J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 349 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Wolbachia Endobacteria in Natural Populations of Culex pipiens of Iran and its Phylogenetic Congruence Mohsen Karami 1, *Seyed Hassan Moosa-Kazemi 1, *Mohammad Ali Oshaghi 1, Hasan Vatandoost 1, Mohammad Mehdi Sedaghat 1, Ramazan Rajabnia 2, Mostafa Hosseini 3, Naseh Maleki-Ravasan 4, Yousef Yahyapour 2, Elaheh Ferdosi- Shahandashti 5 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Infectious Diseases & Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran 3Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 5Department of Advanced Technologies in Medicine (SATiM), Medical Biotechnology,Tehran University of Medical Sciences, Tehran, Iran (Received ١٥ Apr 2015; accepted 3 Oct 2015) Abstract Background: Wolbachia are common intracellular bacteria that infect different groups of arthropods including mos- quitoes. These bacteria modify host biology and may induce feminization, parthenogenesis, male killing and cyto- plasmic incompatibility (CI). Recently Wolbachia is being nominated as a bio-agent and paratransgenic candidate to control mosquito borne diseases. Methods: Here we report the results of a survey for presence, frequency, and phylogenetic congruence of these en- dosymbiont bacteria in Culex pipiens populations in Northern, Central, and Southern parts of Iran using nested-PCR amplification of wsp gene. Results: Wolbachia DNA were found in 227 (87.3%) out of 260 wild-caught mosquitoes. The rate of infection in adult females ranged from 61.5% to 100%, while in males were from 80% to 100%. The Blast search and phyloge- netic analysis of the wsp gene sequence revealed that the Wolbachia strain from Iranian Cx. pipiens was identical to the Wolbachia strains of supergroup B previously reported in members of the Cx. pipiens complex. They had also identical sequence homology with the Wolbachia strains from a group of distinct arthropods including lepidopteran, wasps, flies, damselfly, thrips, and mites from remote geographical areas of the world. Conclusion: It is suggested that Wolbachia strains horizontally transfer between unrelated host organisms over evo- lutionary time. Also results of this study indicates that Wolbachia infections were highly prevalent infecting all Cx. pipiens populations throughout the country, however further study needs to define Wolbachia inter-population repro- ductive incompatibility pattern and its usefulness as a bio-agent control measure. Keywords: Culex pipiens, Wolbachia, cytoplasmic incompatibility, nested-PCR, Iran Introduction Mosquitoes including Culex pipiens com- plex with global distribution are vectors of ar- boviral pathogens and parasites such as West Nile, St Louis, Sindbis, Wuchereria bancrofti, Dirofilaria immitis, D. repens, Plasmodium relictum, and P. gallinaceum (Vinogradova *Corresponding author: Dr Seyed Hassan Moosa- Kazemi, Email: moosakazemii@tums.ac.ir, Dr Moham- mad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 350 http://jad.tums.ac.ir Published Online: January 06, 2016 2000, Pawelek et al. 2014). Among the ‘ne- glected’ mosquito-borne diseases, lymphatic filariasis continues to be a hazard to over a billion people in 83 countries (O'Connor et al. 2012). Culex pipiens is a species complex and comprise Cx. quinquefasciatus and Cx. pipiens in South and North America, Asia and Africa, as well as Cx. globocoxitus and Cx. australicus in Australia (Farajollahi et al. 2011). Culex pipiens and Cx. quinquefascia- tus are distributed in most parts of Iran rang- ing from north to south (Zaim 1986, Azari- Hamidian 2007, Nikookar et al. 2010, Khoshdel-Nezamiha et al. 2013, Banafshi et al. 2013, Dehghan et al. 2013, 2014). The raising of resistance to current insec- ticides by insect vectors (Hemingway and Ranson 2000), the progress of drug re- sistance in parasites (Talisuna et al. 2004) and lack of clinical cures or vaccines for many vector borne diseases have led re- searchers to develop urgently new and ad- vanced approaches to control of the diseases. Paratransgenesis, as a new approach, direct towards reducing vector competence through genetically manipulated symbionts (Coutinho- Abreu et al. 2010). Transformed symbionts are distributed across the insect population via transovarial or transstadial transmision routs (Durvasula et al. 1997, Chavshin