Molecular evidence of Culex pipiens form molestus and hybrids pipiens/molestus in Morocco, North Africa

  • Fadila Amraoui1, 2,

    Affiliated with

    • Mhamed Tijane2,

      Affiliated with

      • Mhammed Sarih1 and

        Affiliated with

        • Anna-Bella Failloux3Email author

          Affiliated with

          Parasites & Vectors20125:83

          DOI: 10.1186/1756-3305-5-83

          Received: 11 March 2012

          Accepted: 27 April 2012

          Published: 27 April 2012

          Abstract

          Background

          Culex pipiens L. is the most widespread mosquito vector in temperate regions including North Africa. Cx. pipiens has two recognized forms or biotypes; pipiens and molestus are morphologically indistinguishable with distinct behavior and physiology that may influence their vectorial status. In our study, we prospected for the different forms of Cx. pipiens in Morocco.

          Methods

          Cx. pipiens larvae were collected in 9 sites throughout Morocco during summer 2010 and reared until imago stage. Cx. pipiens was identified using diagnostic primers designed for the flanking region of microsatellite CQ11.

          Results

          We established the presence of both forms of Cx. pipiens and their hybrids in Morocco.

          Conclusions

          Molecular identification provides the first evidence of the presence of Cx. pipiens form molestus in Morocco and hybrids between pipiens and molestus forms in North Africa. The epidemiological implications of our findings are discussed.

          Keywords

          Culex pipiens complex Microsatellite Molecular taxonomy Morocco North Africa

          Background

          The Culex pipiens complex includes several species; Cx. pipiens pipiens Linnaeus, 1758 and Cx. pipiens quinquefasciatus Say, 1823 are the most ubiquitous mosquitoes in temperate and tropical regions, respectively. Cx. p. pipiens has two distinct forms or biotypes: form pipiens and form molestus which are morphologically indistinguishable and differ in physiology and behavior. Cx. pipiens form pipiens is subjected to diapause (heterodynamic), is anautogeneous (only lays eggs after a blood-meal), and eurygamous (unable to mate in confined spaces). On the other hand, Cx. pipiens form molestus Forskal, 1775 does not diapause (homodynamic), is autogeneous (lays first batch of eggs without taking a blood-meal) and stenogamous (mates in confined spaces) [1, 2]. In addition, the biotypes molestus and pipiens occupy distinct habitats in Russia and the northeastern United States. Indeed, molestus form occurs in underground areas in urban settings while pipiens form lives aboveground [3, 4]. In Europe, sympatric occurrence of both biotypes has been observed in aboveground habitats as well as in underground habitats [57]. The two forms did not seem to be genetically isolated and were reported to hybridize in the United States and Europe [68]. They have different trophic preferences: pipiens biting mainly birds and molestus feeding on mammals, whereas hybrids exhibit an opportunistic behavior and can readily feed on both hosts. These feeding patterns are thought to influence the transmission of avian and mammalian pathogens.

          In North Africa, Cx. p. pipiens is a competent vector of several pathogens infecting animals and humans including West Nile virus [9], Rift Valley Fever virus [1012] and filarial worms [1316]. Based on morphological characters, behavioral and reproductive specializations, the mosquito Cx. p. pipiens was described in the North African region [1725]. Nevertheless, these classical characters present limited value. Therefore, our study aims to identify members of the Cx. pipiens complex present in Morocco based on a molecular identification.

          Methods

          Mosquitoes were collected as larvae using the “dipping” sampling method during summer 2010 from three Moroccan regions (Figure 1). A total of 9 sites were classified according to the habitat (urban, suburban or rural) and the type of breeding site (aboveground or underground). Fourth instar larvae were used for morphological identification [26] and reared until imago stage at 28 ± 1°C with 80% relative humidity and a 16 h:8 h photoperiod. Emerged adults were conserved at −20°C for subsequent molecular characterization.
          http://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-83/MediaObjects/13071_2012_Article_588_Fig1_HTML.jpg
          Figure 1

          Localization of the collection sites in Morocco.

