Molecular identification of Anopheles squamosus (Diptera: Culicidae) using internal transcribed spacer 2

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Nguyen, Renee L. M. N. Ali, Bianca C. Burini, Dalia S. Dryden, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7278578/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2025 Read the published version in Malaria Journal → Version 1 posted 9 You are reading this latest preprint version Abstract Background Anopheles squamosus is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with Plasmodium falciparum and Plasmodium vivax . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish An. squamosus from morphologically similar Anopheles species. Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. We evaluated six sets of primers overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens from Zambia and South Africa. Results A new assay consisting of a forward (ITS2-ASQ-R10, 5’-CCC TCG AAG GGT GCT GTG-3’) and reverse (ITS2-ASQ-R10 5’-AAT CCA CGG TGT GAT GGC-3’) primer reliably (> 94.8%) amplified an ITS2 fragment of 301bp length for An. squamosus . The An. squamosus- specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification. Conclusions The development of this robust PCR assay for An. squamosus is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. In addition, investigating host association and malaria transmission can be facilitated with this assay by correctly identifying An. squamosus. Applying genomic tools to correctly identified anopheline species may lead to the discovery of genetic factors that influence its behavior and new innovations in malaria elimination. Anopheles squamosus molecular diagnosis internal transcribed spacer mosquito malaria Figures Figure 1 Figure 2 Figure 3 Introduction With global concerted efforts to reduce the burden of malaria, the World Health Organization (WHO) created the Global Technical Strategy for Malaria 2016–2030 (GTS) with the goal to reduce malaria incidence and mortality rates by 90% in 35 countries during this period [ 1 ]. These efforts have led several regions within Africa to reach a pre-elimination stage of malaria transmission. Pre-elimination for a region is defined as a population with either a Rapid Diagnostic Test (RDT) positivity rate below 5% annually or a parasite positivity rate lower than 5% among those with fever [ 2 ]. Countries and regions that have met these pivotal milestones towards malaria elimination include Cape Verde, central Senegal, Guinea-Bissau, Isle of Príncipe, and southern Zambia [ 3 – 9 ]. In some cases, secondary malaria vectors have been implicated in transmission of malaria parasites in pre-elimination areas. These secondary vectors include Anopheles vaneedeni and An. parensis in areas of southern Africa and An. coustani and An. ziemanni in central Africa [ 28 – 31 ]. In Zambia, anopheline species like An. squamosus, An. rufipes , and An. coustani are of increasing concern as vectors for malaria in pre-elimination areas after reductions in populations of the primary vector species [ 11 – 15 ]. Anopheles squamosus is an anopheline species that is common and collected in abundance across sub-Saharan Africa [ 16 ] including Zambia. Previous work suggested that this species could be a species complex based on chromosome inversion polymorphism data [ 17 ]. In the adult stage, this species is morphologically identical to An. cydippis and difficult to distinguish molecularly from other unnamed Anopheles species like Anopheles sp. 11 and Anopheles sp. 15, cryptic species related to An. squamosus [ 18 , 19 ]. There is no morphological data for An. sp. 11 and 15 to compare with An. squamosus to date. Therefore, morphological identification for An. squamosus can be unreliable and currently requires sequencing of fragments of the cytochrome c oxidase subunit I (COI) and/or internal transcribed spacer 2 (ITS2) genes for species confirmation [ 4 ]. While COI is often used for species identification [ 20 – 22 ], mitochondrial markers are often insufficient to delineate mosquito species within closely related species groups in anopheline mosquitoes [ 23 – 25 ]. For this reason, ITS2 regions on the X chromosome of Anopheles species are commonly used for molecular species identification [ 26 , 26 – 28 ]. Internal transcribed spacer 2 is used primarily for parsing cryptic species in anopheline complexes, such as species of the An. maculatus complex, An. maculipennis complex, An. quadrimaculatus complex, An. fluviatilis complex, An. crucians complex and the An. nili group [ 25 – 29 ]. The challenge in applying this method to identification of An. squamosus is that the currently available primer sets used in ITS2 identification assays for anopheline species do not reliably produce PCR products from An. squamosus DNA templates [ 13 ]. Inability to accurately identify An. squamosus limits the capacity of routine surveillance to detect the presence of this potential malaria vector and may lead to misidentification with species not implicated in malaria transmission [ 24 ]. The lack of a reliable molecular identification tool for this species also hinders further research. Application of genomic tools to accurately identified species may lead to the discovery of genomic regions responsible for parasite infection, insecticide resistance, host choice, and other traits relevant to pathogen transmission [ 14 – 18 ]. In this study, a new reliable ITS2 PCR assay is introduced that distinguishes An. squamosus from other sympatric Anopheles species. Materials and Methods Sample Collection and DNA Preparation Anopheles squamosus specimens from Zambia and South Africa were used for this study. Specimens from Macha in Choma District, Southern Province, Zambia (16.42181°S, 177.20417°W) were collected in January 2023 using a CDC light trap placed inside animal pens or human dwellings upon permission from homeowners. Specimen collection in Limpopo Province, South Africa (23.4013° S, 29.4179° E) was conducted using CO 2 -baited tent traps and sweep nets. Morphological identification was done using a morphological key to African anopheline species [ 17 ]. Samples were stored in 80% ethanol and refrigerated at 4°C until DNA extraction. To extract DNA from individual Zambian mosquitoes, a magnetic bead-based protocol as described