Sequencing confirms Anopheles stephensi distribution across southern Yemen

Sequencing confirms Anopheles stephensi distribution across southern Yemen | 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 Sequencing confirms Anopheles stephensi distribution across southern Yemen Yasser A Baheshm, Alia Zayed, Abdullah A Awash, Madison Follis, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4783439/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Parasites & Vectors → Version 1 posted 11 You are reading this latest preprint version Abstract The invasion of Anopheles stephensi in Africa warrants investigation of neighboring countries. In this study, genetic analysis was applied to determine the status of An. stephensi in southern Yemen. Cytochrome oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) were sequenced in An. stephensi collected in Aden City, Lahj, Rodoom, Al Mukalla, and Sayhut, and phylogenetic analysis confirmed An. stephensi identity. Our analyses revealed that the ITS2 sequence were identical in all An. stephensi , while COI analysis revealed two haplotypes, one previously reported in northern Horn of Africa and one identified in this study for the first time. Overall, these findings revealed low levels of mitochondrial DNA diversity, which is consistent with a recent population introduction in parts of southern Yemen. Further whole genomic analysis is needed to elucidate the original connection with invasive populations of An. stephensi in the Horn of Africa. Figures Figure 1 Figure 2 Figure 3 Introduction Anopheles stephensi is a malaria vector originally found in South Asia and the Middle East. In 2012, An. stephensi was detected on the African continent in Djibouti City ( 1 ) and three years later in Kebridehar, Ethiopia ( 2 ). With the detection of the vector in East Africa and parts of West Africa ( 3 ), there is rising concern that this invasive vector could spread further. The connection between the presence of An. stephensi and malaria outbreaks in Djibouti City, Djibouti ( 4 ) and Dire Dawa, Ethiopia ( 5 ) heightens the need to track the spread of this invasive species. Given the proximity to the Horn of Africa (HoA), the status of An. stephensi in the Arabian Peninsula is of growing interest. The predicted native range of An. stephensi includes Saudi Arabia and Oman ( 6 ). However, An. stephensi has not been reported in Yemen until recently. Anopheles stephensi was detected in Yemen in 2021 in Aden City, located in the southern part of Yemen ( 7 ). Aden City shares some similarities with locations where An. stephensi is now well-established that serve as major hubs for its onward spread across the HoA ( 8 ). It is an urban area and port city with frequent population movement inward through the flow of internally displaced persons and refugees from the Horn of Africa ( 9 ). It is a major port city connecting with Bosaso, Somalia and Mukalla Port City, another location on the southern coast of Yemen with a recent detection of An. stephensi . Finally, an increase in malaria incidence has also been reported in Aden ( 9 ). Given the detection of An. stephensi in these two locations in the southern part of Yemen, it is important to confirm how far An. stephensi has spread throughout the south. Molecular confirmation of species identification is an important component of the tracking of invasive An. stephensi across its invasive range ( 10 ). It provides a basis to evaluate the successful integration of the updated morphological keys that include An. stephensi into ongoing vector surveillance. In addition, DNA sequence-based molecular identification has the benefit of providing preliminary insight into the relationship between newly identified invasive populations and other reported populations. This study reports the results of molecular analyses of morphologically identified An. stephensi across southern Yemen, including four newly reported locations. Methods Site Descriptions Five sites were surveyed for this study (Table 1 ; Fig. 1 ): Dar Sa’ad (Aden City), Tuban in Lahj governorate, Rodoom in Shabwah governorate, Al Mukalla Port City in Hadramout governorate, and Sayhut in Al Mahrah governorate. Aden is a major port city along an historic maritime route connecting the Horn of Africa and the Arabian Peninsula. The climate in Aden is mostly desert, although there is steady humidity year-round. Lahj is located just under 40 km north of Aden with similar climate. Rodoom coastal city in Shabwah governorate is located