et al. 2012, 2014, 2015, Maleki-Ravasan et al. 2015). Symbionts currently aimed at in par- atransgenesis include fungi (Rasgon 2011), symbiont bacteria of triatomine bugs (Dur- vasula et al. 1997, Durvasula et al. 1999, Durvasula et al. 2008), tsetse flies (Cheng and Aksoy 1999), sandflies (Maleki-Ravasan et al. 2015) and mosquitoes (Favia et al. 2007, Chavshin et al. 2014), and densovi- ruses infecting An. gambiae and Ae. aegypti mosquitoes (Ward et al. 2001, Ren et al. 2008). Recently, paratransgenesis have been successfully employed to reduce vector com- petence of the triatomine bug, Rhodnius pro- lixus, vector of Trypanosoma cruzi, the caus- ative agent of Chagas disease (Durvasula et al. 1997), and Anopheles gambiae and An. stephensi, two main malaria vectors (Rasgon 2011, Wang and Jacobs-Lorena 2013). These data showed that the genetically manipulated symbionts could interfere with the develop- ment of the parasites in the vectors and pro- vide the groundwork for the use of genet- ically modified symbionts as a potent tool to battle vector borne diseases. The bacterium of Wolbachia pipientis is an intracellular organism and inherited ma- ternally. It is established in more than 20% of all insects and a vast majority of other ar- thropods as well as filarial nematodes (Wer- ren 1997a, Dobson 2004, Lo and Evans 2007). Recent studies imply that 20–76% of investigated insects give shelter to Wolbach- ia (Hilgenboecker et al. 2008), as well as many arachnids, terrestrial crustaceans, and mites (Cordaux et al. 2001, Gotoh et al. 2003, Rowley et al. 2004). This unique en- dosymbiont species was originally found in Cx pipiens but later molecular studies have discovered a number of phylogenetically di- verse strains within the species (Lo et al. 2007). This endosymbiont bacterium has significant effects on its arthropod hosts and nominated as a bioagent to control important arthropod pests. Wolbachia is the cause of various modifi- cations in insect reproductive arrangement, comprising male-killing, feminization, cyto- plasmic incompatibility (CI), and partheno- genesis (Werren et al. 2008). When CI oc- curs, sperm and eggs are not able to produce feasible progeny (Werren 1997b, Clark et al. 2003, Beckmann and Fallon 2013). Infected females relative to uninfected ones, partici- pate more in offspring production, which permit Wolbachia to take up by all of host individuals even if it cases fitness costs (Field et al. 1999). The bacterium also can be used as a vector for delivering desirable genetic modifications in insect populations (Werren 1997b). As reviewed by Werren (1997a), Wolbachia have potential roles in J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 351 http://jad.tums.ac.ir Published Online: January 06, 2016 the rapid speciation of their hosts. Also as a pandemic endosymbiont, Wolbachia can be recruited to control of a large number of human infectious diseases (Slatko et al. 2014). In filarial nematodes comprising Wu- chereria bancrofti, Brugia malayi, Brugia timori and Onchocerca volvulus that infect humans, Wolbachia are obligated for proper development, fertility and survival, whereas in arthropods, although they can affect de- velopment and reproduction, but are not re- quired for host survival. So Wolbachia have been a target for drug discovery against fila- riasis. In vivo/ vitro experiments indicate that antibiotics such as doxycycline and tet- racycline can kill both adults and immature nematodes through depletion of Wolbachia (Foster et al. 2013, Taylor et al. 2014). It is also shown that, Wolbachia spp where natu- rally infected or artificially introduced into vector population can affect and decrease the mosquitoes competence carrying of viruses, such as Yellow Fever, Chikungunya, Den- gue, West Nile, as well as ones transmitting of the Plasmodium protozoans and filarial nematodes (Bourtzis et al. 2014). Due to the fact that Wolbachia is an obli- gate endosymbiont that cannot be cultured exterior their hosts, recognition of infection has been based vastly on amplification of Wolbachia DNA using PCR. Until now a number of loci including wsp, 16S rDNA, coxA, ftsZ, hcpA, gatB, groEL, fbpA, gltA and dnaA genes have been studied and eval- uated in the phylogenetic studies (Zhou et al. 1998, Ravikumar et al. 2011). The sequences from Wolbachia surface protein (wsp) gene were extremely mutable and could