Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches

Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 28 February 2026 V1 Latest version Share on Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches Authors : Arvind Sharma , Benjamin Faustino , Isaac Hinne 0000-0001-5959-1958 , Jakub Wudarski , Saransh Beniwal , Nurul Hamidi , Kyla Ercit , Sanjay Basu 0000-0003-2098-7423 , Malimba Lisulo , Mattia Poletto , Kelly Matzen , and Monika Gulia Nuss 0000-0001-7095-1037 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177226397.75634278/v1 159 views 73 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Accurate sex identification of immature ticks is essential for understanding sex-specific ecological dynamics, pathogen transmission, and reproductive biology. However, tick larvae and nymphs lack morphological sexual dimorphisms, limiting the studies. Here, we report the development and validation of copy number variation (CNV)-based molecular sexing approach for two important hard tick species, Ixodes scapularis, a major public health vector of human pathogens, and Rhipicephalus microplus, a pest responsible for significant economic losses in cattle industry. Using newly published chromosomal-level genome assemblies and whole-genome resequencing data, we identified female-enriched CNVs in two genes, calcium/calmodulin-dependent 3’,5’-cyclic nucleotide phosphodiesterase 1A-like (NPD) and rap guanine nucleotide exchange factor 2-like (RAPGEF2), and developed SYBR-Green based and probe-based qPCR, and droplet digital PCR (ddPCR) assays using DNA extracted non-destructively from a single leg, preserving ticks for continued feeding, molting, and behavioral analysis. In I. scapularis, we were able to assign the sex of 87% of nymphs based on results from at least two molecular tests, and these assignments were confirmed when the nymphs later molted into adults. In R. microplus, two qPCR assays revealed clear, sex-linked differences in gene copy number, despite the species having an XX: XO sex system. Together, these findings indicate that CNV-based markers are a reliable and widely applicable method for sex determination in immature ticks. Supplementary Material File (sharma_cnv_ecoevo.docx) Download 2.24 MB Information & Authors Information Version history V1 Version 1 28 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords genetics invertebrate laboratory method development Authors Affiliations Arvind Sharma University of Nevada Reno View all articles by this author Benjamin Faustino University of Nevada Reno View all articles by this author Isaac Hinne 0000-0001-5959-1958 University of Nevada Reno View all articles by this author Jakub Wudarski University of Nevada Reno View all articles by this author Saransh Beniwal University of Nevada Reno View all articles by this author Nurul Hamidi Oxitec Ltd View all articles by this author Kyla Ercit Oxitec Ltd View all articles by this author Sanjay Basu 0000-0003-2098-7423 Oxitec Ltd View all articles by this author Malimba Lisulo The University of Edinburgh The Roslin Institute View all articles by this author Mattia Poletto Oxitec Ltd View all articles by this author Kelly Matzen Oxitec Ltd View all articles by this author Monika Gulia Nuss 0000-0001-7095-1037 [email protected] University of Nevada Reno View all articles by this author Metrics & Citations Metrics Article Usage 159 views 73 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Arvind Sharma, Benjamin Faustino, Isaac Hinne, et al. Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches. Authorea . 28 February 2026. DOI: https://doi.org/10.22541/au.177226397.75634278/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.177226397.75634278/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fde7d887fddad07',t:'MTc3OTE0NTM1NQ=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();