The identification of sex in larvae of insects is usually challenging or even impossible, while in adults the sexual dimorphism is usually evident. Here, we used copy number analysis to develop a method of sex detection in Colorado potato beetle (Leptinotarsa decemlineata), which has an X0 sex determination system. The X linked gene LdVssc and autosomal gene LdUBE3B were identified as appropriate target and reference loci, respectively. The copy numbers (CNV) of LdVssc in males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BAddPCR = 0.709 and BAqPCR = 0.683) with 100% ability to distinguish females from males. The use of qPCR-based sex detection in a broad collection of 448 random CPB adults showed a perfect association (Phi = 1.0, p < 0.05) with the true sexes of adults, with mean CNV in females of 2.032 (SD = 0.227) and 0.989 in males (SD = 0.147). In the collection of 50 random 4th instar larvae, 27 females and 23 males were identified, consistent with the expected 1:1 sex ratio (p = 0.689). The method is suitable for sexing in all stages of ontogenesis. The optimal cost-effective application of the method in large populations requires the DNA extraction using CTAB, the qPCR assay in one biological replicate and three technical replicates of each marker, and the use of one randomly chosen male per run to calibrate calculation of CNV.

Department of Genetics and Breeding Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 16500 Prague 6 Suchdol Czech Republic

Department of Potato Protection Potato Research Institute Havlíčkův Brod Ltd Dobrovského 2366 58001 Havlíčkův Brod Czech Republic

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Mora C, et al. How many species are there on Earth and in the ocean? PLoS Biol. 2011;9(8):e1001127. doi: 10.1371/journal.pbio.1001127. PubMed DOI PMC

Jarne P, Auld JR. Animals mix it up too: The distribution of self-fertilization among hermaphroditic animals. Evolution. 2006;60(9):1816–1824. doi: 10.1111/j.0014-3820.2006.tb00525.x. PubMed DOI

Normark BB. The evolution of alternative genetic systems in insects. Annu. Rev. Entomol. 2003;48:397–423. doi: 10.1146/annurev.ento.48.091801.112703. PubMed DOI

Blackmon H, Ross L, Bachtrog D. Sex determination, sex chromosomes, and karyotype evolution in insects. J. Hered. 2017;108(1):78–93. doi: 10.1093/jhered/esw047. PubMed DOI PMC

Gempe T, Beye M. Function and evolution of sex determination mechanisms, genes and pathways in insects. BioEssays. 2010;33:52–60. doi: 10.1002/bies.201000043. PubMed DOI PMC

Bull JJ. The Evolution of Sex Determining Mechanisms. Benjamin/Cummings Publishing Company; 1983.

Jaquiéry J, et al. Accelerated evolution of sex chromosomes in aphids, an X0 system. Mol. Biol. Evol. 2012;29(2):837–847. doi: 10.1093/molbev/msr252. PubMed DOI

Sawanth SK, et al. The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects. J. Biosci. 2016;41(2):283–294. doi: 10.1007/s12038-016-9609-x. PubMed DOI

Graham P, Penn JKM, Schedl P. Masters change, slaves remain. BioEssays. 2003;25:1–4. doi: 10.1002/bies.10207. PubMed DOI

Blackmon H, Demuth JP. Genomic origins of insect sex chromosomes. Curr. Opin. Insect Sci. 2015;7:45–50. doi: 10.1016/j.cois.2014.12.003. PubMed DOI

Roderick GK, et al. Sperm precedence in Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae): Temporal variation assessed by neutral markers. Ann. Entomol. Soc. Am. 2003;96(5):631–636. doi: 10.1603/0013-8746(2003)096[0631:SPICPB]2.0.CO;2. DOI

Hsiao TH, Hsiao C. A chromosomal analysis of Leptinotarsa and Labidomera species (Coleoptera: Chrysomelidae) Genetica. 1983;60:139–150. doi: 10.1007/BF00127500. DOI

Petitpierre E, Segarra C, Yadav JS, Virkki N. Chromosome Numbers and Meioformulae of Chrysomelidae. In: Jolivet P, Petitpierre E, Hsiao TH, editors. Biology of Chrysomelidae. Springer; 1988. pp. 161–186.

Blackmon H, Demuth JP. Estimating tempo and mode of Y chromosome turnover: Explaining Y chromosome loss with the fragile Y hypothesis. Genetics. 2014;197:561–572. doi: 10.1534/genetics.114.164269. PubMed DOI PMC

Blackmon H, Demuth JP. The fragile Y hypothesis: Y-chromosome aneuploidy as a selective pressure in sex chromosome and meiotic mechanism evolution. BioEssays. 2015;37:942–950. doi: 10.1002/bies.201500040. PubMed DOI

Khidhir AQS, Mustafa RA. Illustrate the morphologic characters of Colorado potato beetle, Leptinotarsa decemlineata say, 1824 (Coleoptera: Chrysomelidae) J. Entomol. Zool. Stud. 2018;6:456–462.