by Chen et al. [ 19 ] was used. The DNA from South African samples was extracted with the Macherey-Nagel NuceloSpin Kit following the manufacturer's instructions (Düren, Germany). Primer Design Previously published ITS2 contig sequences (National Center for Biotechnology Information GenBank Accession number: MK592048, MK592075, OQ241725, MK592071) were obtained for An. sp. 11, An. sp. 15, and An. squamosus for primer design [ 30 ]. These sequences were selected because of their high sequence similarity (between 73.33% and 90.82%) to the available An. squamosus ITS2 sequences in GenBank, which allowed primer design that was specific for the target species. Of note, there is currently no sequence data available for An. cydippus . Multiple sequence alignment of these ITS2 sequences was conducted in Geneious Prime (version 2023.1.2) [ 20 ], which illuminated candidate ITS2 regions where An. squamosus -specific amplificon could be achieved. The consensus sequences of the multiple alignment were used as input sequences for primer design using Primer-BLAST [ 31 ]. A target amplimer range was set between 290 to 315 bp, so it can be multiplexed with the existing ITS2 sequence, which produces bands > 400bp for other Anopheles species. Programs such as MPprimer (version 3.1) [ 21 ] were used to test the primer compatibility for multiplex PCR. Six candidate primer sets (using three forward primers and reverse primers) were identified as compatible primers and used for assay validation (Table 1 ). PCR Validation A 25 µL PCR mixture was prepared for each mosquito specimen to contain 2–5 µL of extracted DNA template, 2.5 µL of 10× PCR buffer, 2.0 µL of 10 mM dNTPs mix, 0.4 µL of Promega GoTaq Taq DNA polymerase (5 U/µl; Madison, WI, USA), 0.3 µL of each forward and reverse primer (10 µM), and 14.5–17.5 µL of PCR-grade water. Assay validation was performed on An. squamosus, An. sp. 11, An. sp. 15, An. stephensi, An. arabiensis, An. gambiae s.s., and An. funestus s.s to screen for primer specificity. The robustness of the PCR amplification for An. squamosus was evaluated on 78 replicates of individual specimens of An. squamosus. PCR conditions were as follows: initial denaturation at 94°C for 2 min followed by 39 cycles of 94°C for 30 s, 57.6ºC for 30 s, and 72ºC for 40 s. Then a final extension step of 72ºC for 10 min before being held at 4ºC. Amplification of a PCR product of the expected size range was confirmed by electrophoresis on a 1.5% agarose gel. A total of four new forward primers (ITS2-ASQ-F1, -F2, -F6, and -F10) and two new reverse primers (ITS2-ASQ-R8 and -R10) were designed (Fig. 1 , Table 1 ). Six combinations of these primers were tested for species-specific amplification. Cocktail PCR Validation The new primer sets were assessed in a multiplexed PCR cocktail with existing Anopheles ITS2 primers (UV [ 32 ] or ITS2A [ 33 ], ITS2b [ 33 ], ITS-ASQ-F10, ITS-ASQ-R10) on individual specimens of An. squamosus, An. sp. 11, An. sp. 15, An. stephensi, An. arabiensis, An. gambiae s.s., and An. funestus s.s. These Anopheles species were selected due to their overlapping distribution across Africa. The PCR conditions were the same as described above. Table 1 A list of candidate primer sets used to assess their specificity to An. squamosus . The bold font indicates the primer set used for further testing based on the reliability of An. squamosus- specific amplification. In the primer name, ITS2A refers to a universal primer for Anopheles [ 33 ] (sometimes denoted as UV [ 32 ]). F and R refer to the forward primer and the reverse primer. Primer pairs Forward primer Reverse primer Amplimer length (bp) ITS2A + ITS2-ASQ-R8 5’-TGTGAACTGCAGGACACAT-3’ 5’-TCAACGTACCACACTTGACG-3’ 301 ITS2-ASQ-F1 + ITS2-ASQ-R8 5’-CATCGGACGTTCTAACACGA-3’ 5’-TCAACGTACCACACTTGACG-3’ 253 ITS2-ASQ-F2 + ITS2-ASQ-R8 5’-TCGACACGTTGAACGCATA-3’ 5’-TCAACGTACCACACTTGACG-3’ 277 ITS2A + ITS2-ASQ-R10 5’-TGTGAACTGCAGGACACAT-3’ 5’-AATCCACGGTGTGATGGC-3’ 436 ITS2-ASQ-F6 + ITS2-ASQ-R10 5’-GTGCTGTGGGACAATCCAC-3’ 5’-AATCCACGGTGTGATGGC-3’ 291 ITS2-ASQ-F10 + ITS2-ASQ-R10 5’- CCCTCGAAGGGTGCTGTG -3’ 5’- AATCCACGGTGTGATGGC -3’ 301 Results Among the primers tested, the combination of ITS2-ASQ-F10 and ITS2-ASQ-R10 produced An. squamosus- specific amplificons (Fig. 2 ) for 74 of 78 individual An. squamosus specimens (72 from Zambia, six from South Africa) evaluated. With only four reactions failed to produce an amplicon (5.1% false negative rate), this assay demonstrated a robust (> 94.9%) success rate. The new ITS2-ASQ-F10/ITS2-ASQ-R10 primer set was successfully used with existing Anopheles ITS2 PCR primers (ITS2A and ITS2B) that have been typically used for Anopheles species identification [ 33 ] (Fig. 3 ). Testing of the multiplexed ITS2 PCR showed unique amplimer sizes for each Anopheles species with An. squamosus distinct from the anopheline mosquitoes included here. Discussion In the absence of morphological discrimination, an An. squamosus -specific molecular tool is needed to positively identify this species. Compatibility and the ability to multiplex with existing PCR-based assays [ 34 ] is ideal to reduce both costs and effort, making the approach viable for implementation as a routine malaria vector surveillance method in Africa. This study resulted in a primer set that can robustly (> 94.9%) amplify a fragment of the An. squamosus ITS2 gene when multiplexed with the standard ITS2A/ITS2B primers. The size of the amplicon was 301 bp, which was specific to An. squamosus against all the other African Anopheles species evaluated within this study. Other Anopheles species routinely caught in collections with An. squamosus produce different sized ITS2 amplicons from the ITS2A and ITS2B primers (Fig. 3 ). For example, members of the An. gambiae complex produce an amplicon near 600 bp, while members of the An. funestus group produce an amplicon of 850 bp. Anopheles rufipes , An. maculipalpis , and An. pretoriensis generate indistinguishable amplicons of approximately 500 bp when using the ITS2A and ITS2B primers. The reaction could be further optimized using a touchdown PCR to reduce the unspecific binding and increase specificity. With an understanding of anopheline community composition, species-specific targeted and effective methods of control may be better implemented. For example, indoor