between Hadramout to the east and Abyan to the west with coastal and semiarid climate. Tuban is located just under 40 km north of Aden with similar climate. Al Mukalla Port City is located over 500 km east of Aden and has a coastal and semi-arid climate. Sayhut is the easternmost site of this collection and has a subtropical climate with peak rains in May. Table 1 Description of sites. Site Governorate Coordinates Date collected Dar Sa’ad Aden 12.78652104214733, 45.01848534347771 October, December 2021 Tuban Lahj 13.05807428525727, 44.88331315931494 December 2021 Rodoom Shabwah 13.841611, 47.59567 June 2022 Al Mukalla Hadramout 14.54943802880376, 49.12458936878869 October 2021, April, September, October 2022 Sayhut Al Mahrah 15.202806, 51.201750 May 2022 Collections Larvae were collected using the dipping method over the course of three days and reared to adult stage. Potential breeding habitats surveyed included domestic containers, air condition drips, accumulated water in brick factories, and cemeteries (Fig. 2 ). Adult Anopheles were identified morphologically using the Afrotropical mosquitoes key ( 11 ). The specimens morphologically identified as An. stephensi were preserved with silica gel and a subsample of specimens was sent to Baylor University for molecular analysis. PCR and sequence analysis Two loci were selected for PCR amplification-based species identifications: cytochrome oxidase subunit 1 ( COI) and internal transcribed spacer 2 ( ITS2) as previously applied ( 2 , 12 ). First, species were confirmed using a PCR species-specific assay targeting the ITS2 region ( 2 , 13 ) and with PCR and sequencing of the ITS2 and an informative portion of COI using methods described previously ( 2 ). The primer sequences for PCR in the ITS2 endpoint assay are 5.8SB (5′-ATCACTCGGCTCGTGGATCG-3ʹ) and 28SC (5ʹ- GTCTCGCGACTGCAACTG-3ʹ) ( 13 ). The primer sequences for the ITS2 PCR for sequencing were 5.8SB (5ʹ-ATCACTCGGCTCGTGGATCG-3ʹ) and 28SB (5ʹ-ATGCTTAAATTTAGGGGGTAGTC-3ʹ) ( 13 ). The primer sequences for COI PCR are LCO1490F (5ʹ-GGTCAACAAATCATAAAGATATTGG-3ʹ) and HCO2198R (5ʹ-TAAACTTCAGGGTGACCAAAAAATCA-3ʹ) ( 14 ). ITS2 and COI PCR products were sequenced using Sanger sequencing technology. Sequences were then trimmed and analyzed using CodonCode Aligner (CodonCode Corporation, Centerville, MA). Final sequences were submitted to the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool (BLAST) (Accession #: PP752283, PP752284, PQ006012). Alignments were generated in CodonCode Aligner that included previously published sequences from NCBI GenBank. ITS2-based sequence identification excluded the microsatellite region found within ITS2 ( 10 ). Likewise, only a portion of the COI previously identified as geographically informative was used for subsequent analysis in order to maximize the overlap with existing datasets for haplotype and phylogenetic comparisons (see Carter et al. 2021). Phylogenetic analysis was conducted using the maximum-likelihood method with RAxML ( 15 ). The best scoring trees under ML with bootstrap values from RAxML were viewed and annotated using Figtree ( 16 ). Results and Discussion A total of forty-four mosquitoes were molecularly characterized and all specimens were confirmed to be An. stephensi with the ITS2 end-point assay. In addition, BLAST analyses of the COI and ITS2 sequences revealed highest scoring matches for An. stephensi . Most of the COI sequences had a 100% sequence identity match with An. stephensi sequences originating from northeastern Ethiopia, Somaliland, Djibouti, and Yemen. The 162 bp segment of ITS2 sequence were identical for all specimens and had a 100% sequence identity match with other An. stephensi sequences. Phylogenetic analysis confirmed An. stephensi identity (ITS2 bs = 100, COI bs = 89) (Fig. 3 , Supplemental Fig.) Supplemental Figure. Phylogenetic analysis of An. stephensi ITS2 sequences from southern Yemen using the maximum likelihood approach. The ITS2 sequence observed in southern Yemen is in blue. Analysis of a specific region of the COI sequence has previously been identified as geographically informative ( 17 ). Using this region of the COI, we identified two haplotypes among the Yemen samples (Fig. 3 ). One haplotype was found in the majority of the mosquitoes (n = 43). This haplotype has been observed in Yemen and in the northeastern Horn of Africa ( 7 , 17 , 18 ) in other studies and designated Hap3 by Carter et al. ( 17 ). The second haplotype (n = 1, found in Sayhut) has not been reported