          DNA extraction from F0 individuals was performed using the method of DNAzol as described in the manufacturer’s protocol. Specimens were identified as belonging to the Culex pipiens complex using a multiplex PCR assay described in Bahnck and Fonseca (2006) [27]. The locus CQ11 was used to distinguish between the two forms of Cx. pipiens. The DNA fragment size amplified varied between pipiens and molestus allowing us to distinguish the two forms in a single PCR reaction (Figure 2). Specimens of Cx. pipiens molestus from Japan were used as control.
          http://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-83/MediaObjects/13071_2012_Article_588_Fig2_HTML.jpg
          Figure 2

          Example of PCR amplification of the flanking region of the CQ11 microsatellite of Culex pipiens collected in an underground site in Casablanca (Morocco). DNA was extracted from individual mosquitoes and identified by PCR amplification of the flanking region of the CQ11 microsatellite. Lane M: 100-bp size marker; Lane 1: control Cx. pipiens form molestus from Japan; Lane 2: molestus form; Lane 3: pipiens form; Lane 4: hybrid form.

          Results and Discussion

          A total of 214 adults were characterized by PCR and frequencies of different forms are represented in Table 1. Overall, 52.3% of adults tested were homozygous for the 200 bp fragment which is characteristic of the pipiens form, 22% were homozygous for the 250 bp fragment identifying the molestus form and the remaining (25.7%) corresponded to hybrids.
          Table 1

          Frequency of forms of the Culex pipiens complex in Morocco

          City

          Habitat

          Breeding site

          Pipiens

          Molestus

          Hybrids

            

          (Ground)

          form (%)

          form (%)

          (%)

          Tanger

          Urban

          Above

          69.6 (16)

          8.7 (2)

          21.7 (5)

           

          Sub-urban

          Above

          52.2 (12)

          34.8 (8)

          13 (3)

           

          Rural

          Above

          62.5 (15)

          8.3 (2)

          29.2 (7)

          Casablanca/

          Urban

          Above

          31 (9)

          17.2 (5)

          51.8(15)

          Mohammedia

          Urban

          Under

          25 (8)

          59.4 (19)

          15.6 (5)

           

          Sub-urban

          Above

          53.6 (15)

          17.8 (5)

          28.6 (8)

          Marrakech

          Sub-urban

          Above

          60.9 (14)

          8.7 (2)

          30.4 (7)

           

          Rural

          Above

          78.3 (18)

          4.3 (1)

          17.4 (4)

           

          Rural

          Under

          55.6 (5)

          33.3 (3)

          11.1 (1)

          Cx.pipiens larvae were collected at various sites in Morocco, reared to adults and identified by PCR amplification of the flanking region of the CQ11 microsatellite. In brackets, number of tested mosquitoes.

          This study provides the first molecular evidence for the presence of Cx. pipiens form molestus in Morocco and hybrids in North Africa.

          The molestus form has been described as a distinct species, Cx. molestus Forskal, 1775 from autogeneous Egyptian specimens. Because Cx. pipiens form molestus is stenogamous and autogenous, it colonizes underground areas in urban settings [3] with limited geographic distribution throughout the world. In our study, Cx. pipiens form pipiens and form molestus were found in urban, suburban and rural habitats. Indistinctly, the two forms co-occur in aboveground and underground breeding sites. Sympatric distribution of the biotypes molestus and pipiens in surface breeding sites has been observed in southern Europe and the United States [5, 6, 8] and in underground breeding sites in North Europe [7].

          Conclusions

          Until now, hybrids were mainly reported in the United States [4, 8] and South Europe [6]. Our findings corroborate the presence of hybrids in all breeding sites sampled. Hybrids between molestus and pipiens forms are considered of great epidemiological importance. They exhibit intermediate physiological and behavioral traits [28] and can readily feed on avian and mammalian hosts [8, 29]. This opportunistic biting behavior will potentiate the role of Cx. pipiens as a bridge-vector for the transmission of pathogens such as West Nile virus, from birds (amplification hosts) to humans [8, 30].