be used to recognition and to re solve the phyloge- netic relationships of different Wolbachia strains (Zhou et al. 1998). In the present study we used a nested PCR assay to detect and investigate the prev- alence of Wolbachia endobacteria using the partial genomic nucleotide sequence of wsp gene in twelve field populations of Culex pipiens in various geographical regions across Iran ranging from north to south. Results of this study will provide fundamental back- ground for understanding ecology, distribu- tion, and potential utility of Wolbachia as bio-control agent of Cx. pipiens. Materials and Methods Study areas The study was conducted in twelve loca- tions belong to three provinces of Iran, Ma- zandaran in the North (six locations), Isfahan in the center (3 locations) and Hormozgan in the South (3 locations) of the country (Fig. 1). Live larvae, pupae, and adult mosquitoes were collected from different biotypes in- cluding plane, jungle, riverside, rice field and human dwellings. Mosquito collection Adult mosquitoes were collected in hu- man dwellings monthly for a period of five months (June to late October, 2014) by hand- catch collection method using mouth aspira- tor. Also live larvae and pupae were col- lected from mosquito breeding sites locating in plane, jungle, riverside and rice field us- ing dipping method, transferred to insectary, and allowed them to grow till adult emer- gence. Adult specimens were keyed to spe- cies level using standard morphological keys (Zaim 1986, Azari-Hamidian and Harbach 2009). The male and female mosquito spec- imens belong to Cx. pipiens were selected and stored individually at -20 ºC for further molecular investigations. Double distilled water and mix of 10 adult male and female specimens of Anopheles maculipennis were collected from Mazanderan Province and used as negative controls. DNA extraction and PCR Totally 260 (120 males and 140 females) Cx. pipiens specimens originated from dif- ferent biotopes from north to south of Iran J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 352 http://jad.tums.ac.ir Published Online: January 06, 2016 were randomly subjected to genomic DNA extraction. Genomic DNA of An. maculi- pennis ss was extracted and used in all PCR assays as negative control. Total DNA of individual mosquitoes was extracted using Collins DNA extraction method (Collins et al. 1987). Previously a PCR based method for the classification of Wolbachia has been described (Zhou et al. 1998). In that method, group-specific wsp PCR primers have been used to identify Wolbachia strains without the need to clone and sequence individual Wolbachia genes. Here in detection of Wolbachia infection in the mosquitoes was performed by a nested-PCR assay on the ba- sis of Zhou introduced primers. Initially, a set of primers including 81F: 5'–TGGTCCA ATAAGTGATGAAGAAAC–3' and 691R: 5'– AAAAATTAAACGCTACTCCA–3' were recruited to amplify 632 bp of partial se- quence of the wsp gene. The PCR product of the first step was applied as a template for second step. In the second step, another pairs of the primers, 183F: 5'–AAGGAACCG AAGTTCATG–3' and 691R: 5'–AAAAA TTAAACGCTACTCCA–3', were used to amplify a 501 bp fragment. The PCR amplification was performed using Maxime PCR PreMix Kit (i-Taq) Cat. No. 25026 in 20 μl reaction mixtures con- taining 2.5 μl of 10 μM both forward and reverse primers and 5 μl (~0.5 μg) of ge- nomic DNA and 2.5 μl PCR product for the first and second step of nested-PCR reac- tions respectively. An individual specimen of Anopheles maculipennis s.s. was used as DNA extraction and PCR negative controls. The PCR conditions were set as an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, followed by a final ex- tension at 72 °C for 7 min. PCR products were visualized on a 1% agarose gel con- taining ethidium bromide and using an UV transilluminator. Wsp gene sequencing and analyzing Representative specimens with clear and sharp wsp gene amplicons of the twelve Cx. pipiens populations were sequenced via the same amplification primers by Bioneer Com- pany (S. Korea). The consensus of confident sequences was analyzed using NCBI (Nu- cleotide collection) database. The wsp gene sequences determined in this study were subjected to molecular phy- logenetic analysis together with 44 wsp gene sequences of Wolbachia from various ar- thropod host species retrieved from the Gen- bank