previously. As the second unique Yemen haplotype, it will be designated HapYem2 in this study. In order to further investigate the genetic similarity between sequences from southern Yemen and other areas of the An. stephensi range, we performed phylogenetic analysis of the COI sequences. Phylogenetic analysis showed support for differentiation from Saudi Arabia for all sequences, although further geographic distinctions were not detected in this phylogenetic tree likely due to the low variation of this marker. Overall, this study revealed a more homogenous population relative to populations in northern Horn of Africa, including Ethiopia, Djibouti, and Somaliland. This evidence provides support for a more recently established An. stephensi population in portions of Yemen compared to the northern Horn of Africa populations. Interestingly, no haplotypes from Saudi Arabia or the United Arab Emirates were observed in any locations, suggesting a stronger connection to the invasive Horn of Africa populations rather than long-established populations in the Arabian Peninsula. This study was limited by the lack of full representative sampling of An. stephensi across the long-established geographic range in the Arabian Peninsula and by the limited resolution of the markers used in this study. Regardless, these findings highlight the importance of population genomic surveillance in this region. Declarations Ethical Approval: The research activity which involved the collection of larvae from outdoor breeding habitats was approved by the National Malaria Control Program, Ministry of Health in Aden. Collectors are qualified entomological technicians from the malaria program staff who conduct collection with the goal of minimizing exposure to adult mosquitoes. This work did not involve human subject research. Funding: This research was funded by a NIH Research Enhancement Award (1R15AI151766) and funding through the Centers for Disease Control and Prevention awarded to TEC. Additional support for the collection of mosquitoes was provided by NAMRU-EURAFCENT project funded by Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance Branch (GEIS) # P0041_22_N3 & P0036_23_N3. Contributions: TEC, AZ, SA, YAB contributed to the conception and design of the project. YAB, YA, AZ, JH organized and led the collection of specimens. MA, PA, JNS generated molecular data. TEC analyzed the data. TEC wrote the first draft of manuscript. All authors read and approved the final manuscript. Disclaimer: The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. Acknowledgments : Our gratitude goes to the World Health Organization for their technical support for implementation of surveillance. The authors would like to thank Dr. Ghasem Zamani (Regional Advisor WHO/EMRO-MVC) for his contribution and technical support for implementation of surveillance and review of the manuscript. References Faulde MK, Rueda LM, Khaireh BA. First record of the Asian malaria vector Anopheles stephensi and its possible role in the resurgence of malaria in Djibouti, Horn of Africa. Acta Trop. 2014;139:39-43. Carter TE, Yared S, Gebresilassie A, Bonnell V, Damodaran L, Lopez K, et al. First detection of Anopheles stephensi Liston, 1901 (Diptera: culicidae) in Ethiopia using molecular and morphological approaches. Acta Trop. 2018;188:180-6. WHO Malaria Threats Map: World Health Organization; [Available from: https://apps.who.int/malaria/maps/threats/. Seyfarth M, Khaireh BA, Abdi AA, Bouh SM, Faulde MK. Five years following first detection of Anopheles stephensi (Diptera: Culicidae) in Djibouti, Horn of Africa: populations established-malaria emerging. Parasitol Res. 2019;118(3):725-32. Emiru T, Getachew D, Murphy M, Sedda L, Ejigu LA, Bulto MG, et al. Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa. Nat Med. 2023;29(12):3203-11. Sinka ME, Bangs MJ, Manguin S, Chareonviriyaphap T, Patil AP, Temperley WH, et al. The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic precis. Parasit Vectors. 2011;4:89. Allan R, Weetman D, Sauskojus H, Budge S, Hawail TB, Baheshm Y. Confirmation of the presence of Anopheles stephensi among internally displaced people's camps and host communities in Aden city, Yemen. Malar J. 2023;22(1):1. Samake JN, Lavretsky P, Gunarathna I, Follis M, Brown JI, Ali S, et al. Population genomic analyses reveal population structure and major hubs of invasive Anopheles stephensi in the Horn of Africa. Mol Ecol. 2023;32(21):5695-708. Al-Eryani SM, Irish SR, Carter TE, Lenhart A, Aljasari A, Montoya LF, et al. Public health impact of the spread of Anopheles stephensi in the WHO Eastern Mediterranean Region countries in Horn of Africa and Yemen: need for integrated vector surveillance and control. Malar J. 2023;22(1):187. Waymire E, Samake JN, Gunarathna I, Carter TE. A decade of invasive Anopheles stephensi sequence-based identification: Toward a global standard. Trends in Parasitology. 