          Declarations

          Acknowledgments

          We thank Laurence Mousson for technical help. FA was supported by the “Division Internationale” of the Institut Pasteur. This work was funded by the Institut Pasteur (ACIP grant A-08-2009) and the European Commission Framework Program Seven Award “InfraVec”.

          Authors’ Affiliations

          (1)
          Institut Pasteur du Maroc, Laboratoire des Maladies Vectorielles
          (2)
          Faculté des Sciences, Laboratoire de Biochimie et Immunologie
          (3)
          Institut Pasteur, Department of Virology, Arboviruses and Insect Vectors

          References

          1. Harbach RE, Harrison BA, Gad AM: Culex (Culex) molestus Forskal (Diptera: Culicidae): Neotype designation, description, variation, and taxonomic status. Proc Entomol Soc Wash. 1984, 86: 521-542.
          2. Harbach RE, Dahl C, White GB: Culex (Culex) pipiens Linnaeus (Diptera, Culicidae) - concepts, type designations, and description. Proc Entomol Soc Wash. 1985, 87: 1-24.
          3. Byrne K, Nichols RA: Culex pipiens in London Underground tunnels: differentiation between surface and subterranean populations. Heredity. 1999, 82: -15-
          4. Huang S, Molaei G, Andreadis TG: Genetic insights into the population structure of Culex pipiens (Diptera: Culicidae) in the Northeastern United States by using microsatellite analysis. AmJTrop Med Hyg. 2008, 79: 518-527.
          5. Chevillon C, Eritja R, Pasteur N, Raymond M: Commensalism, adaptation and gene flow: mosquitoes of the Culex pipiens complex in different habitats. Genet Res. 1995, 66: 147-157. 10.1017/S0016672300034492.View ArticlePubMed
          6. Gomes B, Sousa CA, Novo MT, Freitas FB, Alves R, Côrte-Real AR, Salgueiro P, Donnelly MJ, Almeida AP, Pinto J: Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal. BMC Evol Biol. 2009, 9: 262-10.1186/1471-2148-9-262.PubMed CentralView ArticlePubMed
          7. Reusken CBEM, de Vries A, Buijs J, Braks MAH, den Hartog W, Scholte EJ: First evidence for presence of Culex pipiens biotype molestus in the Netherlands, and of hybrid biotype pipiens and molestus in northern Europe. J Vector Ecol. 2010, 35: 210-212. 10.1111/j.1948-7134.2010.00080.x.View ArticlePubMed
          8. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Mogi M, Fleischer RC, Wilkerson RC: Emerging vectors in the Culex pipiens complex. Science. 2004, 303: 1535-1538. 10.1126/science.1094247.View ArticlePubMed
          9. Krida G, Diancourt L, Bouattour A, Rhim A, Chermiti B, Failloux AB: Assessment of the risk of introduction to Tunisia of the Rift Valley fever virus by the mosquito Culex pipiens. Bull Soc Pathol Exot. 2011, 104: 250-259. 10.1007/s13149-010-0122-4.View ArticlePubMed
          10. Hoogstraal H, Meegan JM, Khalil GM, Adham FK: The Rift Valley fever epizootic in Egypt 1977–78. 2. Ecological and entomological studies. Trans R Soc Trop Med Hyg. 1979, 73: 624-629. 10.1016/0035-9203(79)90005-1.View ArticlePubMed
          11. Meegan JM, Khalil GM, Hoogstraal H, Adham FK: Experimental transmission and field isolation studies implicating Culex pipiens as a vector of Rift Valley fever virus in Egypt. AmJTrop Med Hyg. 1980, 29: 1405-1410.
          12. Moutailler S, Krida G, Schaffner F, Vazeille M, Failloux AB: Potential vectors of Rift Valley fever virus in the Mediterranean Region. Vector Borne Zoonot Dis. 2008, 8: 749-753. 10.1089/vbz.2008.0009.View Article