database (Table 1). A multiple align- ment of the wsp sequences was generated by the program package Clustal W (Thompson et al. 1994). Phylogenetic trees were con- structed using the neighbor-joining method embedded in MEGA5 software. Bootstrap tests were performed with 1,000 replications. Statistics analyzing Wolbachia infection data in Culex pipiens specimens were analyzed using SPSS 22.0 and Chi square (χ2) test to make comparisons and evaluate variation in infection rates be- tween the males and females and among the twelve populations. The P-value more than 5% was considered as significant. Results Wolbachia detection in Cx. pipiens The infection of Wolbachia in different Cx. pipiens populations was detected by the nested-PCR assay using wsp gene. The am- plicons of first and second runs of nested- PCR assay were ~ 650 and 500 bp respec- tively (Fig. 2). Wolbachia infection rate Results of the study demonstrated that in total, 227 (87.3%) out of 260 individual adult mosquitoes belonged to 12 distinct populations were positive against wsp gene (Table 2). All the infected mosquitoes were J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 353 http://jad.tums.ac.ir Published Online: January 06, 2016 found to harbor a single wPip strain. Infec- tion rate in adult females and males were 61.5–100% and 80–100% respectively. There were no significant differences be- tween total infection rates of either sexes (Female= 89.2%, Male = 85.7%, df= 1, P> 0.05) or zones (df= 3, P> 0.05). Wolbachia wsp sequences Seven nested–PCR products the wsp gene of Wolbachia found in different Iranian pop- ulations of Cx. pipiens were successfully se- quenced and submitted to Genbank (Acces- sion Numbers (ANs): KM401551–7). The nested primers we used were only able to amplify fragments from infected specimens and not from uninfected An. maculipennis ss hosts. The sequences were A-T rich (61%) with only 39% GC content. The BLAST re- sults indicated that all the wsp sequences of Wolbachia detected from the Iranian Cx. pipiens were 100% identical to each other and to the Wolbachia strains found in other members of the Cx. pipiens complex includ- ing Cx. pipiens, Cx. pipiens form molestus, Cx. pipiens (syn. pallens), and Cx. quinque- fasciatus from remote geographical areas of the world (Table 3). Since the Wolbachia strain that infects Cx. pipiens complex be- longs to Pip group of B supergroup (wPipB) (Zhou et al. 1998, Pidiyar et al. 2003), we can conclude that the Wolbachia strains from Iranian Cx. pipiens specimens belongs to wPipB strain. In addition, the sequences of Wolbachia wsp gene of Iranian Cx. pipiens were 100% identical to the wsp gene of Wolbachia strains found in divers insect or arthropod groups particularly to the order of Lepidoptera comprising 18 different but- terfly and moth species, as well as to wasps, thrips, damselflies, Aedes mosquito, Three- striped fruit fly, leaf-mining fly, and mite. These Wolbachia host species belong to ge- ographically remote regions of Asian, Euro- pean, and African countries (Table 3). A comparison of the wsp sequences from the arthropod hosts showed up to 30.67% ge- netic diversity between taxa, in which the wsp sequence from bedbug was the most di- verged one. Phylogenetic analysis For phylogenetic analysis a subset of the Wolbachia strains identified in this study were combined with a 44 available sequence data of other Wolbachia strains from Gen- bank. These sequences belonged to twenty different arthropod hosts of Wolbachia in- cluding mosquitoes (Culex and Aedes), fruit flies, blow flies, sand flies, tsetse flies, leaf mining flies, bed bugs, thrips, damselflies, plant hoppers, crickets, termites, butterflies, moths, wasps, ants, beetles, pill woodlouse, spiders, and mites (table 1). Phylogenetic tree was constructed using neighbor-joining meth- od, based on the 445–511 bp of wsp se- quences (Fig. 3). The length variation be- tween sequence data was due to insertion or deletion (indels) events. We also used Diro- filaria immitis wsp sequence as an out-group in the analysis. Phylogenetic analysis showed that Wolbachia strains from Iranian Cx. pipiens specimens were clustered with Wolbachia strains of other members of the Cx. pipiens complex such as Cx. pipiens, Cx. pipiens (syn. pallens), Cx. pipiens form mo- lestus and Cx. quinquefasciatus (Fig. 3). They also associated with Wolbachia