Determining the age of free-living insects, particularly of blood-sucking species, is important for human health because such knowledge critically influences the estimates of biting frequency and vectoring ability. Genetic age determination is currently not available. Pteridines gradually accumulate in the eyes of insects and their concentrations is the prevailing method. Despite of their stability, published extractions differ considerably, including for standards, for mixtures of pteridines and even for light conditions. This methodological inconsistency among studies is likely to influence age estimates severely and to hamper their comparability. Therefore we reviewed methodological steps across 106 studies to identify methodological denominators and results across studies. Second, we experimentally test how different pteridines vary in their age calibration curves in, common bed (Cimex lectularius) and bat bugs (C. pipistrelli). Here we show that the accumulation of particular pteridines varied between a) different populations and b) rearing temperatures but not c) with the impact of light conditions during extraction or d) the type of blood consumed by the bugs. To optimize the extraction of pteridines and measuring concentrations, we recommend the simultaneous measurement of more than one standard and subsequently to select those that show consistent changes over time to differentiate among age cohorts.

Czech University of Life Sciences Prague Faculty of Environmental Science Department of Ecology Prague 16521 Czech Republic

Masaryk University Faculty of Sciences Department of Botany and Zoology Brno 61137 Czech Republic

Masaryk University Research Centre for Toxic Compounds in the Environment Brno 62500 Czech Republic

Technische Universität Dresden Department of Biology Applied Zoology Dresden 01069 Germany

Universität Bayreuth Animal Ecology 1 Animal Population Ecology Bayreuth 95440 Germany

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Lehane, M. J. Feeding preferences of bloodsucking insects in The Biology of Blood-sucking in Insects 12–24 (Cambridge University Press, 2005).

Detinova TS. Age structure of insect populations of medical importance. Annu. Rev. Entomol. 1968;13:427–450.

Tyndale-Biscoe M. Age-grading methods in adult insects: a review. Bull. Entomol. Res. 1984;74:341–377.

McGraw JB, Caswell H. Estimation of individual fitness from life-history data. Am. Nat. 1996;147:47–64.

Saunders DS. Determination of physiological age for female Glossina morsitans. Nature. 1960;186:651–651. PubMed

Hayes EJ, Wall R. Age-grading adult insects: a review of techniques. Physiol. Entomol. 1999;24:1–10.

Robertson CW. The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes. J. Morphol. 1936;59:351–399.

Neville AC. Daily growth layers for determining the age of grasshopper populations. Oikos. 1963;14:1–8.

Schlein J, Gratz NG. Age determination of some flies and mosquitos by daily growth layers of skeletal apodemes. Bull. World Health Organ. 1972;47:71–76. PubMed PMC

Bainbridge SP, Bownes M. Staging the metamorphosis of Drosophila melanogaster. J. Embryol. Exp. Morphol. 1981;66:57–80. PubMed

Tyndale-Biscoe M, Kitching RL. Cuticular bands as age criteria in the sheep blowfly Lucilia cuprina (Wied.) (Diptera, Calliphoridae) Bull. Entomol. Res. 1974;64:161–174.

Reinhardt K, Köhler G, Webb S, Childs D. Field mating rate of female meadow grasshoppers, Chorthippus parallelus, estimated from sperm counts. Ecol. Entomol. 2007;32:637–642.

Detinova, T. S., Bertram, D. S. & World health organization. Age-grouping methods in diptera of medical importance, with special reference to some vectors of malaria / T. S. Detinova; [with] an Annex on the ovary and ovarioles of mosquitos (with glossary) by D. S. Bertram https://apps.who.int/iris/handle/10665/41724 (1962). PubMed

Moon RD, Krafsur ES. Pterin quantity and gonotrophic stage as indicators of age in Musca autumnalis (Diptera: Muscidae) J. Med. Entomol. 1995;32:673–684. PubMed

Ziegler I, Harmsen R. The biology of pteridines in insects. Adv. In Insect Phys. 1970;6:139–203.

Tomic-Carruthers N, Robacker DC, Mangan RL. Identification and age-dependance of pteridines in the head of adult mexican fruit fly, Anastrepha ludens. J. Insect Physiol. 1996;42:359–366.

Brown, D. J. Fused pyrimidines: Pteridines in Chemistry of Heterocyclic Compounds (ed. Brown, D. J.) 1–730 (Wiley, 1988).

Bel Y, Porcar M, Socha R, Němec V, Ferré J. Analysis of pteridines in Pyrrhocoris apterus (L.) (Heteroptera, Pyrrhocoridae) during development and in body-color mutants. Arch. Insect Biochem. Physiol. 1997;34:83–98.

Pfleiderer, W. Pteridines in Comprehensive Heterocyclic Chemistry 263–327 (Elsevier, 1984).

Blau, N. & Thöny, B. Pterins and related enzymes in Laboratory Guide to the Methods in Biochemical Genetics 665–701 (Springer Berlin Heidelberg, 2008).

