The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis

The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis Líbia Zé-Zé, Inês Campos Freitas, Manuel Silva, Patrícia Soares, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4583334/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Sep, 2024 Read the published version in Parasites & Vectors → Version 1 posted 10 You are reading this latest preprint version Abstract Background Aedes albopictus , commonly known as the Asian tiger mosquito, has become one of the most invasive mosquito species, introduced and established in various tropical and temperate regions worldwide over the last five decades. First reported in Europe in 1979 in Albania and later in Italy in 1990, the species is now established in 13 EU/EEA countries and 337 regions (2023). In Portugal, Ae. albopictus was first detected in the Algarve and Penafiel regions in 2017, followed by Alentejo in 2022 and Lisbon in 2023. This mosquito species poses a significant public health risk as a vector for numerous pathogenic viruses, including dengue, Zika and chikungunya. Methods Aedes albopictus collected in Lisbon in 2023, were analyzed using COX gene sequencing to understand their genetic relationships. Results Our data indicate that the Ae. albopictus mosquito populations detected in three different locations in Lisbon in 2023 correspond to recent, but distinct introduction events. Conclusions Although there has been no local transmission of Aedes -transmitted viruses in mainland Portugal to date, the spread of the mosquito and increased international travel increase the risk of Aedes -borne disease outbreaks. The ongoing spread of Ae. albopictus in the country and the confirmed multiple introductions in new locations raise awareness of the need to monitor mosquito vectors in order to control and prevent autochthonous Aedes -borne disease outbreaks. Aedes albopictus population diversity COX Portugal Figures Figure 1 Background First described by Skuse in India in 1894, Aedes (Stegomya) albopictus has been recognized as one of the most invasive mosquito species, successfully colonizing numerous tropical and temperate regions worldwide over the last five decades. Aedes albopictus was first reported in Europe in 1979 in Albania [ 1 ], followed by Italy in 1990 [ 2 ]. Italy is currently considered the most infested country in Europe, with Ae. albopictus established over large areas and thriving particularly in urban areas [ 3 ]. Since its introduction in Italy, Ae. albopictus has steadily spread throughout Europe, particularly to most Mediterranean countries. In 2023, Ae. albopictus was established in 13 EU/EEA countries and 337 regions, while in 2013, it was established only in 8 countries and 114 European regions [ 4 ]. In Portugal, Ae. albopictus was first detected in 2017 through two different introduction events in the Algarve, the southernmost region [ 5 ], and Penafiel, in the Oporto region [ 6 ]. In 2022, this vector was detected in the Alentejo region and in late September 2023, in Lisbon [ 7 ]. The National Vector Surveillance Network-REVIVE (REde de VIgilância de VEctores), which has been running since 2008 under the auspices of the Portuguese Ministry of Health [ 8 ]. REVIVE conducts nationwide surveillance of the most important hematophagous arthropods for public health (mosquitoes, ticks and sandflies). Regular surveillance of mosquito species and screening of field-collected mosquitoes for arboviruses is conducted. Airports, ports, storage areas and certain border regions with Spain are monitored throughout the year with the involvement of local and regional authorities. In addition to the nuisance associated with the establishment of Aedes albopictus , its ability to act as a vector for a wide range of arboviruses remains a major concern. This mosquito species has emerged as a significant global public health threat due to its ability to transmit several pathogenic flaviviruses (such as dengue, Zika and yellow fever) and alphaviruses (especially chikungunya virus). The Algarve, Lisbon and Oporto are the main tourist regions in the country, where the impact of the presence of this vector species can be a major concern due to the higher risk of incoming viremic travelers. Since 2007, cases of autochthonous transmission of chikungunya associated with Ae. albopictus have been documented in Europe [ 9 ]. Dengue has been reported in Europe since 2010, with autochthonous cases transmitted by Ae. albopictus in Croatia, France, Italy and Spain [ 10 ]. To date, local transmission of Aedes -borne viruses has not yet been detected in mainland Portugal. However, the geographic expansion of Ae. albopictus across Portugal, combined with the increasing number of international travelers, often from regions with ongoing Aedes -borne outbreaks, highlights the importance of mosquito vector surveillance and control, and raises public health concerns about the risk of increased introduction and autochthonous transmission of Aedes -borne viral infections. Here, we report a preliminary genetic analysis of the Ae. albopictus mosquitoes detected in Lisbon