residual spraying and long-lasting insecticidal netting are widely used in malaria-endemic areas, as they have been most effective at targeting the behavior of endophilic and endophagic primary vectors (WHO 2023, Bhatt 2015). These efforts have resulted in reductions of principal malaria vectors such as Anopheles gambiae s.l. in Kenya, Tanzania, and Zambia [ 15 , 35 , 36 ]. However, these intervention methods do not consider foraging and resting behaviors of secondary vectors that may be transmitting malaria at low levels [ 10 , 11 ]. This new An. squamosus- specific assay was evaluated on specimens from a relatively narrow geographic range of the known An. squamosus distribution; Zambia and South Africa. Therefore, this assay should be further evaluated on An. squamosus from a much broader geographic region to assess accuracy and robustness, especially for geographically isolated populations such as Madagascar, where An. squamosus is abundant [ 37 ]. Moreover, Coetzee [ 17 ] suggested that An. squamosus is likely to be a species complex based on chromosome inversion polymorphisms. If this is true, the ITS2 PCR alone may not be sufficient to delineate species within this complex and may need further refinement, as has been demonstrated for the An. gambiae complex [ 38 ]. In addition, An. cydippis and additional species that are sympatric to An. squamosus should be evaluated to ensure that this new ITS2 primer set is indeed species-specific. This assay allows the more robust surveillance of An. squamosus in Africa. Though cryptic with other taxa as adults, this species can be morphologically differentiated as 4th instar larvae. Unfortunately, earlier instars may not have developed distinguishable features. In addition, common methods of trapping adult anopheline such as CDC miniature light traps may damage specimens and lose key features that are used to distinguish them morphologically, such as wings, legs, and scales [ 19 ]. DNA-based tools, such as the ITS2 assay, avoid these challenges, providing robust identification regardless of life stage or specimen quality with minimal tissue input [ 39 ]. This assay opens new opportunities for investigating the role of An. squamosus in malaria transmission. By exploiting the genetics of vectors and building resources to study them, we can identify genes that can be tied to vector competence. For example, in Anopheles arabiensis , there was an identified genetic component that influenced its host choice and behavior [ 40 ]. Alleles linked to the 2Rb and/or 3Ra inversions were linked to cattle-feeding preferences in An. arabiensis. The link between genetics and host preference is vital to further assess how An. squamosus is contributing to disease transmission with a new and accurate method of identification of this species. The development of this ITS2 primer for An. squamosus , using the methods detailed here, can be applied to other understudied species and secondary vectors of malaria that do not have reliable methods of identification. For example, An. pharoensis is also found to be infected with P. falciparum , but does not reliably amplify in currently available ITS2. This assay shows repeatability, reproducibility, specificity, and a high limit of detection (> 95% amplification of biological replicates used), showing these primer sets provide a robust method for the detection of An. squamosus. This framework will allow for accurate and robust monitoring of secondary vectors of malaria. Declarations Ethics approval and consent to participate The study involves collection of mosquito specimen within individual households in Zambia as part of the project that had been approved by National Health Research Authority, Zambia: Approval No: NHRA00016/18/08/2021. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding We acknowledge funding support from the United States National Institute of Health as part of the International Centers of Excellence for Malaria Research (2U19AI089680), Small Grant Program (R03AI178041), and T32 Institutional Training Grant (T32AI0074717). Additional support was provided by the United States Department of Agriculture National Institute of Food and Agriculture Multistate Hatch Project (1025565 and 7007941), the Bloomberg Philanthropies, the Johns Hopkins Malaria Research Institute, the College of Agricultural and Life Sciences Dean’s Award at the University of Florida, and Global Fellows Program at the University of Florida International Center. Author Contribution VTN and YL conceptualized the study. VTN, BCB, and YL provided methodology. VTN, RLMNA, BCB, DSD, MAR, KS, LER, and YL conducted investigation. VTN curated data. VTN, RLMNA, and YL conducted data analysis and data visualization. VTN, RLMNA, ES, DEN, and YL acquired funding and provided resources for the study. YL carried out project administration and supervision. VTN, DSD, and YL wrote original draft. All authors contributed to review and editing. Acknowledgement We thank the entomology staff at Macha Research Trust for assisting with mosquito collection and sample processing. We thank Mr. Sangwoo Seok (University of Florida) for reviewing our manuscript. Availability of data and materials All data generated or analyzed during this study are included in this article. References World Health Organization. Global technical strategy for malaria 2016–2030 [Internet]. World Health Organization. 2015 [cited 2024 Dec 9]. Available from: https://iris.who.int/handle/10665/176712 Moonasar D, Nuthulaganti T, Kruger PS, Mabuza A, Rasiswi ES, Benson FG, et al. Malaria control in South Africa 2000–2010: Beyond MDG6. Malar J. 2012;11:1–7. 