Hevener KE, et al. Structural studies of pterin-based inhibitors of dihydropteroate synthase. J. Med. Chem. 2010;53:166–177. PubMed PMC

Penilla RP, Rodríguez MH, López AD, Viader-Salvadó JM, Sánchez CN. Pteridine concentrations differ between insectary-reared and field-collected Anopheles albimanus mosquitoes of the same physiological age. Med. Vet. Entomol. 2002;16:225–234. PubMed

Bernhardt V, et al. Quantitative pteridine fluorescence analysis: A possible age-grading technique for the adult stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) J. Insect Physiol. 2017;98:356–359. PubMed

Harmsen R. Identification of fluorescing and u.v. absorbing substances in Pieris brassicae L. J. Insect Physiol. 1966;12:23–30.

Krafsur ES, Rosales AL, Robison-Cox JF, Turner JP. Age structure of horn fly (Diptera: Muscidae) populations estimated by pterin concentrations. J. Med. Entomol. 1992;29:678–686. PubMed

Tomic-Carruthers N, Mangan R, Carruthers R. Age estimation of mexican fruit fly (Diptera: Tephritidae) based on accumulation of pterins. J. Econ. Entomol. 2002;95:1319–1325. PubMed

Parravani A, et al. Seasonal abundance of the stable fly Stomoxys calcitrans in southwest England. Med. Vet. Entomol. 2019;33:485–490. PubMed

Pfleiderer, W. Bicyclic 6-6 systems: Pteridines in Comprehensive Heterocyclic Chemistry II 679–736 (Elsevier, 1996).

Mail TS, Chadwick J, Lehane MJ. Determining the age of adults of Stomoxys calcitrans (L.) (Diptera: Muscidae) Bull. Entomol. Res. 1983;73:501–525.

Zhu GH, Ye GY, Li K, Hu C, Xu XH. Determining the age of adult flesh flies, Boettcherisca peregrina, using pteridine fluorescence. Med. Vet. Entomol. 2013;27:59–63. PubMed

Lehane MJ, Mail TS. Determining the age of adult male and female Glossina morsitans morsitans using a new technique. Ecol. Entomol. 1985;10:219–224.

McIntyre GS, Gooding RH. Pteridine accumulation in Musca domestica. J. Insect Physiol. 1995;41:357–368.

Nisshanthini, S. D. Evaluation of anticancer efficacy of 6-propinyl pterin characterized from cyanide untilizing bacterium Bacillus subtilis. (Bharathiar University, 2012).

Handschin G. Entwicklungs- und organspezifisches verteilungsmuster der pterine bei einem wildstamm und bei der mutante rosy2 von Drosophila melanogaster. Dev. Biol. 1961;3:115–139. PubMed

Mail TS, Lehane MJ. Characterisation of pigments in the head capsule of the adult stablefly Stomoxys calcitrans. Entomol. Exp. Appl. 1988;46:125–131.

Lardeux F, Ung A, Chebret M. Spectrofluorometers are not adequate for aging Aedes and Culex (Diptera: Culicidae) using pteridine fluorescence. J. Med. Entomol. 2000;37:769–773. PubMed

Bartel AH, Hudson BW, Craig R. Pteridines in the milkweed bug, Oncopeltus fasciatus (Dallas): I. Identification and localization. J. Insect Physiol. 1958;2:348–354.

Descimon H. Les ptérines des Pieridae (Lepidoptera) et leur biosynthèse: I — Identification des principales ptérines de Colias croceus (Fourcroy) et de quelques autres espèces de Pieridae. Biochimie. 1971;53:407–418. PubMed

Ferré J, Silva FJ, Real MD, Ménsua JL. Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster. Biochem. Genet. 1986;24:545–569. PubMed

Porcar M, Bel Y, Socha R, Němec V, Ferré J. Identification of pteridines in the firebug, Pyrrhocoris apterus (L.) (Heteroptera, Pyrrhocoridae) by high-performance liquid chromatography. J. Chromatogr. A. 1996;724:193–197.

Merlini L, Nasini G. Insect pigments—IV. Pteridines and colour in some Hemiptera. J. Insect Physiol. 1966;12:123–127.

Murata, S., Ichinose, H. & Urano, F. Tetrahydrobiopterin and related biologically important pterins in Bioactive Heterocycles II 127–171 (Springer Berlin Heidelberg, 2007).

Krajíček J, et al. Capillary electrophoresis of pterin derivatives responsible for the warning coloration of Heteroptera. J. Chromatogr. A. 2014;1336:94–100. PubMed

Becker E. Über das pterinpigment bei insekten und die färbung und zeichnung von vespa im besonderem. Zeitschrift für Morphol. und Ökologie der Tiere. 1937;32:672–751.

Egelhaaf A. Photolabile fluoreszenzstoffe bei Ephestia kuhniella. Naturwissenschaften. 1956;43:309.

