to determine and explain spe- cies composition of the group. Several methods including studies of egg morphology, ecology, hybridization, larval and pupal chaetotaxy, wing characteristics, cuticular hydrocarbons, DNA sequences, chromosome, and zymotaxonomy have been used for identifi- cation of this group (Linton et al. 2003). These works contributed to the current rec- ognition of twelve formal species in the Palae- arctic region, including An. artemievi, An. at- roparvus, An. daciae, An. beklemishevi, An. labranchiae, An. lewisi, An. maculipennis, An. martinius, An. melanoon, An. messeae, An. per- siensis and An. sacharovi (White 1978, de Zu- lueta et al. 1983, Sedaghat et al. 2003 a, Harbach 2004, Nicolescu et al. 2004, Sedaghat and Har- bach, 2005, Gordeyev et al. 2005). The Maculipennis Group currently divided to three subgroups including Maculipennis, Quadri- maculatus and Freeborni Subgroups. The Macu- lipennis Subgroup (which previously named Maculipennis Complex) comprises all Palaearc- tic members of the group except An. beklem- ishevi (Harbach 2004). Seven taxa of the members of the subgroup previously reported from Iran. These species in- clude: An. Atroparvus (Dinparast-Jadid et al. 2001, 2007), An. labranchiae (Gholizadeh et al. 2004, Dinparast-Jadid et al. 2007), An. maculi- pennis s.s. (Dow 1953, Shahgudian 1960, Din- parast-Jadid et al. 1990, Glick 1992, Momeni et al. 1992, Dinparast-Jadid et al. 2001, Azari- Hamidian et al. 2003, Oshaghi et al. 2003, Se- daghat et al. 2003a, Dinparast-Jadid et al. 2007), An. melanoon (Dow 1953, Faghih 1969, as An. subalpinus, Dinparast-Jadid et al. 1990, Momeni 1991, Glick 1992, Azari-Hamidian et al. 2003) An. messeae (de Zulueta et al. 1957, Minar 1974, Momeni 1991, Momeni et al. 1992, Dinparast- Jadid et al. 2001, Azari-Hamidian et al. 2003, Dinparast-Jadid et al. 2007), An. Persiensis (Se- daghat et al. 2003a, Sedaghat and Harbach 2005, Dinparast-Jadid et al. 2007) and An. sa- charovi (Dow 1953, Etherington and Sellick 1946, Shahgudian 1960, Dinparast-Jadid et al. 1990, Glick 1992, Momeni et al. 1992, Din- parast-Jadid et al. 2001, Oshaghi et al. 2003, Sedaghat et al. 2003a,b, Sedaghat and Harbach 2005, Dinparast-Jadid et al. 2007). Although, Glick (1992) listed An. martinius among Ira- nian anopheline, the occurrence of this taxon has not been confirmed in the country (Sedaghat and Harbach 2005). Among the members of An. maculipennis subgroup, five species of An. Atroparvus, An. labranchiae, An. maculipennis, An. messeae and An. sacharovi have been considered as malaria vectors in Eurasia (Jaenson et al. 1986, Ribeiro et al. 1988, Kasap 1990, Jetten and Takken 1994, Romi 1999, Romi et al. 2001, Alten et al. 2000, Faghih 1969). To date, Anopheles maculipennis s.s. has been discriminated as the major vector of ma- laria in the Caspian Sea littoral, and An. sa- charovi is considered to be the principal vector in the central plateau (Faghih 1969, Manouche- hri et al. 1992). Early studies on this group were based on morphological methods. Falleroni (1926) and van Thiel (1927), as first attempts to understand complexity of the Maculipennis Group, consid- ered long- and short-winged forms of An. macu- lipennis sensu lato. The other morphological studies include: study on eggs (Missiroli et al. 1933); male genitalia (Martini 1933); wing scales (Ungureanu and Shute 1947); larval or pupal chaetotaxy (Bates 1939, Pichot and Deruaz 1981, Suzzoni-Blatger and Sevin 1981, Boccolini et al. 1986, Suzzoni-Blatger et al. 1990, Deruaz et al. 1991). In the larval stage, situation of seta 3-C (outer clypeal hair) in comparison to lateral pala- tal brush in addition number and situation of seta 1-II was considered as a useful method (Abul-hab 1955). However, morphometric varia- tion of seta 2-IV and V was regarded as a useful and reliable morphologic character to differ- entiate in the larval stage by some medical en- tomologists (Bates 1939, Romi et al. 2002). There is little information on vector capaci- ties and species distributions of these sibling Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 30 species in Iran. Besides, there were disagree- ments with the occurrence of some