strains found in distinct groups of arthropods not obtained from the same insect genus, family, or even order. In other word, Wolbachia strains obtained from the same insect genus or families were not clustered into distinct groups but were scattered throughout the phy- logenetic tree. Except for the congenic clus- ters of mosquitoes, sand flies, and tsetse flies, there were no other congenic clusters in- dicating little congruence between Wolbach- ia phylogeny and host systematics. The phy- logenetic analysis revealed six main clades for the wsp sequences of Wolbachia strains analysed (Fig. 3). The first clade was com- J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 354 http://jad.tums.ac.ir Published Online: January 06, 2016 posed of all mosquitoes (eight Culex spp and two Aedes spp) and ten wsp sequences from lepidopteran, wasp, Thrips, damselfly, Three- striped fruit fly, leaf-mining fly, leaf beetle, and mite, all belonged to the known super- group B of Wolbachia. The second lineage was composed of nine wsp sequences from blowfly, plant hopper, cricket, moth, wasp, fire ant, flour beetle, and mite. Eleven wsp sequences from fruit flies, sand flies (2 spe- cies), tsetse flies (2 species), termite, moth, wasps (2 species), ant, and spider, consti- tuted an isolated lineage. The wsp sequences from one of each wasp, plant hopper, and moth formed a distinct clade. Most of strains of second and third clades belong to the known supergroup A of Wolabachia. Notably the bedbug and one termite wsp sequences associated together and formed a well-defined clade, and finally pill wood louse constituted a diverse clade well separated from other five clades. Except for four nodes with 57– 71% support, all of the nodes had very high (82–100) bootstrap support values (Fig. 3). Fig. 1. Map of study areas for collection of Cu- lex pipiens specimens in Iran. Nos, 1–2: Ramsar, 3–4: Amol, 5–6: Behshahr in Mazandaran Prov- ince, 7: Vinicheh, 8: Dizicheh, 9: Dorcheh in Isfahan Province, 10: Hormodar, 11: Siahoo, and 12: Shamil in Hormozgan Province Fig. 2. Species-specific nested-PCR products (~ 500 bp) of Wolbachia wsp gene of Culex pipiens specimens. Lanes: M, 1 Kbp molecular weight marker (Fermentas), 1–2: Mazandaran Provine, 3: Isfahan Provine, 4–5: Hormozgan Provine, 6: Anopheles maculipennis as negative control J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 355 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 1. Description of Wolbachia strains used for phylogenetic analysis in this study No Wolbachia Strain Host Common name Accession number References 1 wPip/B Culex pipiens Mosquito KM401552 This study 2 wPip/B Cx. pipiens Mosquito KM401553 This study 3 wPip/B Cx. pipiens Mosquito KM401556 This study 4 wPip/B Cx. pipiens Mosquito JX474753 Direct Submission 5 wPip/B Cx. pipiens (syn. pallens) Mosquito AF216860 Direct Submission 6 wPip/B Cx. pipiens form molestus Mosquito HG428761 (Pinto et al. 2013) 7 wPip/B Cx. quinquefasciatus Mosquito AF020060 (Zhou et al. 1998) 8 wPip/B Cx. quinquefasciatus Mosquito KJ140126 Direct Submission 9 wAlbB/B Aedes albopictus Mosquito AF020059 (Zhou et al. 1998) 10 wPip/B Ae. punctor Mosquito AJ311040 (Ricci et al. 2002) 11 w AlbA/A Ae. albopictus Mosquito AF020059 (Zhou et al. 1998) 12 wNo/B Drosophila simulans Fruit Fly AF020074 (Zhou et al. 1998) 13 wMel/A D. melanogaster Fruit Fly AF020072 (Zhou et al. 1998) 14 wAus/A Glossina austeni Tsetse fly AF020077 (Zhou et al. 1998) 15 wMors/A G. morsitans morsitans Tsetse fly AF020079 (Zhou et al. 1998) 16 N.S Protocalliphora sialia Blow fly DQ842482 (Baldo et al. 2006) 17 wPak-B1 Hydrellia pakistanae Leaf mining fly AF217718 (Jeyaprakash and Hoy, 2000) 18 papa01/A Phlebotomus papatasi Sand fly EU780683 (Parvizi et al. 2013) 19 Turk 07 Ph. mongolensis Sand Fly KC576916 (Parvizi et al. 2013) 20 wCon/B Tribolium confusum Flour Beetle AF020083 (Zhou et al. 1998) 21 N.S Chelymorpha alternans Leaf Beetle DQ842458 (Baldo et al., 2006) 22 wOri/B Tagosodes orizicolus Plant hopper AF020085 (Zhou et al. 1998) 23 wStri/B Laodelphax striatellus Plant hopper AF020080 (Zhou et al. 1998) 24 F Cimex lectularius Bed Bug DQ842459 (Baldo et al. 2006) 25 wDei/B Trichogramma deion Wasp AF020084 (Zhou et al. 1998) 26 wTde-HEB T. dendrolimi Wasp JX027991 Direct Submission 27 wkue/A Spalangia cameroni Wasp AF289668 Direct Submission 28 N.S Encarsia formosa Wasp DQ842471 (Baldo et al. 2006) 29 wNPan/A Nomada panzeri Red Wasp KC798315 (Gerth et al. 2013) 30 A Solenopsis invicta Fire Ant DQ842483 (Baldo et al. 2006) 31 A Formica truncorum Ant AF326978 (Wenseleers et al. 2002) 32 wCauB/B Ephestia cautella Moth AF020076 (Zhou et al. 1998) 33 wCauA/A Ephestia cautella Moth AF020075 (Baldo et al. 2006) 34 B Ostrinia scapulalis Moth DQ842481 (Baldo et al. 2006) 35 NS Eurema hecabe Butterfly AB285478 (Narita et al. 2007) 36 NS Udaspes folus Butterfly JN236179 (Salunke et al. 2012) 37 NS Agriocnemis femina Damselfly AY173939 (Thipaksorn et al. 2003) 38 NS Gryllus firmus Cricket DQ842474 (Baldo et al. 2006) 39 A Incisitermes snyderii Termite DQ842475 (Baldo et al. 2006) 40 F Coptotermes acinaciformis Termite AJ833931 (Baldo et al. 2006) 41 NS Hercinothrips femoralis Thrips AB245521 Direct Submission 42 NS Nephila clavata Spider EF612772 Direct Submission 43 NS Oxyopes sertatus Spider EF612771 Direct Submission 44 NS Eriovixia cavaleriei Spider DQ778738 Direct Submission 45 NS Tetranychus urticae Two-spotted spi- der mite AJ437290 Direct Submission 46 NS Bryobia berlesei Mite JN572865 (Ros et al. 2012) 47 NS Armadillidium vulgare Pill woodlouse DQ842457 (Baldo et al. 2006) 48 Outgroup Dirofilaria immitis Nematode AJ252062 (Bazzocchi et al. 2000) NS: Not stated. J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 356 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 2. Prevalence of Wolbachia pipientis infection in the Culex pipiens collected from North, Center and South of Iran, 2014 Province Location Biotope Males tested (% P +) Females tested (% P+) Total (% P+) Mazandaran (North) Amol 1 Plane 10(90) 13(61.5) 74 Amol 2 Jungle 10(80) 10(100) 90 Behshar 1 Plane 10(100) 10(100) 100 Behshar 2 Jungle 10(90) 10(90) 90 Ramsar 1 Plane 10(90) 10(80) 85 Ramsar 2 Jungle 10(100) 14(100) 100 Isfahan (Center) Dizicheh Rice fields 10(90) 10(90) 90 Vinicheh Rice fields 10(80) 10(70) 75 Dorcheh Rice fields 10(100) 15(100) 100 Hormozgan (South) Shamil Date Groves 10(80) 13(61.5) 70 Siahoo Riverside 10(80) 10(90) 85 Hormoodar Date Groves 10(90) 15(86.7) 88 Total 120(89.2) 140(85.7) 87.3 (260) Table 3. Details of arthropods have identical Wolbachia wsp sequences with the Iranian Culex pipiens Arthropod group Species Accession Number Country Reference Mosquito Culex pipiens Cx. pipiens form molestus Cx. pipiens (Syn. pallens) Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Aedes punctor JX474753 HG428761 AF216860 KJ140126 EU194487 AF397413, AF397412 AY462861 AM999887 AJ311040 Turkey NS China China India India India Taiwan NS Italy Direct Submission (Pinto et al. 2013) Direct Submission Direct Submission Direct Submission Direct Submission Direct Submission (Tsai et al. 2004) (Klasson et al. 2008) (Ricci et al. 2002) Butterfly Udaspes folus Hypolimnas bolina Castalius rosimon Eurema hecabe Ypthima asterope Papilio demoleus Zizeeria knysna Colotis amata Pseudozizeeria maha Leptidea sinapis Pararge aegeria Polygonia calbum Hypolimnas bolina JN236179 JN236180 JN236182 JN236189 JN236192 JN236193 JN236194 JN236195 JN236205 KC137222 KC137224 JN093149 AJ307076 India India India India India India India India India NS NS NS Fiji (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Salunke et al. 2012) (Russell et al. 2012) (Russell et al. 2012) (Kodandaramaiah et al. 2011) (Dyson et al. 2002) Moth Corcyra cephalonica Epirrita autumnata Spodoptera exempta Corcyra cephalonica Acraea encedon KC844060 JX310335 JN656943 AY634679 AJ271198 China NS Tanzania China Tanzania Direct Submission (Kvie et al. 2012) Direct Submission Direct Submission Direct Submission Wasp Trichogramma chilonis T. dendrolimi T. brassicae T. dendrolimi T. japonicum Tropobracon schoenobii AY311486 JX027991 AF452646 DQ017751 KC161917 AF481194 China China China China China NS Direct Submission Direct Submission Direct Submission Direct Submission Direct Submission (Kittayapong et al. 2003) Thrips Hercinothrips femoralis AB245521 Japan Direct Submission Damselfly Agriocnemis femina Coenagrionidae sp AY173939 KC161926 NS China (Thipaksorn et al. 2003) Direct Submission J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 357 http://jad.tums.ac.ir Published Online: January 06, 2016 Fruit fly Bactocera diversa AF295353 NS (Jamnongluk et al. 2002) Leaf-mining fly Hydrellia pakistanae AF217718) NS (Jeyaprakash and Hoy 2000) Mite Bryobia berlesei JN572865 