2024;40:5. Coetzee M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). Malar J. 2020;19(1):70. Ali S, Samake JN, Spear J, Carter TE. Morphological identification and genetic characterization of Anopheles stephens i in Somaliland. Parasit Vectors. 2022;15(1):247. Djadid ND, Gholizadeh S, Aghajari M, Zehi AH, Raeisi A, Zakeri S. Genetic analysis of rDNA-ITS2 and RAPD loci in field populations of the malaria vector, Anopheles stephensi (Diptera: Culicidae): implications for the control program in Iran. Acta Trop. 2006;97(1):65-74. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3(5):294-9. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312-3. Rambaut A. Figtree Version. 1.4. 4 ed. Institute of Evolutionary Biology, Univ2018. Carter TE, Yared S, Getachew D, Spear J, Choi SH, Samake JN, et al. Genetic diversity of Anopheles stephensi in Ethiopia provides insight into patterns of spread. Parasit Vectors. 2021;14(1):602. Assada M, Al-Hadi M, Esmail MA, Al-Jurban J, Alkawri A, Shamsan A, et al. Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022. Emerg Infect Dis. 2024;30(7):1467-71. Additional Declarations No competing interests reported. Supplementary Files SupplementalInformation.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 29 Aug, 2024 Reviews received at journal 29 Aug, 2024 Reviews received at journal 23 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers agreed at journal 05 Aug, 2024 Reviewers agreed at journal 05 Aug, 2024 Reviewers invited by journal 05 Aug, 2024 Editor assigned by journal 02 Aug, 2024 Submission checks completed at journal 01 Aug, 2024 First submitted to journal 22 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Map were created using MapChart and edited with Microsoft PowerPoint.\u003c/p\u003e","description":"","filename":"Figure1Map.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4783439/v1/049a86c321b1c98bcd28bb1c.jpg"},{"id":63467615,"identity":"2ea842c0-79e9-4b56-aad6-24921278c1a1","added_by":"auto","created_at":"2024-08-28 12:35:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219646,"visible":true,"origin":"","legend":"\u003cp\u003eExample of breeding habitats where \u003cem\u003eAn. stephensi \u003c/em\u003ewere collected, including (A) water reservoir, (B) flower beds on grave filled with water, and (C) brick factory. Photos captured by National Malaria Control Programme in Aden, Yemen.\u003c/p\u003e","description":"","filename":"Figure2Breeding.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4783439/v1/6df7757c86d39a51be62c062.jpg"},{"id":63467617,"identity":"4381c043-4efe-4456-b9a5-162f22a692a9","added_by":"auto","created_at":"2024-08-28 12:35:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2820173,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003eAn. stephensi\u003c/em\u003e COI sequences from southern Yemen using the maximum likelihood approach. Yemen sequences are in color. \u0026nbsp;Rodoom = purple, Sayhut = red, Mukulla = light green, Dar Sa’ad (Aden) = blue, Tuban= teal.\u003c/p\u003e","description":"","filename":"Figure3COI.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4783439/v1/d45cd2a2c36ba48ba4f061ac.jpg"},{"id":72202011,"identity":"488a2f6c-83f9-480d-8e17-d72ead444cee","added_by":"auto","created_at":"2024-12-23 16:13:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3906772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4783439/v1/c68d6b56-7211-4e1d-82c2-de57af1b3998.pdf"},{"id":63467619,"identity":"4983dc21-b949-4c16-afda-6fea3f8c9a32","added_by":"auto","created_at":"2024-08-28 12:35:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":696856,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4783439/v1/201d7fc716e41b40b6d504a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sequencing confirms Anopheles stephensi distribution across southern Yemen","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAnopheles stephensi\u003c/em\u003e is a malaria vector originally found in South Asia and the Middle East. In 2012, \u003cem\u003eAn. stephensi\u003c/em\u003e was detected on the African continent in Djibouti City (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and three years later in Kebridehar, Ethiopia (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). With the detection of the vector in East Africa and parts of West Africa (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), there is rising concern that this invasive vector could spread further. The connection between the presence of \u003cem\u003eAn. stephensi\u003c/em\u003e and malaria outbreaks in Djibouti City, Djibouti (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and Dire Dawa, Ethiopia (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) heightens the need to track the spread of this invasive species.