in 2023, using the primary barcode sequence for members of the animal kingdom, a partial sequence of the cytochrome c oxidase I (COX) gene, widely used to study the genetic relationships of Ae. albopictus [ 11 – 14 ] and previously used in Portuguese mosquito populations [ 15 ]. Materials and Methods Mosquito samples and DNA extraction Immature mosquito samples were collected in natural and artificial water containers at three sites in the Lisbon region (Algés, Alvalade and São Domingos de Benfica, see Table 1 ). Mosquito samples from the Algarve and Oporto populations, collected in 2022 and 2023, were also submitted for analysis. All mosquito samples were collected by the national REVIVE surveillance network [ 6 ], in public and private properties, with the knowledge and permission of the respective responsibles/owners. The collected mosquitoes were reared to adults in the insectary (by collection site and date), and 10 adult mosquitoes were randomly selected from each collection site for analysis. Larval and adult mosquitoes were morphologically confirmed as Ae. albopictus [ 16 , 17 ]. Sampled mosquitoes collected in the Lisbon region were ground individually by grinding with a mortar and pestle with liquid nitrogen and further ground after the addition of 500 µL lysis buffer (NUCLISENS® easyMAG, Biomérieux). Nucleic acid extraction was performed with the prepared lysate suspensions in the automated platform NUCLISENS® easyMAG (Biomérieux), as previously described [ 6 ]. Table 1 Aedes albopictus samples collected in Portugal and included in this study. Original Designation COX GenBank ID Collection Date Collection Place Region Number of Mosquitoes COI Haplotype Reference PoMo1076 MF990905 04/09/2017 Penafiel Oporto 1 1 [ 6 ] PoMo2608 MK995309 12/09/2017 Penafiel Oporto 1 1 [ 15 ] PoMo2708 MK995312 27/09/2018 Almancil Algarve 1 1 [ 15 ] PoMo2725A MK995316 17/10/2018 Penafiel Oporto 1 1 [ 15 ] PoMo3075 PP825977 01/06/2022 Penafiel Oporto 1 1 This study PoMo3197 PP825978 28/09/2022 Penafiel Oporto 7 1 This study PoMo3198 PP825979 19/09/2022 Olhão Algarve 6 1 This study PoMo3266B PP825980 08/08/2023 Faro Algarve 6 1 This study PoMoF506 MK995319 12/07/2018 Quarteira Algarve 1 2 [ 15 ] PoMo2710 MK995320 26/09/2018 Almancil Algarve 1 2 [ 15 ] PoMo3266A PP825981 08/08/2023 Faro Algarve 6 2 This study PoMoAlbALV_6 PP825982 26/10/2023 Alvalade Lisbon 1 2 This study PoMo2709 MK995326 27/09/2018 Almancil Algarve 3 3 [ 15 ] PoMoAlbALV_1 PP825983 26/10/2023 Alvalade Lisbon 9* 2 This study PoMoF505 MK995331 11/07/2018 Penafiel Oporto 1 4 [ 15 ] PoMoF503 MK995332 11/07/2018 Penafiel Oporto 1 5 [ 15 ] PoMoAlbTA02 PP825984 26/10/2023 Algés Lisbon 10* 6 This study PoMoAlbCN01 PP825985 26/10/2023 São Domingos Benfica Lisbon 10* 7 This study *Number of individually analyzed mosquitoes from the same collection site and date with identical COX sequence. Molecular analysis Molecular identification was performed using the COX gene of mitochondrial DNA with primers LCO1490 and HCO 2198 [ 18 ], as previously described [ 6 ]. Aedes albopictus haplotype diversity for COX sequences was estimated using DnaSP v.6.10.01 [ 19 ]. To integrate the mosquitoes circulating in Portugal and into the global Ae. albopictus genetic diversity, the consensus nucleotide partial sequences of the COX gene were aligned against several sequences available in GenBank (20 COX previously reported at the global level; Fig. 1 ) using BioEdit version 7.2.5 [ 20 ] and further used to construct a phylogenetic tree using the UPGMA method (1000 bootstraps) in MEGA X [ 21 ]. Results The 10 partial COX sequences obtained for Ae. albopictus mosquitoes collected at three sites in Lisbon (30 sequences in total) show low genetic diversity, with only one sequence type in Algés (GenBank accession number PP825984) and São Domingos de Benfica (GenBank accession number PP825985). In Alvalade, two sequence types were identified, one corresponding to nine of the mosquito specimens collected (GenBank accession number PP825983) and the other corresponding to only one mosquito (GenBank accession number PP825982). Sequences from mosquitoes collected in 2022 and 2023 in the Algarve and Oporto regions are identical to previously detected sequence types for these regions (Table 1 and Fig. 1 ), namely haplotype 1 (H1, Algarve and Oporto) and H2 for the Algarve, as previously defined [ 15 ]. The haplotype diversity analysis for the sequence types detected in the Lisbon region shows, in Alvalade, two haplotypes, H2 and H3, previously detected in the Algarve, and two new haplotypes for the other sites, H6 in Algés (with similarity to sequences from widespread sites) and H7 in São Domingos de Benfica, identical to sequences detected in Spain (Table 1 and Fig. 1 ). Discussion and Conclusion The observed low diversity of COX sequences in mosquitoes collected in Lisbon is consistent with recent introductions. Although the collection of immature mosquitoes may bias the population diversity, collections were made in heavily infested containers, corresponding to multiple female mosquito ovipositions. For most of these sites, the water containers