France (Ros et al. 2012) NS: Not stated. Fig. 3. The phylogenetic tree inferred from 445–511 bp of wsp sequences of Wolbachia pipientis hosts using the neighbor- Joining method embedded in MEGA 5.0. C.p1–3 (Culex pipiens from this study), C.p4 (Culex pipiens), C.pm (Culex pipiens form molestus), C.q and C.q2 (Culex quinquefasciatus), C.pa (Culex pipiens, syn.: pallens), A.a (Aedes albopictus), D.m (Drosophila melanogaster), D.s (Drosophila simulans), G.m (Glossina morsitans morsitans), G.a (Glossina austeni), P.s (Protocalliphora sialia), P.p (Phlebotomus papatasi), P.m (Phlebotomus mongolensis), T.c (Tribolium confusum), Ch.a (Chelymorpha alternans), L.s (Laodelphax striatellus), T.o (Tagosodes orizicolus), C.l (Cimex lectularius), T.d (Trichogramma deion), T.dr (T.dendrolimi), S.c (Spalangia cameroni), E.f (Encarsia formosa), N.p (Nomada panzeri), S.i (Solenopsis invicta), , F.t (Formica truncorum), E.c1–2 (Ephestia cautella), O.s (Ostrinia scapulalis), E.h (Eurema hecabe), G.f (Gryllus firmus), I.s (Incisitermes snyderii), C.a (Coptotermes acinaciformis), N.c (Nephila clavata), Ox.s (Oxyopes sertatus), E.c (Eriovixia cavaleriei), T.u (Tetranychus urti- cae), A.v (Armadillidium vulgare), A.f (Agriocnemis femina), H.f (Hercinothrips femoralis), B.b (Bryobia berlesei), A.p (Aedes punctor), U.f (Udaspes folus), H.p (Hydrellia pakistanae), and D.i (Dirofilaria immitis). The bootstrap values are shown as num- bers on the nodes Table 3. Continued… J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 358 http://jad.tums.ac.ir Published Online: January 06, 2016 Discussion This is the first report on Wolbachia in- fection from Cx. pipiens populations of Iran. In our study, 260 specimens of Cx. pipiens collected from the 12 villages were individ- ually assayed for Wolbachia, and the overall rate of infection was determined to be 87.3%. This result is in agreement with pre- vious study conducted in South West Iran revealed 100 percent Wolbachia infection in Cx. quinquefasciatus specimens (Behbahani 2012). In California, Wolbachia infection frequency in Cx. pipiens complex during 1999 and 2000 was 99.4% (Rasgon and Scott, 2003). Also Sunish et al. (2011) found an overall prevalence of 91.2% Wolbachia infections in Cx. quinquefasciatus mosqui- toes from south India. Study of Chen et al (2013) revealed that three Cx. pipiens (Syn. pallens) populations of China were all in- fected with Wolbachia. This rate was re- ported between 10–100% in members of Cx. pipiens complex mosquitoes from the Upper Rhine Valley in Germany and Cebu City in Philippines (Mahilum et al. 2003). In this study we found no Wolbachia in- fection in An. maculippenis ss specimens which is in concurrence of study of Rasgon and Scott (2004) who tested five genera of mosquito (Aedes, Anopheles, Culiseta, Culex, and Ochlerotatus) for Wolbachia, and infec- tions was only detected in members of the Cx. pipiens complex. Also study of Kitta- yapong et al. (2000) detected Wolbachia in- fection in all main disease vector genera ex- cluding Anopheles. In our study, the percent- age prevalence in adult males was 80–100%, while in females were 61.5–100%. However the difference was not significant between males and females. In contrast, in the study of Sunish et al (2011) the rate of Wolbachia in- fection in females of Cx. quinquefasciatus was found slightly higher than in males but like our study it was not statistically significant. This study showed no sequence variation in wsp gene of Wolbachia from Cx. pipiens populations across geographical regions of Iran, which is similar to the results of Morais et al. (2012) which showed that both Cx. quinquefasciatus and Cx. pipiens × Cx. quinquefasciatus hybrids collected Brazil and Argentina were infected with a single Wolbachia strain. The genetic similarity de- tected among Wolbachia samples in the Cu- lex mosquitoes from geographically scattered regions may be explained by either Wolbachia host-endosymbiont specificity (Werren et al. 2008) or recently Wolbachia infection in Cu- lex populations (Morais et al. 2012). High sequence homology and close phy- logenetic relationships of Wolbachia strains from mosquitoes, spider, wasp, mite, dam- selfly, butterfly, thrips, fruit fly, and leaf mining fly indicate that Wolbachia endo- symbionts not only are maternally transmit- ted through host generations by vertical transmission but also