\u003c/p\u003e \u003cp\u003eGiven the proximity to the Horn of Africa (HoA), the status of \u003cem\u003eAn. stephensi\u003c/em\u003e in the Arabian Peninsula is of growing interest. The predicted native range of \u003cem\u003eAn. stephensi\u003c/em\u003e includes Saudi Arabia and Oman (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, \u003cem\u003eAn. stephensi\u003c/em\u003e has not been reported in Yemen until recently. \u003cem\u003eAnopheles stephensi\u003c/em\u003e was detected in Yemen in 2021 in Aden City, located in the southern part of Yemen (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Aden City shares some similarities with locations where \u003cem\u003eAn. stephensi\u003c/em\u003e is now well-established that serve as major hubs for its onward spread across the HoA (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It is an urban area and port city with frequent population movement inward through the flow of internally displaced persons and refugees from the Horn of Africa (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It is a major port city connecting with Bosaso, Somalia and Mukalla Port City, another location on the southern coast of Yemen with a recent detection of \u003cem\u003eAn. stephensi\u003c/em\u003e. Finally, an increase in malaria incidence has also been reported in Aden (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Given the detection of \u003cem\u003eAn. stephensi\u003c/em\u003e in these two locations in the southern part of Yemen, it is important to confirm how far \u003cem\u003eAn. stephensi\u003c/em\u003e has spread throughout the south.\u003c/p\u003e \u003cp\u003eMolecular confirmation of species identification is an important component of the tracking of invasive \u003cem\u003eAn. stephensi\u003c/em\u003e across its invasive range (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It provides a basis to evaluate the successful integration of the updated morphological keys that include \u003cem\u003eAn. stephensi\u003c/em\u003e into ongoing vector surveillance. In addition, DNA sequence-based molecular identification has the benefit of providing preliminary insight into the relationship between newly identified invasive populations and other reported populations. This study reports the results of molecular analyses of morphologically identified \u003cem\u003eAn. stephensi\u003c/em\u003e across southern Yemen, including four newly reported locations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSite Descriptions\u003c/h2\u003e \u003cp\u003eFive sites were surveyed for this study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): Dar Sa\u0026rsquo;ad (Aden City), Tuban in Lahj governorate, Rodoom in Shabwah governorate, Al Mukalla Port City in Hadramout governorate, and Sayhut in Al Mahrah governorate. Aden is a major port city along an historic maritime route connecting the Horn of Africa and the Arabian Peninsula. The climate in Aden is mostly desert, although there is steady humidity year-round. Lahj is located just under 40 km north of Aden with similar climate. Rodoom coastal city in Shabwah governorate is located between Hadramout to the east and Abyan to the west with coastal and semiarid climate. Tuban is located just under 40 km north of Aden with similar climate. Al Mukalla Port City is located over 500 km east of Aden and has a coastal and semi-arid climate. Sayhut is the easternmost site of this collection and has a subtropical climate with peak rains in May.