containing immature mosquitoes were the only ones found with Ae. albopictus in the surveyed area. Some sites were analyzed following citizen science reports of mosquito presence, and others were the result of detailed inspections by the Lisbon and Tagus Valley Health Authority following the initial detection event. As the COX sequences from the three Lisbon sites differed, these introductions can be considered as separate events. Although preliminary, these data suggest that, at least in Alvalade, the introduction may have come from the Algarve. However, direct external introductions cannot be excluded, especially in São Domingos de Benfica and Algés. Nevertheless, more data is needed on the current mosquito populations circulating in Oporto, the Algarve and the Alentejo. A finer genetic analysis, namely by mitogenome sequencing, with a broader sampling of mosquitoes in the coming season (2024) is underway to obtain more details. Nevertheless, the identification of three independent introduction events in one mosquito season highlights the potential of this species to invade new geographic areas in a short period of time. It also means that these events are most likely currently underway and that they indicate naive regions that could be rapidly colonized. In 2022, over 19 million travelers entered Europe from dengue-affected areas [ 22 ]. Between 2012 and 2022, our team at the National Reference Laboratory of the Portuguese National Institute of Health (INSA) detected 142 Aedes -borne infections in viremic travelers. However, the high rates of asymptomatic infection in humans and the relatively short viremic window of symptomatic patients suggest that many traveler infections may be under-recognized and the actual number of viremic travelers is much higher. Europe is experiencing a warming trend, with more frequent and severe heat waves and floods, and longer and warmer summers [ 10 ]. This creates more favorable conditions for invasive mosquito species such as Ae. albopictus and Ae. aegypti . The geographical spread of invasive mosquito species to previously unaffected areas in the EU/EEA is an ongoing reality. In most European countries, cold winters do not allow year-round transmission [ 10 ], but in the southernmost region of Portugal, the Algarve, adult Ae. albopictus mosquitoes are already present year-round, although at lower population levels in winter. Given climate change, with continued increases in temperature and subsequent milder winters, the conditions for virus transmission will certainly increase. Given the ongoing spread of Ae. albopictus in mainland Portugal, it is essential to raise awareness of mosquito-borne diseases among the general public, healthcare professionals and travelers. Abbreviations COX cytochrome c oxidase I EU/EEA European Union/ European Economic Area FCT Portuguese Foundation for Science and Technology. Declarations Ethics approval and consent to participate No special permits were required for the field studies. Consent was obtained from the property owners for mosquito collection in private residential areas, and with the knowledge and permission of the relevant authority in public areas. No sites were legally protected and no endangered or protected species were involved in this study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was funded by the Institute of Environmental Health of the Faculty of Medicine of the University of Lisbon (ISAMB), project references FCT (UIDB/04295/2020 and UIDP/04295/2020). This work was partially funded by the MOBVEC - Mobile Bio-Lab to support first response in Arbovirus outbreaks (2023–2026) project, reference HORIZON-EIC-2022-PATHFINDEROPEN-01 under the Pathfinderopen program of the European Innovation Council (CEI), and PhD fellowship reference 2022.13476.BDANA (FCT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution Conceived and designed the experiments: LZZ and HCO. Performed the experiments: LZZ, ICF, MS and HCO. Analyzed the data: LZZ, ICF and PS. Drafting of the manuscript: LZZ and PS. Revised the manuscript: LZZ, ICF, MS, MJA, and HCO. All authors read and approved the final version of the manuscript. Acknowledgement We are grateful to Marco Brustolin and Carles Aranda for providing evidence of the collection date and location in Spain of the sampled mosquitoes corresponding to sequences KU319443, KU319444, KU319446 and KU319447. We are also grateful to the REVIVE team for collecting mosquitoes throughout the country, especially Lisbon and Tagus Valley Health Authority. This work received support by FCT (reference: CEECINST/00049/2021/CP2817/CT0001, DOI: 10.54499/CEECINST/00049/2021/CP2817/CT0001; reference: FCT/MCTES UIB/00211/2020, DOI: 10.54499/UIDB/00211; reference: FCT/MCTES UIP/00211/2020, DOI: 10.54499/UIDP/00211). Data Availability The nucleotide sequence data reported in this paper have been deposited in the NBCI GenBank database under the accession numbers: PP825977- PP825985. References Adhami J, Reiter P. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4583334","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":317719330,"identity":"57846dd6-a323-4d3b-9354-28d54fa4d1c4","order_by":0,"name":"Líbia Zé-Zé","email":"data:image/png;base64,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","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":true,"prefix":"","firstName":"Líbia","middleName":"","lastName":"Zé-Zé","suffix":""},{"id":317719331,"identity":"5f48d75a-46ac-4702-9e78-b1dcd99557b5","order_by":1,"name":"Inês Campos Freitas","email":"","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":false,"prefix":"","firstName":"Inês","middleName":"Campos","lastName":"Freitas","suffix":""},{"id":317719332,"identity":"4e75364f-e822-4b20-8d63-8d18983a226b","order_by":2,"name":"Manuel Silva","email":"","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Silva","suffix":""},{"id":317719333,"identity":"652b21b1-eb64-4ea7-a291-1959daefdcb2","order_by":3,"name":"Patrícia Soares","email":"","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":false,"prefix":"","firstName":"Patrícia","middleName":"","lastName":"Soares","suffix":""},{"id":317719334,"identity":"7061a8ca-32e8-488a-9938-5cb8d721ef06","order_by":4,"name":"Maria João Alves","email":"","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"João","lastName":"Alves","suffix":""},{"id":317719335,"identity":"75472ece-a1a6-459c-9119-3e20e62dbf29","order_by":5,"name":"Hugo Costa Osório","email":"","orcid":"","institution":"National Institute of Health Doutor Ricardo Jorge (INSA)","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"Costa","lastName":"Osório","suffix":""}],"badges":[],"createdAt":"2024-06-14 16:45:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4583334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4583334/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-024-06460-w","type":"published","date":"2024-09-13T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58967711,"identity":"05c867c3-f82d-4c83-ae5f-dee475a49f47","added_by":"auto","created_at":"2024-06-24 19:20:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6840520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eUPGMA phylogenetic tree constructed from 18 (9 novel) COX sequences obtained from mosquitoes circulating in Portugal (Table 1) and 20 sequences available in GenBank. Bootstrap values (1000 replicates) greater than 65 are shown above the branches, and branch lengths (in the same units as those of the evolutionary distances used to infer the phylogenetic tree) are shown below the branches. Evolutionary distances were calculated using maximum composite likelihood. Sequences are identified by GenBank accession numbers, country region, country, and year of collection (if available). Tree nodes are identified by the COX haplotype. Colored circles indicate the regions where mosquitoes from Portugal were collected. For better visualization of the tree, the KX383935 sequence was used as an outgroup.\u003cstrong\u003e (B) \u003c/strong\u003eMap showing the locations of mosquito sample collection and the most likely origin of introduction in Lisbon, based on available data. Composite figure created using BioRender.com.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4583334/v1/3c71abaa8299de4871f2b224.jpeg"},{"id":64619241,"identity":"91fa5395-7dba-434f-a67c-1feba7278d83","added_by":"auto","created_at":"2024-09-16 16:13:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7311667,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4583334/v1/39e14245-24c8-41f1-800a-d1bfeae6a2ab.pdf"},{"id":58967712,"identity":"3238eeac-ce33-4497-892d-ba1cd450ef75","added_by":"auto","created_at":"2024-06-24 19:20:51","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3852021,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4583334/v1/f87a9b6d6207822a94a88e73.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe spread of the invasive mosquito \u003cem\u003eAedes albopictus\u003c/em\u003e (Diptera: Culicidae) in Portugal: a first genetic analysis\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eFirst described by Skuse in India in 1894, \u003cem\u003eAedes (Stegomya) albopictus\u003c/em\u003e has been recognized as one of the most invasive mosquito species, successfully colonizing numerous tropical and temperate regions worldwide over the last five decades. \u003cem\u003eAedes albopictus\u003c/em\u003e was first reported in Europe in 1979 in Albania [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], followed by Italy in 1990 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Italy is currently considered the most infested country in Europe, with \u003cem\u003eAe. albopictus\u003c/em\u003e established over large areas and thriving particularly in urban areas [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since its introduction in Italy, \u003cem\u003eAe. albopictus\u003c/em\u003e has steadily spread throughout Europe, particularly to most Mediterranean countries. In 2023, \u003cem\u003eAe. albopictus\u003c/em\u003e was established in 13 EU/EEA countries and 337 regions, while in 2013, it was established only in 8 countries and 114 European regions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In Portugal, \u003cem\u003eAe. albopictus\u003c/em\u003e