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Cite Share Download PDF Status: Published Journal Publication published 16 Oct, 2025 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Revision requested 06 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviews received at journal 26 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 05 Aug, 2025 Submission checks completed at journal 05 Aug, 2025 First submitted to journal 02 Aug, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Nguyen","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Valerie","middleName":"T.","lastName":"Nguyen","suffix":""},{"id":500086258,"identity":"828c1c6a-85ca-4a19-91b5-5e9e29aaebc5","order_by":1,"name":"Renee L. M. N. Ali","email":"","orcid":"","institution":"The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Renee","middleName":"L. M. N.","lastName":"Ali","suffix":""},{"id":500086259,"identity":"387c4d4d-7f58-40d4-a59f-0d6663388da2","order_by":2,"name":"Bianca C. Burini","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"C.","lastName":"Burini","suffix":""},{"id":500086260,"identity":"fcc169ef-8cda-461d-bde3-e7c29ff05642","order_by":3,"name":"Dalia S. Dryden","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"S.","lastName":"Dryden","suffix":""},{"id":500086261,"identity":"345b1321-2af6-451b-b819-e641d183bff1","order_by":4,"name":"Megan A. Riddin","email":"","orcid":"","institution":"University of Pretoria","correspondingAuthor":false,"prefix":"","firstName":"Megan","middleName":"A.","lastName":"Riddin","suffix":""},{"id":500086262,"identity":"b0178d23-7c2f-4328-8691-23b8dee29224","order_by":5,"name":"Kochelani Saili","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Kochelani","middleName":"","lastName":"Saili","suffix":""},{"id":500086263,"identity":"85de0af5-0f02-473c-bfa3-76007529930a","order_by":6,"name":"Edgar Simulundu","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"","lastName":"Simulundu","suffix":""},{"id":500086264,"identity":"8f6626a0-8d53-4191-ac5c-3834c0ee8383","order_by":7,"name":"Lawrence E. Reeves","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Lawrence","middleName":"E.","lastName":"Reeves","suffix":""},{"id":500086265,"identity":"95d1fe3d-119e-46fe-a362-9a3f0458c77d","order_by":8,"name":"Douglas E. Norris","email":"","orcid":"","institution":"The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"E.","lastName":"Norris","suffix":""},{"id":500086266,"identity":"fa297e5a-3a95-4731-a886-fca0a97720c9","order_by":9,"name":"Yoosook Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYPCCBAY+BgbGBww2FmAOcVrYGBiYDRjSJEjTwiZBlBaD482HP3yoSGNgY28+Vs2TIMHAz55jgF/LmWNpkjPO5DCw8RxLuw3SItnzhoCWGzlmzLxtFUBX5Zjd5v0hARIhqMX4M1iL/PtvxSBb7InQYiDN2wZ0mAQPGzNIi4EEAS2SEL+k8bDxpBlLzkmQ4JE486wArxY+SIgly/GzH3744U2CjRx/e/IGvFoUDkBoHpgADw6FCCDfQFDJKBgFo2AUjHgAAMybPdbt4BxJAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Yoosook","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-08-02 13:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7278578/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7278578/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-025-05600-6","type":"published","date":"2025-10-16T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89373847,"identity":"55d53dc1-1d87-479b-9d00-fb2a95d4d186","added_by":"auto","created_at":"2025-08-19 10:39:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79908,"visible":true,"origin":"","legend":"\u003cp\u003ePrimer position relative to the consensus ITS2 sequences of \u003cem\u003eAn. squamosus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7278578/v1/c6443c4c44ee0b35b89aa59d.png"},{"id":89373838,"identity":"6e9344dd-0b30-4a5c-8113-a40666fd841e","added_by":"auto","created_at":"2025-08-19 10:39:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183728,"visible":true,"origin":"","legend":"\u003cp\u003eITS2 PCR results for \u003cem\u003eAn. squamosus \u003c/em\u003eamplification ~300 bp. Lane 1: ladder. Lanes 2-9: eight different individual \u003cem\u003eAn. squamosus \u003c/em\u003especimens (one specimen per lane). Lane 10: negative control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7278578/v1/4b9f9c1fc3c898caac24211a.png"},{"id":89373833,"identity":"e2ace701-aafa-4d40-bd2d-480cfc7f5fcf","added_by":"auto","created_at":"2025-08-19 10:39:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241804,"visible":true,"origin":"","legend":"\u003cp\u003eMultiplexed ITS2 PCR results showingITS-ASQ-F10 and ITS-ASQ-R10 primers used together with ITS2A and ITS2B primers. Lanes 1-2: \u003cem\u003eAn. squamosus. \u003c/em\u003eLane 3: \u003cem\u003eAn. \u003c/em\u003esp. 11.\u003cem\u003e \u003c/em\u003eLane 4: \u003cem\u003eAn. \u003c/em\u003esp. 15.\u003cem\u003e \u003c/em\u003eLane 5: \u003cem\u003eAn. stephensi. \u003c/em\u003eLane 6: \u003cem\u003eAn. arabiensis. \u003c/em\u003eLane 7: \u003cem\u003eAn. gambiae \u003c/em\u003es.s.\u003cem\u003e \u003c/em\u003eLane 8: \u003cem\u003eAn. funestus \u003c/em\u003es.s\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7278578/v1/47591b37524badaa3de8ee63.png"},{"id":93956195,"identity":"755a2f42-908d-4ac9-bea0-1203e73a0323","added_by":"auto","created_at":"2025-10-20 16:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1161100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7278578/v1/029c5392-1a57-421c-b6b3-a21c9516f80b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMolecular identification of \u003cem\u003eAnopheles squamosus \u003c/em\u003e(Diptera: Culicidae) using internal transcribed spacer 2\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith global concerted efforts to reduce the burden of malaria, the World Health Organization (WHO) created the Global Technical Strategy for Malaria 2016\u0026ndash;2030 (GTS) with the goal to reduce malaria incidence and mortality rates by 90% in 35 countries during this period [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These efforts have led several regions within Africa to reach a pre-elimination stage of malaria transmission. Pre-elimination for a region is defined as a population with either a Rapid Diagnostic Test (RDT) positivity rate below 5% annually or a parasite positivity rate lower than 5% among those with fever [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Countries and regions that have met these pivotal milestones towards malaria elimination include Cape Verde, central Senegal, Guinea-Bissau, Isle of Pr\u0026iacute;ncipe, and southern Zambia [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn some cases, secondary malaria vectors have been implicated in transmission of malaria parasites in pre-elimination areas. These secondary vectors include \u003cem\u003eAnopheles vaneedeni\u003c/em\u003e and \u003cem\u003eAn. parensis\u003c/em\u003e in areas of southern Africa and \u003cem\u003eAn. coustani\u003c/em\u003e and \u003cem\u003eAn. ziemanni\u003c/em\u003e in central Africa [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In Zambia, anopheline species like \u003cem\u003eAn. squamosus, An. rufipes\u003c/em\u003e, and \u003cem\u003eAn. coustani\u003c/em\u003e are of increasing concern as vectors for malaria in pre-elimination areas after reductions in populations of the primary vector species [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles squamosus\u003c/em\u003e is an anopheline species that is common and collected in abundance across sub-Saharan Africa [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] including Zambia. Previous work suggested that this species could be a species complex based on chromosome inversion polymorphism data [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the adult stage, this species is morphologically identical to \u003cem\u003eAn. cydippis\u003c/em\u003e and difficult to distinguish molecularly from other unnamed \u003cem\u003eAnopheles\u003c/em\u003e species like \u003cem\u003eAnopheles\u003c/em\u003e sp. 11 and \u003cem\u003eAnopheles\u003c/em\u003e sp. 15, cryptic species related to \u003cem\u003eAn. squamosus\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. There is no morphological data for \u003cem\u003eAn. sp. 11\u003c/em\u003e and \u003cem\u003e15\u003c/em\u003e to compare with \u003cem\u003eAn. squamosus\u003c/em\u003e to date. Therefore, morphological identification for \u003cem\u003eAn. squamosus\u003c/em\u003e can be unreliable and currently requires sequencing of fragments of the cytochrome c oxidase subunit I (COI) and/or internal transcribed spacer 2 (ITS2) genes for species confirmation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile COI is often used for species identification [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], mitochondrial markers are often insufficient to delineate mosquito species within closely related species groups in anopheline mosquitoes [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For this reason, ITS2 regions on the X chromosome of \u003cem\u003eAnopheles\u003c/em\u003e species are commonly used for molecular species identification [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Internal transcribed spacer 2 is used primarily for parsing cryptic species in anopheline complexes, such as species of the \u003cem\u003eAn. maculatus\u003c/em\u003e complex, \u003cem\u003eAn. maculipennis\u003c/em\u003e complex, \u003cem\u003eAn. quadrimaculatus\u003c/em\u003e complex, \u003cem\u003eAn. fluviatilis\u003c/em\u003e complex, \u003cem\u003eAn. crucians\u003c/em\u003e complex and the \u003cem\u003eAn. nili\u003c/em\u003e group [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe challenge in applying this method to identification of \u003cem\u003eAn. squamosus\u003c/em\u003e is that the currently available primer sets used in ITS2 identification assays for anopheline species do not reliably produce PCR products from \u003cem\u003eAn. squamosus\u003c/em\u003e DNA templates [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Inability to accurately identify \u003cem\u003eAn. squamosus\u003c/em\u003e limits the capacity of routine surveillance to detect the presence of this potential malaria vector and may lead to misidentification with species not implicated in malaria transmission [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The lack of a reliable molecular identification tool for this species also hinders further research. Application of genomic tools to accurately identified species may lead to the discovery of genomic regions responsible for parasite infection, insecticide resistance, host choice, and other traits relevant to pathogen transmission [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, a new reliable ITS2 PCR assay is introduced that distinguishes \u003cem\u003eAn. squamosus\u003c/em\u003e from other sympatric \u003cem\u003eAnopheles\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSample Collection and DNA Preparation\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles squamosus\u003c/em\u003e specimens from Zambia and South Africa were used for this study. Specimens from Macha in Choma District, Southern Province, Zambia (16.42181\u0026deg;S, 177.20417\u0026deg;W) were collected in January 2023 using a CDC light trap placed inside animal pens or human dwellings upon permission from homeowners. Specimen collection in Limpopo Province, South Africa (23.4013\u0026deg; S, 29.4179\u0026deg; E) was conducted using CO\u003csub\u003e2\u003c/sub\u003e-baited tent traps and sweep nets. Morphological identification was done using a morphological key to African anopheline species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Samples were stored in 80% ethanol and refrigerated at 4\u0026deg;C until DNA extraction. To extract DNA from individual Zambian mosquitoes, a magnetic bead-based protocol as described by Chen et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was used. The DNA from South African samples was extracted with the Macherey-Nagel NuceloSpin Kit following the manufacturer's instructions (D\u0026uuml;ren, Germany).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePrimer Design\u003c/span\u003e\u003c/p\u003e\u003cp\u003ePreviously published ITS2 contig sequences (National Center for Biotechnology Information GenBank Accession number: MK592048, MK592075, OQ241725, MK592071) were obtained for \u003cem\u003eAn.\u003c/em\u003e sp. 11, \u003cem\u003eAn.\u003c/em\u003e sp. 15, and \u003cem\u003eAn. squamosus\u003c/em\u003e for primer design [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These sequences were selected because of their high sequence similarity (between 73.33% and 90.82%) to the available \u003cem\u003eAn. squamosus\u003c/em\u003e ITS2 sequences in GenBank, which allowed primer design that was specific for the target species. Of note, there is currently no sequence data available for \u003cem\u003eAn. cydippus\u003c/em\u003e. Multiple sequence alignment of these ITS2 sequences was conducted in Geneious Prime (version 2023.1.2) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which illuminated candidate ITS2 regions where \u003cem\u003eAn. squamosus\u003c/em\u003e-specific amplificon could be achieved. The consensus sequences of the multiple alignment were used as input sequences for primer design using Primer-BLAST [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A target amplimer range was set between 290 to 315 