\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\u003eDescription of sites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGovernorate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoordinates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDate collected\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDar Sa\u0026rsquo;ad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAden\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.78652104214733, 45.01848534347771\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOctober, December 2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTuban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLahj\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.05807428525727, 44.88331315931494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDecember 2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRodoom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShabwah\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.841611, 47.59567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJune 2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAl Mukalla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHadramout\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.54943802880376, 49.12458936878869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOctober 2021, April, September, October 2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSayhut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAl Mahrah\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.202806, 51.201750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCollections\u003c/h2\u003e \u003cp\u003eLarvae were collected using the dipping method over the course of three days and reared to adult stage. Potential breeding habitats surveyed included domestic containers, air condition drips, accumulated water in brick factories, and cemeteries (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Adult \u003cem\u003eAnopheles\u003c/em\u003e were identified morphologically using the Afrotropical mosquitoes key (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The specimens morphologically identified as \u003cem\u003eAn. stephensi\u003c/em\u003e were preserved with silica gel and a subsample of specimens was sent to Baylor University for molecular analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePCR and sequence analysis\u003c/h2\u003e \u003cp\u003eTwo loci were selected for PCR amplification-based species identifications: cytochrome oxidase subunit 1 (\u003cem\u003eCOI)\u003c/em\u003e and internal transcribed spacer 2 (\u003cem\u003eITS2)\u003c/em\u003e as previously applied (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). First, species were confirmed using a PCR species-specific assay targeting the \u003cem\u003eITS2\u003c/em\u003e region (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and with PCR and sequencing of the ITS2 and an informative portion of \u003cem\u003eCOI\u003c/em\u003e using methods described previously (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The primer sequences for PCR in the \u003cem\u003eITS2\u003c/em\u003e endpoint assay are 5.8SB (5\u0026prime;-ATCACTCGGCTCGTGGATCG-3ʹ) and 28SC (5ʹ- GTCTCGCGACTGCAACTG-3ʹ) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The primer sequences for the \u003cem\u003eITS2\u003c/em\u003e PCR for sequencing were 5.8SB (5ʹ-ATCACTCGGCTCGTGGATCG-3ʹ) and 28SB (5ʹ-ATGCTTAAATTTAGGGGGTAGTC-3ʹ) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The primer sequences for \u003cem\u003eCOI\u003c/em\u003e PCR are LCO1490F (5ʹ-GGTCAACAAATCATAAAGATATTGG-3ʹ) and HCO2198R (5ʹ-TAAACTTCAGGGTGACCAAAAAATCA-3ʹ) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). ITS2 and \u003cem\u003eCOI\u003c/em\u003e PCR products were sequenced using Sanger sequencing technology. Sequences were then trimmed and analyzed using CodonCode Aligner (CodonCode Corporation, Centerville, MA). Final sequences were submitted to the National Center for Biotechnology Information\u0026rsquo;s (NCBI) Basic Local Alignment Search Tool (BLAST) (Accession #: PP752283, PP752284, PQ006012). Alignments were generated in CodonCode Aligner that included previously published sequences from NCBI GenBank. ITS2-based sequence identification excluded the microsatellite region found within ITS2 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Likewise, only a portion of the COI previously identified as geographically informative was used for subsequent analysis in order to maximize the overlap with existing datasets for haplotype and phylogenetic comparisons (see Carter et al. 2021). Phylogenetic analysis was conducted using the maximum-likelihood method with RAxML (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The best scoring trees under ML with bootstrap values from RAxML were viewed and annotated using Figtree (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eA total of forty-four mosquitoes were molecularly characterized and all specimens were confirmed to be \u003cem\u003eAn. stephensi\u003c/em\u003e with the ITS2 end-point assay. In addition, BLAST analyses of the COI and ITS2 sequences revealed highest scoring matches for \u003cem\u003eAn. stephensi\u003c/em\u003e. Most of the COI sequences had a 100% sequence identity match with \u003cem\u003eAn. stephensi\u003c/em\u003e sequences originating from northeastern Ethiopia, Somaliland, Djibouti, and Yemen. The 162 bp segment of ITS2 sequence were identical for all specimens and had a 100% sequence identity match with other \u003cem\u003eAn. stephensi\u003c/em\u003e sequences. Phylogenetic analysis confirmed \u003cem\u003eAn. stephensi\u003c/em\u003e identity (ITS2 bs\u0026thinsp;=\u0026thinsp;100, COI bs\u0026thinsp;=\u0026thinsp;89) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Fig.)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSupplemental Figure.\u003c/b\u003e Phylogenetic analysis of \u003cem\u003eAn. stephensi\u003c/em\u003e ITS2 sequences from southern Yemen using the maximum likelihood approach. The ITS2 sequence observed in southern Yemen is in blue.\u003c/p\u003e \u003cp\u003eAnalysis of a specific region of the COI sequence has previously been identified as geographically informative (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Using this region of the COI, we identified two haplotypes among the Yemen samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). One haplotype was found in the majority of the mosquitoes (n\u0026thinsp;=\u0026thinsp;43). This haplotype has been observed in Yemen and in the northeastern Horn of Africa (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) in other studies and designated Hap3 by Carter et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The second haplotype (n\u0026thinsp;=\u0026thinsp;1, found in Sayhut) has not been reported previously. As the second unique Yemen haplotype, it will be designated HapYem2 in this study. In order to further investigate the genetic similarity between sequences from southern Yemen and other areas of the \u003cem\u003eAn. stephensi\u003c/em\u003e range, we performed phylogenetic analysis of the COI sequences. Phylogenetic analysis showed support for differentiation from Saudi Arabia for all sequences, although further geographic distinctions were not detected in this phylogenetic tree likely due to the low variation of this marker.\u003c/p\u003e \u003cp\u003eOverall, this study revealed a more homogenous population relative to populations in northern Horn of Africa, including Ethiopia, Djibouti, and Somaliland. This evidence provides support for a more recently established \u003cem\u003eAn. stephensi\u003c/em\u003e population in portions of Yemen compared to the northern Horn of Africa populations. Interestingly, no haplotypes from Saudi Arabia or the United Arab Emirates were observed in any locations, suggesting a stronger connection to the invasive Horn of Africa populations rather than long-established populations in the Arabian Peninsula. This study was limited by the lack of full representative sampling of \u003cem\u003eAn. stephensi\u003c/em\u003e across the long-established geographic range in the Arabian Peninsula and by the limited resolution of the markers used in this study. Regardless, these findings highlight the importance of population genomic surveillance in this region.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research activity which involved the collection of larvae from outdoor breeding habitats was approved by the National Malaria Control Program, Ministry of Health in Aden. Collectors are qualified entomological technicians from the malaria program staff who conduct collection with the goal of minimizing exposure to adult mosquitoes. This work did not involve human subject research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by a NIH Research Enhancement Award (1R15AI151766) and funding through the Centers for Disease Control and Prevention awarded to TEC. Additional support for the collection of mosquitoes was provided by NAMRU-EURAFCENT project funded by Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance Branch (GEIS) # P0041_22_N3 \u0026amp; P0036_23_N3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEC, AZ, SA, YAB contributed to the conception and design of the project. YAB, YA, AZ, JH organized and led the collection of specimens. MA, PA, JNS generated molecular data. TEC analyzed the data. TEC wrote the first draft of manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eDisclaimer:\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eOur gratitude goes to the World Health Organization for\u0026nbsp;their technical support for implementation of surveillance.