was first detected in 2017 through two different introduction events in the Algarve, the southernmost region [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and Penafiel, in the Oporto region [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In 2022, this vector was detected in the Alentejo region and in late September 2023, in Lisbon [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The National Vector Surveillance Network-REVIVE (REde de VIgil\u0026acirc;ncia de VEctores), which has been running since 2008 under the auspices of the Portuguese Ministry of Health [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. REVIVE conducts nationwide surveillance of the most important hematophagous arthropods for public health (mosquitoes, ticks and sandflies). Regular surveillance of mosquito species and screening of field-collected mosquitoes for arboviruses is conducted. Airports, ports, storage areas and certain border regions with Spain are monitored throughout the year with the involvement of local and regional authorities.\u003c/p\u003e \u003cp\u003eIn addition to the nuisance associated with the establishment of \u003cem\u003eAedes albopictus\u003c/em\u003e, its ability to act as a vector for a wide range of arboviruses remains a major concern. This mosquito species has emerged as a significant global public health threat due to its ability to transmit several pathogenic flaviviruses (such as dengue, Zika and yellow fever) and alphaviruses (especially chikungunya virus). The Algarve, Lisbon and Oporto are the main tourist regions in the country, where the impact of the presence of this vector species can be a major concern due to the higher risk of incoming viremic travelers. Since 2007, cases of autochthonous transmission of chikungunya associated with \u003cem\u003eAe. albopictus\u003c/em\u003e have been documented in Europe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Dengue has been reported in Europe since 2010, with autochthonous cases transmitted by \u003cem\u003eAe. albopictus\u003c/em\u003e in Croatia, France, Italy and Spain [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, local transmission of \u003cem\u003eAedes\u003c/em\u003e-borne viruses has not yet been detected in mainland Portugal. However, the geographic expansion of \u003cem\u003eAe. albopictus\u003c/em\u003e across Portugal, combined with the increasing number of international travelers, often from regions with ongoing \u003cem\u003eAedes\u003c/em\u003e-borne outbreaks, highlights the importance of mosquito vector surveillance and control, and raises public health concerns about the risk of increased introduction and autochthonous transmission of \u003cem\u003eAedes\u003c/em\u003e-borne viral infections. Here, we report a preliminary genetic analysis of the \u003cem\u003eAe. albopictus\u003c/em\u003e mosquitoes detected in Lisbon in 2023, using the primary barcode sequence for members of the animal kingdom, a partial sequence of the cytochrome c oxidase I (COX) gene, widely used to study the genetic relationships of \u003cem\u003eAe. albopictus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and previously used in Portuguese mosquito populations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito samples and DNA extraction\u003c/h2\u003e \u003cp\u003eImmature mosquito samples were collected in natural and artificial water containers at three sites in the Lisbon region (Alg\u0026eacute;s, Alvalade and S\u0026atilde;o Domingos de Benfica, see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mosquito samples from the Algarve and Oporto populations, collected in 2022 and 2023, were also submitted for analysis. All mosquito samples were collected by the national REVIVE surveillance network [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], in public and private properties, with the knowledge and permission of the respective responsibles/owners. The collected mosquitoes were reared to adults in the insectary (by collection site and date), and 10 adult mosquitoes were randomly selected from each collection site for analysis. Larval and adult mosquitoes were morphologically confirmed as \u003cem\u003eAe. albopictus\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Sampled mosquitoes collected in the Lisbon region were ground individually by grinding with a mortar and pestle with liquid nitrogen and further ground after the addition of 500 \u0026micro;L lysis buffer (NUCLISENS\u0026reg; easyMAG, Biom\u0026eacute;rieux). Nucleic acid extraction was performed with the prepared lysate suspensions in the automated platform NUCLISENS\u0026reg; easyMAG (Biom\u0026eacute;rieux), as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eAedes albopictus\u003c/em\u003e samples collected in Portugal and included in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOriginal Designation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOX GenBank ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCollection Date\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCollection Place\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e 