bp, so it can be multiplexed with the existing ITS2 sequence, which produces bands\u0026thinsp;\u0026gt;\u0026thinsp;400bp for other \u003cem\u003eAnopheles\u003c/em\u003e species. Programs such as MPprimer (version 3.1) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were used to test the primer compatibility for multiplex PCR. Six candidate primer sets (using three forward primers and reverse primers) were identified as compatible primers and used for assay validation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePCR Validation\u003c/span\u003e\u003c/p\u003e\u003cp\u003eA 25 \u0026micro;L PCR mixture was prepared for each mosquito specimen to contain 2\u0026ndash;5 \u0026micro;L of extracted DNA template, 2.5 \u0026micro;L of 10\u0026times; PCR buffer, 2.0 \u0026micro;L of 10 mM dNTPs mix, 0.4 \u0026micro;L of Promega GoTaq Taq DNA polymerase (5 U/\u0026micro;l; Madison, WI, USA), 0.3 \u0026micro;L of each forward and reverse primer (10 \u0026micro;M), and 14.5\u0026ndash;17.5 \u0026micro;L of PCR-grade water. Assay validation was performed on \u003cem\u003eAn. squamosus, An. sp. 11, An. sp. 15, An. stephensi, An. arabiensis, An. gambiae\u003c/em\u003e s.s., and \u003cem\u003eAn. funestus\u003c/em\u003e s.s to screen for primer specificity. The robustness of the PCR amplification for \u003cem\u003eAn. squamosus\u003c/em\u003e was evaluated on 78 replicates of individual specimens of \u003cem\u003eAn. squamosus.\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePCR conditions were as follows: initial denaturation at 94\u0026deg;C for 2 min followed by 39 cycles of 94\u0026deg;C for 30 s, 57.6\u0026ordm;C for 30 s, and 72\u0026ordm;C for 40 s. Then a final extension step of 72\u0026ordm;C for 10 min before being held at 4\u0026ordm;C. Amplification of a PCR product of the expected size range was confirmed by electrophoresis on a 1.5% agarose gel.\u003c/p\u003e\u003cp\u003eA total of four new forward primers (ITS2-ASQ-F1, -F2, -F6, and -F10) and two new reverse primers (ITS2-ASQ-R8 and -R10) were designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Six combinations of these primers were tested for species-specific amplification.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCocktail PCR Validation\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe new primer sets were assessed in a multiplexed PCR cocktail with existing \u003cem\u003eAnopheles\u003c/em\u003e ITS2 primers (UV [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] or ITS2A [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], ITS2b [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], ITS-ASQ-F10, ITS-ASQ-R10) on individual specimens of \u003cem\u003eAn. squamosus, An. sp. 11, An. sp. 15, An. stephensi, An. arabiensis, An. gambiae\u003c/em\u003e s.s., and \u003cem\u003eAn. funestus\u003c/em\u003e s.s. These \u003cem\u003eAnopheles\u003c/em\u003e species were selected due to their overlapping distribution across Africa. The PCR conditions were the same as described above.\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\u003eA list of candidate primer sets used to assess their specificity to \u003cem\u003eAn. squamosus\u003c/em\u003e. The bold font indicates the primer set used for further testing based on the reliability of \u003cem\u003eAn. squamosus-\u003c/em\u003especific amplification. In the primer name, ITS2A refers to a universal primer for \u003cem\u003eAnopheles\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] (sometimes denoted as UV [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]). F and R refer to the forward primer and the reverse primer.\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer pairs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward primer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse primer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmplimer length (bp)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITS2A +\u003c/p\u003e\u003cp\u003eITS2-ASQ-R8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-TGTGAACTGCAGGACACAT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-TCAACGTACCACACTTGACG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e301\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITS2-ASQ-F1 +\u003c/p\u003e\u003cp\u003eITS2-ASQ-R8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-CATCGGACGTTCTAACACGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-TCAACGTACCACACTTGACG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e253\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITS2-ASQ-F2 +\u003c/p\u003e\u003cp\u003eITS2-ASQ-R8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-TCGACACGTTGAACGCATA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-TCAACGTACCACACTTGACG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e277\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITS2A +\u003c/p\u003e\u003cp\u003eITS2-ASQ-R10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-TGTGAACTGCAGGACACAT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-AATCCACGGTGTGATGGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e436\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITS2-ASQ-F6 +\u003c/p\u003e\u003cp\u003eITS2-ASQ-R10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-GTGCTGTGGGACAATCCAC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-AATCCACGGTGTGATGGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e291\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eITS2-ASQ-F10 +\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eITS2-ASQ-R10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-\u003cb\u003eCCCTCGAAGGGTGCTGTG\u003c/b\u003e-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-\u003cb\u003eAATCCACGGTGTGATGGC\u003c/b\u003e-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e301\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the primers tested, the combination of ITS2-ASQ-F10 and ITS2-ASQ-R10 produced \u003cem\u003eAn. squamosus-\u003c/em\u003especific amplificons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) for 74 of 78 individual \u003cem\u003eAn. squamosus\u003c/em\u003e specimens (72 from Zambia, six from South Africa) evaluated. With only four reactions failed to produce an amplicon (5.1% false negative rate), this assay demonstrated a robust (\u0026gt;\u0026thinsp;94.9%) success rate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe new ITS2-ASQ-F10/ITS2-ASQ-R10 primer set was successfully used with existing \u003cem\u003eAnopheles\u003c/em\u003e ITS2 PCR primers (ITS2A and ITS2B) that have been typically used for \u003cem\u003eAnopheles\u003c/em\u003e species identification [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Testing of the multiplexed ITS2 PCR showed unique amplimer sizes for each \u003cem\u003eAnopheles\u003c/em\u003e species with \u003cem\u003eAn. squamosus\u003c/em\u003e distinct from the anopheline mosquitoes included here.