species in the country and some records need to be veri- fied by classical morphology study, molecular method and using other advanced tools. Identi- fication of species complex which show differ- ent biology, ecology, behaviour, host prefer- ence and vector potential aspect despite of their resemblance, is very important in malaria con- trol programmes. It is important to make statistical compari- son of the amounts of branching of particular larval setae. This paper provides preliminary information on differentiation of species com- position of An. maculipennis subgroup in the larval stage based on the morphometric varia- tion of seta 2-IV and V. MATERIALS AND METHODS This study was carried out to separate the An. maculipennis subgroup larvae based on seta 2 (Ante palmate hair) in Iran. Two hundred and ninety one larval specimens which were depos- ited in the Medical Arthropods Museum, School of Public Health, Tehran University of Medical Sciences, Iran were examined. These specimens initially had been identified based on egg mor- phology. The number of seta 2 braches (ante palmate hair) of the forth and fifth abdominal segments of each larva was counted using light microscope. For compared mean of seta 2-IV and V, those collection localities which had at least 5 specimens were considered in this study. The mean numbers of larval setae 2-IV and V of the specimens belong to different locations of Iran, were calculated by SPSS (11.5) soft- ware package, followed by cluster analysis. RESULTS In this investigation 149 larval specimens from 17 different locations of Iran were studied to identify the species composition of the Maculipennis Subgroup based on seta 2. There was not statistically significant between the number of the seta 2 branches in abdominal segments IV and V among the specimens. Means and Standard Deviations (SD) of differ- ent locations are shown in Table 1. Total mean of the seta 2 branches in segments 4 and 5 in different locations were statistically significant (P< 0.0001). The mean numbers of the larval seta 2-IV and V of the specimens, were calcu- lated by SPSS (11.5) software package. After cluster analyzing, four different clusters were identified. These four groups include: Group 1 with codes 4, 5, 6, 7, 9, 11, 12, 15, and 16 that they belong to Esmail Tarkhan (Es- fahan Province), Takht Olia (West Azarbaijan), Dehriz (East Azarbaijan), Band Rezaie (East Azar-baijan), Ghagharalu (East Azarbaijan), Sar- velayat (Khorassan), Marsuk (Khorassan), Ghey- bisur (Kurdestan), Sarik (West Azarbaijan), group 2 with code 17 includes Fuman (Gilan), group 3 with codes 1 and 10 includes Dasht Khezri (Fars) and Dehjabal (East Azarbaijan), and group 4 with codes 2, 3, 8 and 13 includes Taher Abad (Qaz- vin), Nazar Abad (Qazvin), Jabal (East Azarbai- jan) and Kaldusakh (Khuzestan) (Fig. 2, 3). The mean numbers of the seta 2 branches for each of these clusters were compared with similar information from Bates (1939) and Romi et al. (2002). The first cluster was in ten locations in Esfahan, East Azarbaijan, West Azarbaijan, Khorassan, Kurdestan, and Mazan- daran Provinces with mean and standard devia- tion (SD) 14.89±1.13, n= 79 was fitted to An. messeae, the second group with one location in Gilan Province (11±1.58, no= 5) fitted to An. atroparvus, the third one with two location in Fars and West Azarbaijan Provinces (27.43± 031, n= 20) fitted to An. melanoon and the final group with four locations in Khuzestan, West Azarbaijan, Qazvin Provinces (36.84±1.91, n= 45) fitted to An. sacharovi (Table 1). Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 31 (b) An. sacharovi (a) (b) An. maculipennis sensu lato (a) Fig. 1. Dorsal (a) and ventral (b) view the of the IV- V abdominal segments of larvae in An. sacharovi and An. maculipen- nis s.l. (setae 2 is shown by arrow) Table 1. Location, mean and standard deviation (SD) of seta2 branches, and the number of larval specimens studied among Iranian An. maculipennis subgroup. Mean of seta2 branches ±SD Species Location No. larvae Province 27.21±4.28 An. melanoon Dasht khezri 14 Fars 36.92±3.79 An. sacharovi Taherabad 13 Qazvin 34.55±7.55 An. sacharovi Nazarabad 9 Qazvin 14.40±2.51 An. messeae Esmailtarkhan 5 Esfahan 14.84±2.91 An. messeae Takhtolia 13 East Azarbaijan 16.57±4.57 An. messeae Dehriz 7 West Azarbaijan 16.16±2.92 An. messeae Band Rezaie 6 West Azarbaijan 36.66±8.47 An .sacharovi A marsh near Jabal 6 West Azarbaijan 14.71±1.70 An. messeae Ghagharalu 7 West Azarbaijan 27.66±7.22 An. melanoon Dehjabal 6 West Azarbaijan 15.47±4.001 An. messeae Sarvelayat 17 Khorassan 15.66±2.80 An. messeae Marsuk 6 Khorassan 39.23±6.15 An. sacharovi Kaldusakh 17 Khuzestan 13.20±1.30 An. messeae Gheybisur 5 Kurdistan 14.80±3.42 An. messeae Sarik 5 East Azarbaijan 13.12±1.80 An. messeae Abdangeh 8 Mazandaran 11±1.58 An. atroparvus Fuman 5 Gilan Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 32 Fig. 2. Geographical distribution of An. sacharovi and An. maculipennis s.l. in Iran based on the mean number of seta 2 branches of available larval slides in the Medical Entomology Museum of School of Public Health, Tehran University of Medical Sciences, Iran. An. sacharovi, An. messeae, ▲An. melanoon, An. atroparvus. 1. East Azarbaijan, 2. West Azarbaijan, 3.Kurdistan, 4.Guilan, 5.Mazandaran, 6.Qazvin, 7.Esfahan, 8. Khorassan, 9.Khuzestan, 10.Fars. An. messeae An. atroparvus An. melanoon An. sacharovi (A) An. messeae An. atroparvus An. melanoon An. sacharovi (B) Fig. 3. (A) The simple dendogram based on the mean number of seta 2 branches from this study (A) in comparison to the dendogram (B) which taken from Marinochi et al. (1999) . Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 33 DISCUSSION Although advanced tools provide diagnostic criteria, egg morphology has remained as a tra- ditional and useful technique to identify the members of the complex for many years. How- ever, seasonal variation in characters such as presence and absence of egg floats (Mer 1937), and intra-specific polymorphic egg surface pat- terns, indicate that recognition by egg morphol- ogy is not completely accurate (Linton et al. 2002b). Recently, DNA sequencing of the mito- chondrial cytochrome c oxidase gene (COI) (Linton et al. 2003, Sedaghat et al. 2003b), and the nuclear ITS2 rDNA have been employed to differentiate members of the complex (Mari- nucci et al. 1999, Proft et al. 1999, Romi et al. 2000, Linton et al. 2002a,b, 2003, Sedaghat et al. 2003a,b). DNA-based techniques are reliable and not limited to specific developmental stages or to specific sex (Collins and Paskewitz 1996). Nuclear ITS2 region has also been used to gen- erate phylogeny of members of the complex (Marinucci et al. 1999, Kampen 2005, Dinparast- Jadid et al. 2007). In spite of the role of the members of Macu- lipennis Subgroup in transmission of malaria and reporting different species of the Subgroup in Iran, so far they have not been differentiated in the larval stage. The information provided in this study is the first one on An. maculipennis subgroup in the larval stage in the country which compared with those available in the world. Bates (1939) and Romi et al. (2002) used the number of branches of setae 2-IV and V to separate the species composition of An. macu- lipennis complex. In this study, An. sacharovi was found in Qazvin, Khuzestan, and East Azarbaijan. Be- fore, this species was reported from this prov- inces based on the morphological character of egg and adult and/or molecular method (Dow 1953, Dinparast-Jadid et al. 1990, Momeni et al. 1992, Dinparast-Jadid et al. 2001, Sedaghat et al. 2003a). Recently, Dinparast-Jadid et al. reported this species based on the molecular information from Gilan and Ardebil Povinces (Dinparast-Jadid et al. 2007). Anopheles atroparvus has been reported from Gilan and Mazandaran based on egg pat- tern and/or molecular method (Dinparast-Jadid et al. 2001, 2007). An. melanoon was found in East Azarbai- jan and Fars Provinces in this study. Although, this species previously was reported in north and north-eastern areas of the country based on the egg morphology (Dow 1953, Dinparast- Jadid et al. 1990, Azari-Hamidian et al. 2003), there is no information about the distribution of this taxon in Fars Province; different authors only mentioned the occurrence of An. sacharovi and An. maculipennis sensu lato