Ledón-Rettig CC, Zattara EE, Moczek AP. Asymmetric interactions between doublesex and tissue and sex-specific target genes mediate sexual dimorphism in beetles. Nat. Commun. 2017;8:14593. doi: 10.1038/ncomms14593. PubMed DOI PMC

Flaherty L, Régnière J, Sweeney J. Number of instars and sexual dimorphism of Tetropium fuscum (Coleoptera: Cerambycidae) larvae determined by maximum likelihood. Can. Entomol. 2012;144:720–726. doi: 10.4039/tce.2012.60. DOI

Moczek AP, Nijhout HF. A method for sexing final instar larvae of the genus Onthophagus Latreille (Coleoptera: Scarabaeidae) Coleopts. Bull. 2002;56(2):279–284. doi: 10.1649/0010-065X(2002)056[0279:AMFSFI]2.0.CO;2. DOI

Feuk L, Carson AR, Scherer SW. Structural variation in the human genome. Nat. Rev. Genet. 2006;7:85–97. doi: 10.1038/nrg1767. PubMed DOI

Hawthorne DJ. AFLP-based genetic linkage map of the Colorado potato beetle Leptinotarsa decemlineata: Sex chromosomes and a pyrethroid-resistance candidate gene. Genetics. 2001;158(2):695–700. doi: 10.1093/genetics/158.2.695. PubMed DOI PMC

Itokawa K, et al. Highthroughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations. PLoS Neglect. Trop. D. 2019;13(11):e0007818. doi: 10.1371/journal.pntd.0007818. PubMed DOI PMC

Silva JJ, Scott JG. Conservation of the voltage-sensitive sodium channel protein within the Insecta. Insect Mol. Biol. 2020;29(1):9–18. doi: 10.1111/imb.12605. PubMed DOI

Kim HS, et al. BeetleBase in 2010: Revisions to provide comprehensive genomic information for Tribolium castaneum. Nucleic Acids Res. 2010;2010(38):D437–442. doi: 10.1093/nar/gkp807. PubMed DOI PMC

Martins WFS, et al. Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus. Sci. Rep. 2017;7:5821. doi: 10.1038/s41598-017-06080-8. PubMed DOI PMC

Chen H, et al. Evaluation of five methods for total dna extraction from western corn rootworm beetles. PLoS ONE. 2010;5(8):e11963. doi: 10.1371/journal.pone.0011963. PubMed DOI PMC

Milligan BG. Total DNA Isolation. In: Hoelzel AR, editor. Molecular Genetic Analysis of Population: A Practical Approach. Oxford University Press; 1998. pp. 29–64.

Rozen S, Skaletsky H. Primer3 on the WWW for General Users and for Biologist Programmers. In: Misener S, Krawetz SA, editors. Bioinformatics Methods and Protocols. Humana Press; 1999. pp. 365–386. PubMed

Larionov A, Krause A, Miller W. A standard curve-based method for relative real time PCR data processing. BMC Bioinformatics. 2005;6(1):1–16. doi: 10.1186/1471-2105-6-62. PubMed DOI PMC

Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30(9):e36. doi: 10.1093/nar/30.9.e36. PubMed DOI PMC

Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999;41:95–98.

Zhang C, Mapes B, Soden B. Bimodality in tropical water vapour. Q. J. Roy. Meteor. Soc. 2003;129(594):2847–2866. doi: 10.1256/qj.02.166. DOI

R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria (2021).

Svec D, et al. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessment. Biomol. Detect. Quantif. 2015;3:9–16. doi: 10.1016/j.bdq.2015.01.005. PubMed DOI PMC

Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 2002;29:23–39. doi: 10.1677/jme.0.0290023. PubMed DOI

Baker JR. Development and sexual dimorphism of larvae of the bee genus Coelioxys. J. Kansas Entomol. Soc. 1971;44(2):225–235.

Sánchez E, Castillo D, Liria J. Pupal shape and size dimorphism in Aedes albopictus (Skuse, 1894) (Diptera: Culicidae) J. Threat. Taxa. 2017;9(6):10314–10319. doi: 10.11609/jott.3059.9.6.10314-10319. DOI

Ramya RS, Mohan M, Sunil J. A simple method for sexing live larvae of pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) Anim. Biol. 2020;70:97–100. doi: 10.1163/15707563-20191136. DOI

Kim HJ, et al. Application of DNA-based genotyping techniques for the detection of kdr-like pyrethroid resistance in field populations of Colorado potato beetle. Pestic. Biochem. Phys. 2005;81:85–96. doi: 10.1016/j.pestbp.2004.10.002. DOI

Lee SH, et al. Molecular analysis of kdr-like resistance in a permethrin-resistant strain of Colorado potato beetle. Pestic. Biochem. Phys. 1999;63:63–75. doi: 10.1006/pest.1999.2395. DOI

Rinkevich FD. Multiple evolutionary origins of knockdown resistance (kdr) in pyrethroid-resistant Colorado potato beetle Leptinotarsa decemlineata. Pestic. Biochem. Phys. 2012;104:192–200. doi: 10.1016/j.pestbp.2012.08.001. DOI

Martins AJ. Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti. Evol. Med. Public Health. 2013;2013(1):148–160. doi: 10.1093/emph/eot012. PubMed DOI PMC

Investigation of genetic diversity and polyandry of Leptinotarsa decemlineata using X-linked microsatellite markers

Bias in sex ratios and polyandry rate in reproduction of Leptinotarsa decemlineata