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\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoF506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMK995319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12/07/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQuarteira\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlgarve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMo2710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMK995320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/09/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlmancil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlgarve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMo3266A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP825981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e08/08/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlgarve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoAlbALV_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP825982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/10/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlvalade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLisbon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMo2709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMK995326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27/09/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlmancil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlgarve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoAlbALV_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP825983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/10/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlvalade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLisbon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoF505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMK995331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/07/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePenafiel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOporto\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoF503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMK995332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/07/2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePenafiel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOporto\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoAlbTA02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP825984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/10/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlg\u0026eacute;s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLisbon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoMoAlbCN01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP825985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/10/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u0026atilde;o Domingos Benfica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLisbon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e*Number of individually analyzed mosquitoes from the same collection site and date with identical COX sequence.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMolecular analysis\u003c/h2\u003e \u003cp\u003eMolecular identification was performed using the COX gene of mitochondrial DNA with primers LCO1490 and HCO 2198 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eAedes albopictus\u003c/em\u003e haplotype diversity for COX sequences was estimated using DnaSP v.6.10.01 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo integrate the mosquitoes circulating in Portugal and into the global \u003cem\u003eAe. albopictus\u003c/em\u003e genetic diversity, the consensus nucleotide partial sequences of the COX gene were aligned against several sequences available in GenBank (20 COX previously reported at the global level; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using BioEdit version 7.2.5 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and further used to construct a phylogenetic tree using the UPGMA method (1000 bootstraps) in MEGA X [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe 10 partial COX sequences obtained for \u003cem\u003eAe. albopictus\u003c/em\u003e mosquitoes collected at three sites in Lisbon (30 sequences in total) show low genetic diversity, with only one sequence type in Alg\u0026eacute;s (GenBank accession number PP825984) and S\u0026atilde;o Domingos de Benfica (GenBank accession number PP825985). In Alvalade, two sequence types were identified, one corresponding to nine of the mosquito specimens collected (GenBank accession number PP825983) and the other corresponding to only one mosquito (GenBank accession number PP825982). Sequences from mosquitoes collected in 2022 and 2023 in the Algarve and Oporto regions are identical to previously detected sequence types for these regions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), namely haplotype 1 (H1, Algarve and Oporto) and H2 for the Algarve, as previously defined [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The haplotype diversity analysis for the sequence types