\u0026nbsp;The authors would like to thank Dr. Ghasem Zamani (Regional Advisor WHO/EMRO-MVC) for his contribution and technical support for implementation of surveillance and review of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFaulde MK, Rueda LM, Khaireh BA. 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Bioinformatics. 2014;30(9):1312-3.\u003c/li\u003e\n\u003cli\u003eRambaut A. Figtree Version. 1.4. 4 ed. Institute of Evolutionary Biology, Univ2018.\u003c/li\u003e\n\u003cli\u003eCarter TE, Yared S, Getachew D, Spear J, Choi SH, Samake JN, et al. Genetic diversity of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Ethiopia provides insight into patterns of spread. Parasit Vectors. 2021;14(1):602.\u003c/li\u003e\n\u003cli\u003eAssada M, Al-Hadi M, Esmail MA, Al-Jurban J, Alkawri A, Shamsan A, et al. Molecular Confirmation of \u003cem\u003eAnopheles stephensi\u003c/em\u003e Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022. Emerg Infect Dis. 2024;30(7):1467-71.\u003c/li\u003e\n\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":"","lastPublishedDoi":"10.21203/rs.3.rs-4783439/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4783439/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe invasion of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Africa warrants investigation of neighboring countries. In this study, genetic analysis was applied to determine the status of \u003cem\u003eAn. stephensi\u003c/em\u003e in southern Yemen. Cytochrome oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) were sequenced in \u003cem\u003eAn. stephensi\u003c/em\u003e collected in Aden City, Lahj, Rodoom, Al Mukalla, and Sayhut, and phylogenetic analysis confirmed \u003cem\u003eAn. stephensi\u003c/em\u003e identity. Our analyses revealed that the ITS2 sequence were identical in all \u003cem\u003eAn. stephensi\u003c/em\u003e, while COI analysis revealed two haplotypes, one previously reported in northern Horn of Africa and one identified in this study for the first time. Overall, these findings revealed low levels of mitochondrial DNA diversity, which is consistent with a recent population introduction in parts of southern Yemen. Further whole genomic analysis is needed to elucidate the original connection with invasive populations of \u003cem\u003eAn. stephensi\u003c/em\u003e in the Horn of Africa.\u003c/p\u003e","manuscriptTitle":"Sequencing confirms Anopheles stephensi distribution across southern Yemen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 12:35:45","doi":"10.21203/rs.3.rs-4783439/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-29T18:45:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-29T18:37:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-23T13:41:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89782255952664257347685112551977943025","date":"2024-08-08T17:54:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309428229122777160187080669986725688281","date":"2024-08-07T09:35:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47895595104946975441915901574816988773","date":"2024-08-05T11:14:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55304669031330951927280532478385754094","date":"2024-08-05T08:34:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-05T08:09:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-02T05:37:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-02T03:50:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-07-22T16:49:30+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":"c031346b-800f-4092-bb54-0a42d33e5521","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:06:14+00:00","versionOfRecord":{"articleIdentity":"rs-4783439","link":"https://doi.org/10.1186/s13071-024-06601-1","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-12-18 15:58:35","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-08-28 12:35:45","video":"","vorDoi":"10.1186/s13071-024-06601-1","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06601-1","workflowStages":[]},"version":"v1","identity":"rs-4783439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4783439","identity":"rs-4783439","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}