Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe José Vicente, Carla Sousa, Bulent Alten, Selim Caglar, Elena Falcutá, José Latorre, Céline Toty, Hélène Barré, Berna Demirci, Marco Luca, et al. To cite this version: José Vicente, Carla Sousa, Bulent Alten, Selim Caglar, Elena Falcutá, et al.. Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe. Malaria Journal, BioMed Central, 2011, 10, pp.5. 10.1186/1475-2875-10-5. hal-03059510 HAL Id: hal-03059510 https://hal.archives-ouvertes.fr/hal-03059510 Submitted on 12 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Malaria Journal 2011, 10:5 http://www.malariajournal.com/content/10/1/5 RESEARCH Open Access Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe José L Vicente1, Carla A Sousa2, Bulent Alten3, Selim S Caglar3, Elena Falcutá4, José M Latorre5, Celine Toty6, Hélène Barré7, Berna Demirci3, Marco Di Luca8, Luciano Toma8, Ricardo Alves1,2, Patrícia Salgueiro1, Teresa L Silva1, Maria D Bargues5, Santiago Mas-Coma5, Daniela Boccolini8, Roberto Romi8, Gabriela Nicolescu4, Virgílio E do Rosário1, Nurdan Ozer3, Didier Fontenille6, João Pinto1,2* Abstract Background: There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. Methods: Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. Results: Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004 <FST <0.086) and phenotypic differentiation were detected among An. atroparvus populations spanning over 3,000 km distance. Genetic differentiation (0.202 <FST <0.299) was higher between the sibling species An. atroparvus and Anopheles maculipennis s.s. Differentiation between sibling species was not so evident at the phenotype level. Conclusions: Levels of population differentiation within An. atroparvus were low and not correlated with geographic distance or with putative physical barriers to gene flow (Alps and Pyrenées). While these results may suggest considerable levels of gene flow, other explanations such as the effect of historical population perturbations can also be hypothesized. Background still not clear, however, if the impact of climate change Under the present scenario of human-driven environ- will be beneficial or adverse. Mosquito populations may mental changes, global climate change is one the most tend to expand with warming and changes in rainfall pat- relevant concerns. Climatic predictions point to a signifi- terns, which will tend to increase disease transmission. cant increase of summer droughts in south-western Eur- On the other hand, mosquito reproduction and survival opean regions over the next 60 years, but there is also an could be impaired by altered rainfall and increased aridity increased risk for more frequent flash floods during the leading to a reduction in transmission [2]. Nonetheless, same period [1]. Since the life cycles and distribution of theoveralleffectofanthropogenicclimatechangeon many insect vector species are directly influenced by cli- vector-borne diseases remains debated, and the outcome matological conditions, climate change has the potential may vary regionally [3]. to affect the incidence, seasonal transmission and geo- Malaria is the vector-borne disease with the highest graphic range of several vector-borne diseases [2]. It is impact in the World’s human population. In 2008, there were ca. 243 million cases, and an estimated 863,000 * Correspondence: [email protected] deaths attributed to malaria [4]. Although at present 1Centro de Malária e outras Doenças Tropicais/UEI Malária, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa. Rua da Junqueira malaria endemic areas are mainly restricted to tropical 100, 1349-008 Lisbon, Portugal and subtropical regions, several models project a Full list of author information is available at the end of the article © 2011 Vicente et al; licensee 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. Vicente et al. Malaria Journal 2011, 10:5 Page 2 of 9 http://www.malariajournal.com/content/10/1/5 geographical expansion of potential malaria transmission summer time (June to October) between 2006 and in the next few decades, and more substantial changes 2008 in 9 collection sites from five European countries later this century [2]. (Figure 1). All sites were rural. The localities in Roma- MalariawasendemicinEuropeuntilthemid20th nia, Italy, France, Spain and three sites in Portugal (2, 3 century [5]. The eradication of malaria in the European and 5, Figure 1) are located in coastal areas character- region was largely due to a combination of changes in ized by wetlands with the presence of rice fields. Sites 1 farming and husbandry, improvement in house con- and 4 (Figure 1) are located, respectively, in drier moun- struction and vector control. However, in recent years, tainous and plain inland regions of Portugal. Mosquitoes the disease re-emerged in residual foci in Eastern Eur- were identified to species or species complex by stereo- ope (Azerbaijan, Georgia, Kyrgyzstan, Tajikistan, Turkey microscopic observation of morphologic characters and Uzbekistan), resulting in more than 30,000 malaria using identification keys [9,10]. All the specimens were cases in the year 2000 [4]. Since then, intensive control preserved individually at 4°C or room temperature in activities have been re-implemented throughout the tubes filled with silica gel, until further analysis. affected region, and the number of reported cases has been reduced substantially to 660 in 2008 [4]. Molecular identification of sibling species Although the risk of malaria re-emergence is uncer- Genomic DNA was extracted from single mosquitoes tain for Western/Southern European countries, the pre- following a phenol-chloroform procedure [11]. Species sent climate change situation gave rise to some concern. identification of four members of the An. maculipennis One of the reasons was a predicted increase in mosquito complex was carried out by PCR-RFLP using protocols vectorial capacity, especially in the southern countries of derived from those described by Proft et al [12]. The Europe and the Mediterranean [6]. This in conjunction Internal Transcribed Spacer 2 (ITS2) of the ribosomal with the increasing intercontinental human movement DNA was amplified using primers for the conserved may favour the re-establishment of autochthonous regions 5.8S and 28S [13]. PCR was carried out in a 25 ® malaria transmission. μl volume containing 1X GoTaq Flexi Buffer (Promega, The former European malaria vectors were mainly USA), 2.5 mM MgCl2, 200 μM dNTP’s (Promega, USA), members of the Anopheles maculipennis complex that 5 μM of each primer and 1U of Go Taq polymerase are still widely distributed throughout the continent [7]. (Promega, USA). The thermal cycling profile was as fol- This complex comprises 13 Palearctic sibling species, of lows: 94°C for 5 min; 34 cycles of 94°C for 30 sec, 53°C which Anopheles atroparvus, Anopheles labranchiae and for 30 sec and 72°C for 30 sec, followed by a final exten- Anopheles sacharovi were the main malaria vectors in sion at 72°C for 7 min. A RFLP protocol allowed the the European region. In Europe, the distribution of identification of four sibling species of the An. maculi- An. atroparvus ranges from Britain to Russia (north pennis complex (An. atroparvus, Anopheles labranchiae, Caucasus). It is absent in some Mediterranean regions, Anopheles maculipennis s.s. and Anopheles melanoon). such as southern Italy, Greece and Turkey [8]. For the restriction reaction, 5 μl of each ITS2 PCR pro- Because its importance as a disease vector has declined, duct was added to 1X restriction enzyme buffer (buffer research on the biology of An. atroparvus and its sibling L, Roche Diagnostics, Germany) and 1.25U of Cfo 1 species has decreased in the last decades. However, the (Roche Diagnostics, Germany) enzyme, in a total volume concern with malaria re-emergence has resulted in a revi- of 20 μl, followed by incubation for 3 h at 37°C. valofinterestinEuropeanAnopheles mosquitoes. In this Digested fragments were