          13. Harb M, Faris R, Gad AM, Hafez ON, Ramzi R, Buck AA: The resurgence of lymphatic filariasis in the Nile Delta. Bull WHO. 1993, 71: 49-54.PubMed CentralPubMed
          14. Krida G, Bouattour A, Rodhain F, Failloux AB: Variability among Tunisian populations of Culex pipiens: genetic structure and susceptibility to a filarial parasite, Brugia pahangi. Parasitol Res. 1998, 84: 139-142.View ArticlePubMed
          15. Abdel-Hamid YM, Soliman MI, Allam KM: Spatial distribution and abundance of culicine mosquitoes in relation to the risk of filariasis transmission in El Sharqiya Governorate, Egypt. Egypt Acad J Biolog Sci. 2009, 1: 39-48.
          16. Abdel-Hamid YM, Soliman MI, kenawy MA: Mosquitoes (Diptera: Culicidae) in relation to the risk of disease transmission in El Ismailia governorate, Egypt. J Egypt Soc Parasitol. 2011, 41: 109-118.PubMed
          17. Roubaud E: Le pouvoir autogène chez le biotype nord-africain du moustique commun Culex pipiens (L.). Bull Soc Path Exot. 1939, 36: 172-175.
          18. Knight KL, Malek AA: A morphological and biological study of Culex pipiens in the Cairo area of Egypt. Bull Soc Fouad I Entomol. 1951, 35: 175-185.
          19. Gaud J: Notes biogéographiques sur les Culicidés du Maroc. Arch Inst Pasteur Maroc. 1953, 4: 443-490.
          20. Vermeil C: Nouvelle contribution à l’étude du complexe Culex pipiens en Tunisie. Bull Soc Pathol Exot. 1954, 47: 841-843.
          21. Rioux JA: Les culicidés du “midi” méditerranéen. 1958, Lechevalier, Paris
          22. Senevet G, Andarelli L, Graells R: A propos de Culex pipiens en Algérie. Arch Inst Pasteur Algérie. 1958, 36: 70-74.PubMed
          23. Rioux JA, Juminer B, Kchouk M, Croset H: Présence du caractère autogène chez Culex pipiens pipiens L. dans un biotope épigé de l’Ile de Djerba. Arch Inst Pasteur Tunis. 1965, 42: 1-8.
          24. Pasteur N, Rioux JA, Guilvard E, Pech-Perières J: Nouvelle mention, pour le “Midi” méditerranéen, de populations naturelles anautogènes et sténogames de Culex pipiens pipiens L. Ann Parasitol Hum Comp. 1977, 52: 205-210.PubMed
          25. Himmi O, Dakki M, Trari B, El Agbani MA: Les Culicidae du Maroc: clés d’identification, avec données biologiques et écologiques. Trav Inst Sci, Série Zool Rabat. 1995, 44: 51-
          26. Brunhes J, Rhaim A, Geoffroy B, Angel G, Hervy JP: Les moustiques de l’Afrique méditerranéenne. Logiciel d’identification et d’enseignement. 2000, IRD & IPT, CD-Rom collection didactique, Éditions IRD, Montpellier, France
          27. Bahnk CM, Fonseca DM: Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L. (Diptera: Culicidae) and hybrid populations. AmJTrop Med Hyg. 2006, 75: 251-255.
          28. Spielman A: Structure and seasonality of Nearctic Culex pipiens populations. Ann NY Acad Sci. 2001, 951: 220-234.View ArticlePubMed
          29. Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Daszak P, Fonseca DM: Genetic influences on mosquito feeding behavior and the emergence of zoonotic pathogens. AmJTrop Med Hyg. 2007, 77: 667-671.
          30. Hamer GL, Kitron UD, Brawn JD, Loss SR, Ruiz MO, Goldberg TL, Walker ED: Culex pipiens (Diptera: Culicidae): a bridge vector of West Niles Virus to humans. J Med Entomol. 2008, 45: 125-128. 10.1603/0022-2585(2008)45[125:CPDCAB]2.0.CO;2.View ArticlePubMed

          Copyright

          © Amraoui et al.; licensee BioMed Central Ltd. 2012

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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