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the absence of morphological discrimination, an \u003cem\u003eAn. squamosus\u003c/em\u003e-specific molecular tool is needed to positively identify this species. Compatibility and the ability to multiplex with existing PCR-based assays [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] is ideal to reduce both costs and effort, making the approach viable for implementation as a routine malaria vector surveillance method in Africa.\u003c/p\u003e\u003cp\u003eThis study resulted in a primer set that can robustly (\u0026gt;\u0026thinsp;94.9%) amplify a fragment of the \u003cem\u003eAn. squamosus\u003c/em\u003e ITS2 gene when multiplexed with the standard ITS2A/ITS2B primers. The size of the amplicon was 301 bp, which was specific to \u003cem\u003eAn. squamosus\u003c/em\u003e against all the other African \u003cem\u003eAnopheles\u003c/em\u003e species evaluated within this study. Other \u003cem\u003eAnopheles\u003c/em\u003e species routinely caught in collections with \u003cem\u003eAn. squamosus\u003c/em\u003e produce different sized ITS2 amplicons from the ITS2A and ITS2B primers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For example, members of the \u003cem\u003eAn. gambiae\u003c/em\u003e complex produce an amplicon near 600 bp, while members of the \u003cem\u003eAn. funestus\u003c/em\u003e group produce an amplicon of 850 bp. \u003cem\u003eAnopheles rufipes\u003c/em\u003e, \u003cem\u003eAn. maculipalpis\u003c/em\u003e, and \u003cem\u003eAn. pretoriensis\u003c/em\u003e generate indistinguishable amplicons of approximately 500 bp when using the ITS2A and ITS2B primers. The reaction could be further optimized using a touchdown PCR to reduce the unspecific binding and increase specificity.\u003c/p\u003e\u003cp\u003eWith an understanding of anopheline community composition, species-specific targeted and effective methods of control may be better implemented. For example, indoor residual spraying and long-lasting insecticidal netting are widely used in malaria-endemic areas, as they have been most effective at targeting the behavior of endophilic and endophagic primary vectors (WHO 2023, Bhatt 2015). These efforts have resulted in reductions of principal malaria vectors such as \u003cem\u003eAnopheles gambiae s.l.\u003c/em\u003e in Kenya, Tanzania, and Zambia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, these intervention methods do not consider foraging and resting behaviors of secondary vectors that may be transmitting malaria at low levels [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis new \u003cem\u003eAn. squamosus-\u003c/em\u003especific assay was evaluated on specimens from a relatively narrow geographic range of the known \u003cem\u003eAn. squamosus\u003c/em\u003e distribution; Zambia and South Africa. Therefore, this assay should be further evaluated on \u003cem\u003eAn. squamosus\u003c/em\u003e from a much broader geographic region to assess accuracy and robustness, especially for geographically isolated populations such as Madagascar, where \u003cem\u003eAn. squamosus\u003c/em\u003e is abundant [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, Coetzee [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] suggested that \u003cem\u003eAn. squamosus\u003c/em\u003e is likely to be a species complex based on chromosome inversion polymorphisms. If this is true, the ITS2 PCR alone may not be sufficient to delineate species within this complex and may need further refinement, as has been demonstrated for the \u003cem\u003eAn. gambiae\u003c/em\u003e complex [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, \u003cem\u003eAn. cydippis\u003c/em\u003e and additional species that are sympatric to \u003cem\u003eAn. squamosus\u003c/em\u003e should be evaluated to ensure that this new ITS2 primer set is indeed species-specific.\u003c/p\u003e\u003cp\u003eThis assay allows the more robust surveillance of \u003cem\u003eAn. squamosus\u003c/em\u003e in Africa. Though cryptic with other taxa as adults, this species can be morphologically differentiated as 4th instar larvae. Unfortunately, earlier instars may not have developed distinguishable features. In addition, common methods of trapping adult anopheline such as CDC miniature light traps may damage specimens and lose key features that are used to distinguish them morphologically, such as wings, legs, and scales [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. DNA-based tools, such as the ITS2 assay, avoid these challenges, providing robust identification regardless of life stage or specimen quality with minimal tissue input [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis assay opens new opportunities for investigating the role of \u003cem\u003eAn. squamosus\u003c/em\u003e in malaria transmission. By exploiting the genetics of vectors and building resources to study them, we can identify genes that can be tied to vector competence. For example, in \u003cem\u003eAnopheles arabiensis\u003c/em\u003e, there was an identified genetic component that influenced its host choice and behavior [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Alleles linked to the 2Rb and/or 3Ra inversions were linked to cattle-feeding preferences in \u003cem\u003eAn. arabiensis.\u003c/em\u003e The link between genetics and host preference is vital to further assess how \u003cem\u003eAn. squamosus\u003c/em\u003e is contributing to disease transmission with a new and accurate method of identification of this species.