in Fars. An. messeae was found in Esfahan, Kurde- stan, East Azarbaijan, West Azarbaijan, Khorassan and Mazandaran in this study. Before, An. messeae has been reported from Gilan or Mazandaran Provinces based on the molecular method, adult morphology (wing scale index) and egg morphol- ogy (Minar 1974, Momeni et al. 1992, Dinparast- Jadid et al. 2001, Azari-Hamidian et al. 2003, Dinparast-Jadid et al. 2007). This species was reported based on the egg morphology from Esfahan by de Zulueta et al. (1957). However, the occurrence of An. messeae has not been confirmed in Esfahan by other authors. This study showed that the means of the seta 2 branches of An. messeae from different areas was more similar than An. maculipennis from different areas. The results of this study can be used as preliminary information in the future works on sibling species. Based on the comparison of two dendograms, A from this study and B, taken from Romi et al. (2002); it showed that there was conformity to separate An. atroparvus and An. sacharovi in two divide monophyletic groups, but there was no conformity for An. melanoon and An. messeae in two dendograms (Fig. 3). In dendogram B, which was based on ITS2 sequence data, An. sacharovi placed in a basal position within the rest of taxa. Previous studies Iranian J Arthropod-Borne Dis, (2007), 1(1): 28-37 S Doosti et al.: Applying Morphometric Variation of… 34 derived from rDNA sequence data isolated An. sacharovi from the other siblings (Kampen 2005, Dinparast-Jadid et al. 2007). This posi- tion also supported the phylogenetic relation- ships inferred from the previous analyses of polytene chromosome banding patterns (White 1978, Stegnii 1981). Moreover a parsimony analy- sis of mtDNA data generated by Sedaghat sup- ported this position of An. sacharovi (Sedaghat 2003). In this study, An. atroparvus + An. messeae formed a separate clade. This position is in agreement with the phylogenetic relationships based on ITS2 sequences defined by Kampen (2005). However, Dinparast-Jadid et al. could not show such relation between An. atroparvus + An. messeae (Dinparast-Jadid et al. 2007). The method which employed in this paper can be applied in integrated systematics studies. It is not possible to provide a meaningful mor- phological diagnosis of An. maculipennis s.l. at this time because the other members of the sub- group have not been studied in detail. To date, the life stages of only An. maculipennis, An. sacharovi and An. daciae from this subgroup have been described in detail (Linton et al. 2003, Sedaghat et al. 2003b, Nicolescu et al. 2004, respectively). However, despite these studies there are not enough publications that give a detailed morphological description of each taxon, while a thorough morphological knowledge of all Anopheles species is essential. The taxonomic recognition of these species should be based on a combination of morpho- logical, biological and molecular methods on the adults, egg, larva and pupa of the members of the group. A detailed comparative studies of associated all life stages of unambiguously iden- tified samples using of progeny broods, is an important resource for systematic studies. Although malaria transmission in north and central areas of Iran was completely controlled, travelling from endemic southern areas of the country and neighbouring countries and also presence An. maculipennis subgroup as poten- tial vectors, always are threats of recurrence of the disease in these areas. In conclusion, considering the medical im- portance of the Maculipennis Subgroup, and dif- ferences in vector capacities and species dis- tributions, it is important to elucidate reliable methods to unambiguously identify the species group. ACKNOWLEDGEMENTS The authors are grateful to Dr S Tirgari, Miss M Jedari and Ms F Faghih-Naini, Depart- ment of Medical Entomology and Vector Con- trol and Medical Arthropods Museum, School of Public Health and Institute of Public Health Tehran University of Medical Sciences, for providing the specimens. The project was fi- nancially supported by the Education Office of the School of Public Health. 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