detected in the Lisbon region shows, in Alvalade, two haplotypes, H2 and H3, previously detected in the Algarve, and two new haplotypes for the other sites, H6 in Alg\u0026eacute;s (with similarity to sequences from widespread sites) and H7 in S\u0026atilde;o Domingos de Benfica, identical to sequences detected in Spain (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion and Conclusion","content":"\u003cp\u003eThe observed low diversity of COX sequences in mosquitoes collected in Lisbon is consistent with recent introductions. Although the collection of immature mosquitoes may bias the population diversity, collections were made in heavily infested containers, corresponding to multiple female mosquito ovipositions. For most of these sites, the water containers containing immature mosquitoes were the only ones found with \u003cem\u003eAe. albopictus\u003c/em\u003e in the surveyed area. Some sites were analyzed following citizen science reports of mosquito presence, and others were the result of detailed inspections by the Lisbon and Tagus Valley Health Authority following the initial detection event.\u003c/p\u003e \u003cp\u003eAs the COX sequences from the three Lisbon sites differed, these introductions can be considered as separate events. Although preliminary, these data suggest that, at least in Alvalade, the introduction may have come from the Algarve. However, direct external introductions cannot be excluded, especially in S\u0026atilde;o Domingos de Benfica and Alg\u0026eacute;s. Nevertheless, more data is needed on the current mosquito populations circulating in Oporto, the Algarve and the Alentejo. A finer genetic analysis, namely by mitogenome sequencing, with a broader sampling of mosquitoes in the coming season (2024) is underway to obtain more details. Nevertheless, the identification of three independent introduction events in one mosquito season highlights the potential of this species to invade new geographic areas in a short period of time. It also means that these events are most likely currently underway and that they indicate naive regions that could be rapidly colonized.\u003c/p\u003e \u003cp\u003eIn 2022, over 19\u0026nbsp;million travelers entered Europe from dengue-affected areas [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Between 2012 and 2022, our team at the National Reference Laboratory of the Portuguese National Institute of Health (INSA) detected 142 \u003cem\u003eAedes\u003c/em\u003e-borne infections in viremic travelers. However, the high rates of asymptomatic infection in humans and the relatively short viremic window of symptomatic patients suggest that many traveler infections may be under-recognized and the actual number of viremic travelers is much higher. Europe is experiencing a warming trend, with more frequent and severe heat waves and floods, and longer and warmer summers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This creates more favorable conditions for invasive mosquito species such as \u003cem\u003eAe. albopictus\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e. The geographical spread of invasive mosquito species to previously unaffected areas in the EU/EEA is an ongoing reality. In most European countries, cold winters do not allow year-round transmission [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], but in the southernmost region of Portugal, the Algarve, adult \u003cem\u003eAe. albopictus\u003c/em\u003e mosquitoes are already present year-round, although at lower population levels in winter. Given climate change, with continued increases in temperature and subsequent milder winters, the conditions for virus transmission will certainly increase.\u003c/p\u003e \u003cp\u003eGiven the ongoing spread of \u003cem\u003eAe. albopictus\u003c/em\u003e in mainland Portugal, it is essential to raise awareness of mosquito-borne diseases among the general public, healthcare professionals and travelers.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytochrome c oxidase I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEU/EEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEuropean Union/ European Economic Area\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePortuguese Foundation for Science and Technology.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNo special permits were required for the field studies. Consent was obtained from the property owners for mosquito collection in private residential areas, and with the knowledge and permission of the relevant authority in public areas. No sites were legally protected and no endangered or protected species were involved in this study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the Institute of Environmental Health of the Faculty of Medicine of the University of Lisbon (ISAMB), project references FCT (UIDB/04295/2020 and UIDP/04295/2020). This work was partially funded by the MOBVEC - Mobile Bio-Lab to support first response in Arbovirus outbreaks (2023\u0026ndash;2026) project, reference HORIZON-EIC-2022-PATHFINDEROPEN-01 under the Pathfinderopen program of the European Innovation Council (CEI), and PhD fellowship reference 2022.13476.BDANA (FCT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceived and designed the experiments: LZZ and HCO. Performed the experiments: LZZ, ICF, MS and HCO. Analyzed the data: LZZ, ICF and PS. Drafting of the manuscript: LZZ and PS. Revised the manuscript: LZZ, ICF, MS, MJA, and HCO. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to Marco Brustolin and Carles Aranda for providing evidence of the collection date and location in Spain of the sampled mosquitoes corresponding to sequences KU319443, KU319444, KU319446 and KU319447. We are also grateful to the REVIVE team for collecting mosquitoes throughout the country, especially Lisbon and Tagus Valley Health Authority. This work received support by FCT (reference: CEECINST/00049/2021/CP2817/CT0001, DOI: 10.54499/CEECINST/00049/2021/CP2817/CT0001; reference: FCT/MCTES UIB/00211/2020, DOI: 10.54499/UIDB/00211; reference: FCT/MCTES UIP/00211/2020, DOI: 10.54499/UIDP/00211).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe nucleotide sequence data reported in this paper have been deposited in the NBCI GenBank database under the accession numbers: PP825977- PP825985.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdhami J, Reiter P. Introduction and establishment of \u003cem\u003eAedes (Stegomyia) albopictus\u003c/em\u003e skuse (Diptera: Culicidae) in Albania. 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Accessed 13 Jun 2024.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aedes albopictus, population diversity, COX, Portugal","lastPublishedDoi":"10.21203/rs.3.rs-4583334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4583334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAedes albopictus\u003c/em\u003e, commonly known as the Asian tiger mosquito, has become one of the most invasive mosquito species, introduced and established in various tropical and temperate regions worldwide over the last five decades. First reported in Europe in 1979 in Albania and later in Italy in 1990, the species is now established in 13 EU/EEA countries and 337 regions (2023). In Portugal, \u003cem\u003eAe. albopictus\u003c/em\u003e was first detected in the Algarve and Penafiel regions in 2017, followed by Alentejo in 2022 and Lisbon in 2023. This mosquito species poses a significant public health risk as a vector for numerous pathogenic viruses, including dengue, Zika and chikungunya.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAedes albopictus\u003c/em\u003e collected in Lisbon in 2023, were analyzed using COX gene sequencing to understand their genetic relationships.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur data indicate that the \u003cem\u003eAe. albopictus\u003c/em\u003e mosquito populations detected in three different locations in Lisbon in 2023 correspond to recent, but distinct introduction events.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAlthough there has been no local transmission of \u003cem\u003eAedes\u003c/em\u003e-transmitted viruses in mainland Portugal to date, the spread of the mosquito and increased international travel increase the risk of \u003cem\u003eAedes\u003c/em\u003e-borne disease outbreaks. The ongoing spread of \u003cem\u003eAe. albopictus\u003c/em\u003e in the country and the confirmed multiple introductions in new locations raise awareness of the need to monitor mosquito vectors in order to control and prevent autochthonous \u003cem\u003eAedes\u003c/em\u003e-borne disease outbreaks.\u003c/p\u003e","manuscriptTitle":"The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 19:20:46","doi":"10.21203/rs.3.rs-4583334/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-18T18:42:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T16:29:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305171828272652084505691823798265829830","date":"2024-07-16T15:32:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48691144205602055895818512119942621927","date":"2024-07-15T15:34:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T15:38:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254719993604665895687979946555309120931","date":"2024-07-03T15:08:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-24T21:00:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-17T13:25:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-17T13:18:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-06-14T16:44:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3b69038d-51a0-436d-b326-986b5eddf38c","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:05:29+00:00","versionOfRecord":{"articleIdentity":"rs-4583334","link":"https://doi.org/10.1186/s13071-024-06460-w","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-09-13 15:58:12","publishedOnDateReadable":"September 13th, 2024"},"versionCreatedAt":"2024-06-24 19:20:46","video":"","vorDoi":"10.1186/s13071-024-06460-w","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06460-w","workflowStages":[]},"version":"v1","identity":"rs-4583334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4583334","identity":"rs-4583334","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}