\u003c/p\u003e\u003cp\u003eThe development of this ITS2 primer for \u003cem\u003eAn. squamosus\u003c/em\u003e, using the methods detailed here, can be applied to other understudied species and secondary vectors of malaria that do not have reliable methods of identification. For example, \u003cem\u003eAn. pharoensis\u003c/em\u003e is also found to be infected with \u003cem\u003eP. falciparum\u003c/em\u003e, but does not reliably amplify in currently available ITS2. This assay shows repeatability, reproducibility, specificity, and a high limit of detection (\u0026gt;\u0026thinsp;95% amplification of biological replicates used), showing these primer sets provide a robust method for the detection of \u003cem\u003eAn. squamosus.\u003c/em\u003e This framework will allow for accurate and robust monitoring of secondary vectors of malaria.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eThe study involves collection of mosquito specimen within individual households in Zambia as part of the project that had been approved by National Health Research Authority, Zambia: Approval No: NHRA00016/18/08/2021.\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\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eWe acknowledge funding support from the United States National Institute of Health as part of the International Centers of Excellence for Malaria Research (2U19AI089680), Small Grant Program (R03AI178041), and T32 Institutional Training Grant (T32AI0074717). Additional support was provided by the United States Department of Agriculture National Institute of Food and Agriculture Multistate Hatch Project (1025565 and 7007941), the Bloomberg Philanthropies, the Johns Hopkins Malaria Research Institute, the College of Agricultural and Life Sciences Dean\u0026rsquo;s Award at the University of Florida, and Global Fellows Program at the University of Florida International Center.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVTN and YL conceptualized the study. VTN, BCB, and YL provided methodology. VTN, RLMNA, BCB, DSD, MAR, KS, LER, and YL conducted investigation. VTN curated data. VTN, RLMNA, and YL conducted data analysis and data visualization. VTN, RLMNA, ES, DEN, and YL acquired funding and provided resources for the study. YL carried out project administration and supervision. VTN, DSD, and YL wrote original draft. All authors contributed to review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the entomology staff at Macha Research Trust for assisting with mosquito collection and sample processing. We thank Mr. Sangwoo Seok (University of Florida) for reviewing our manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. Global technical strategy for malaria 2016\u0026ndash;2030 [Internet]. World Health Organization. 2015 [cited 2024 Dec 9]. 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J Vector Ecol. 2015;40:16\u0026ndash;27.\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":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anopheles squamosus, molecular diagnosis, internal transcribed spacer, mosquito, malaria","lastPublishedDoi":"10.21203/rs.3.rs-7278578/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7278578/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles squamosus\u003c/em\u003e is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e. Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish \u003cem\u003eAn. squamosus\u003c/em\u003e from morphologically similar \u003cem\u003eAnopheles\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultiple alignments of previously published ITS2 contig sequences in NCBI from \u003cem\u003eAn. squamosus\u003c/em\u003e and \u003cem\u003eAn.\u003c/em\u003e species 11 and 15, were used to identify candidate ITS2 regions for primer design. We evaluated six sets of primers overall for specificity of species identification. The one set with \u003cem\u003eAn. squamosus\u003c/em\u003e species-specific amplification was tested using 78 specimens from Zambia and South Africa.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA new assay consisting of a forward (ITS2-ASQ-R10, 5\u0026rsquo;-CCC TCG AAG GGT GCT GTG-3\u0026rsquo;) and reverse (ITS2-ASQ-R10 5\u0026rsquo;-AAT CCA CGG TGT GAT GGC-3\u0026rsquo;) primer reliably (\u0026gt;\u0026thinsp;94.8%) amplified an ITS2 fragment of 301bp length for \u003cem\u003eAn. squamosus\u003c/em\u003e. The \u003cem\u003eAn. squamosus-\u003c/em\u003especific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe development of this robust PCR assay for \u003cem\u003eAn. squamosus\u003c/em\u003e is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. In addition, investigating host association and malaria transmission can be facilitated with this assay by correctly identifying \u003cem\u003eAn. squamosus.\u003c/em\u003e Applying genomic tools to correctly identified anopheline species may lead to the discovery of genetic factors that influence its behavior and new innovations in malaria elimination.\u003c/p\u003e","manuscriptTitle":"Molecular identification of Anopheles squamosus (Diptera: Culicidae) using internal transcribed spacer 2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 10:39:10","doi":"10.21203/rs.3.rs-7278578/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-06T18:20:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T13:41:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-26T09:59:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222520809523701139978515029707392069692","date":"2025-08-12T04:44:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309428229122777160187080669986725688281","date":"2025-08-11T15:34:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-11T14:43:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T08:17:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-05T08:16:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-08-02T13:23:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ffe7d52-fbc1-457f-8e01-f1e3f5ddf2af","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-7278578","link":"https://doi.org/10.1186/s12936-025-05600-6","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2025-10-16 15:56:59","publishedOnDateReadable":"October 16th, 2025"},"versionCreatedAt":"2025-08-19 10:39:10","video":"","vorDoi":"10.1186/s12936-025-05600-6","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05600-6","workflowStages":[]},"version":"v1","identity":"rs-7278578","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7278578","identity":"rs-7278578","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}