horizontally transfer between unrelated host organisms (i.e. shift host species or “jumping”) (Van Meer et al. 1999, Baldo et al. 2005). Although the mech- anisms of jumping are still unclear, it is be- lieved that parasitoids may involve (Heath et al. 1999, Huigens et al. 2000, Noda et al. 2001, Kikuchi and Fukatsu 2003). Recombi- nation in wsp gene of Wolbachia strains has been evidenced by other researchers (Werren and Bartos 2001, Jiggins 2002, Reuter and Keller 2003). For example, Werren and Bar- tos (2001) reported recombination within supergroup B, occurring between the two Wolbachia strains of a parasitoid wasp and the fly it parasitizes. More recently it is shown that hypervariable regions of wsp gene of Wolbachia strains have got a com- plex mosaic structure, suggesting a clear in- tragenic recombination of segments among several divergent strains, both within and J Arthropod-Borne Dis, September 2016, 10(3): 349–365 M Karami et al.: Wolbachia Endobacteria … 359 http://jad.tums.ac.ir Published Online: January 06, 2016 between the arthropod supergroups (Baldo et al. 2005). The phylogenetic analysis of wsp se- quences of Wolbachia from 20 different ar- thropod hosts scattered the sequences into five main clades that in some parts, topo- graphically matched well with the tree of Zhou et al. (1998). Based on Wolbachia ftsZ gene sequences, two major supergroups A and B were reported within the Wolbachia strains (Werren and Jaenike 1995) where the type strain from Cx. pipiens was placed within supergroup B. In the tree we ob- tained in this study, two main clades repre- sent supergroups A and B (Fig. 3). In addi- tion to the Wolbachia strains from mosqui- toes, the strains from spider, wasp, mite, damselfly, butterfly, thrips, fruit fly, and leaf mining fly also placed in supergroup B. In- terestingly the Wolbachia strain from bed- bug was associated with the one from ter- mite of supergroup F or H. As reviewed by Lo et al. (2007), currently the genus Wolbachia was divided into eight taxonomic supergroups (A to H) where A and B are the two major groups established in arthropods, C and D are found in filarial nematodes, E infecting springtails and F contains Wolbachia bacteria that infect termites and filarial species. Supergroup G and H were reported in spiders and termites respectively. In addition other divergent lineages, such as those from various flea species and the fi- larial nematode Dirofilaria repens, might be added to the list of supergroups. Therefore, as more sequence information becomes available the number of clades, groups, or supergroups might be increased. For exam- ple, in our analysis the Wolbachia from woodlouse construct a single clade and might be considered as a separate clade. Conclusion In this study we found a single Wolbachia strain from Cx. pipiens populations across the country. Although it is suggested that a large set of compatible Wolbachia strains are always locally dominate within mosquito populations (Duron et al. 2011), however, several studies have showed that some wPip strains are reciprocally incompatible but also that some others, although genetically dis- tinct, are fully compatible (Duron et al. 2006, Duron et al. 2007, Atyame et al. 2011). Therefore, it is worth to test cyto- plasmic incompatibility (CI) between the Iranian populations. In case of having CI, it can be used as a form of sterile-insect tech- nique (SIT), to suppress, to replace, or to reduce the survival of mosquito populations and thereby control them or reduce their ability to transmit the infection (Townson 2002). Acknowledgements Tehran University of Medical Sciences (TUMS) financially supported this work (Grant No. 22738). Also it is noteworthy that this research has been done by support of the Babol University of Medical Sciences (BUMS). Special thanks to Roghayeh Pour- bagher, Seyedeh Narges Mousavi Kani, Ze- inab Abedian, and Seyed Mohsen Agha- janpour Mir for helping in Cellular and Mo- lecular Laboratory. The authors appreciate Mr Bagheri and Mr Hosseintabar for their cooperation in the laboratory. We would like to thank Mr Pakari, Mr Salari and Mr Aran- dian for helping in field collections. References Atyame CM, Delsuc F, Pasteur N, Weill M, Duron O (2011) Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito. Mol Biol Evol. 28 (10): 2761–2772. 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