Edalat H, Akhoundi M, Basseri H. Age-dependance of pteridines in the malaria vector, Anopheles stephensi. Pteridines. 2017;28:157–161.

Ikan R, Ishay J. Pteridines and purines of the queens of the oriental hornet, Vespa orientalis F. J. Insect Physiol. 1967;13:159–162.

Robson S, Crozier RH. An evaluation of two biochemical methods of age determination in insects (pteridines and lipofuscins) using the ant Polyrhachis sexpinosa Latrielle (Hymenoptera: Formicidae) Aust. J. Entomol. 2009;48:102–106.

Martín Tornero E, Durán Merás I, Espinosa-Mansilla A. HPLC determination of serum pteridine pattern as biomarkers. Talanta. 2014;128:319–326. PubMed

Martín-Tornero E, Gómez DG, Durán-Merás I, Espinosa-Mansilla A. Development of an HPLC-MS method for the determination of natural pteridines in tomato samples. Anal. Methods. 2016;8:6404–6414.

Basu P, Burgmayer SJN. Pterin chemistry and its relationship to the molybdenum cofactor. Coord. Chem. Rev. 2011;255:1016–1038. PubMed PMC

Murr C, et al. Neopterin is an independent prognostic variable in females with breast cancer. Clin. Chem. 1999;45:1998–2004. PubMed

Plata-Nazar K, et al. Reference standard of serum neopterin concentration in healthy children. Pteridines. 2007;18:19–24.

Lehane MJ, Chadwick J, Howe MA, Mail TS. Improvements in the pteridine method for determining age in adult male and female Stomoxys calcitrans (Diptera: Muscidae) J. Econ. Entomol. 1986;79:1714–1719.

Vargas-Lowman A, et al. Cooption of the pteridine biosynthesis pathway underlies the diversification of embryonic colors in water striders. Proc. Natl. Acad. Sci. 2019;116:19046–19054. doi: 10.1073/pnas.1908316116. PubMed DOI PMC

Robson SK, Vickers M, Blows MW, Crozier RH. Age determination in individual wild-caught Drosophila serrata using pteridine concentration. J. Exp. Biol. 2006;209:3155–3163. PubMed

Sheehan MJ, Jinn J, Tibbetts EA. Coevolution of visual signals and eye morphology in polistes paper wasps. Biol. Lett. 2014;10:20140254. doi: 10.1098/rsbl.2014.0254. PubMed DOI PMC

Perl CD, Niven JE. Differential scaling within an insect compound eye. Biol. Lett. 2016;12:20160042. doi: 10.1242/jeb.082818. PubMed DOI PMC

Tomšíková H, Tomšík P, Solich P, Nováková L. Determination of pteridines in biological samples with an emphasis on their stability. Bioanalysis. 2013;5:2307–2326. PubMed

Zvarik M, Martinicky D, Hunakova L, Sikurova L. Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals. J. Photochem. Photobiol. B Biol. 2015;153:191–197. PubMed

Bel Y, Ferré J. Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation in Drosophila melanogaster. Biochem. Genet. 1989;27:59–76. PubMed

Hudson BW, Bartel AH, Craig R. Pteridines in the milkweed bug, Oncopeltus fasciatus (Dallas)—II: Quantitative determination of pteridine content of tissues during growth. J. Insect Physiol. 1959;3:63–73.

Noble RM, Walker PW. Pteridine compounds in adults of the pink-spotted bollworm, Pectinophora scutigera. Entomol. Exp. Appl. 1990;57:77–83.

Balvín O, Munclinger P, Kratochvíl L, Vilímová J. Mitochondrial DNA and morphology show independent evolutionary histories of bedbug Cimex lectularius (Heteroptera: Cimicidae) on bats and humans. Parasitol. Res. 2012;111:457–469. PubMed

Roth S, et al. Bedbugs evolved before their bat hosts and did not co-speciate with ancient humans. Curr. Biol. 2019;29:1847–1853.e4. doi: 10.1016/j.cub.2019.04.048. PubMed DOI

Wawrocka K, Bartonička T. Two different lineages of bedbug (Cimex lectularius) reflected in host specificity. Parasitol. Res. 2013;112:3897–3904. PubMed

Aak A, Rukke BA. Bed bugs, their blood sources and life history parameters: a comparison of artificial and natural feeding. Med. Vet. Entomol. 2014;28:50–59. PubMed

Ferré, J., Silva, F. J., Real, M. D. & Ménsua, J. L. Comparative study of the eye colour mutants of Drosophila melanogaster: quantification of the eye-pigment and related metabolites in Chemistry and Biology of Pteridines: Proceedings (eds. Kisliuk, R. L. & Brown, G. M.) 669–673 (Elsevier/North-Holland, 1979).

Allegri G, et al. Determination of six pterins in urine by LC-MS/MS. Bioanalysis. 2012;4:1739–1746. PubMed

Bílková Z, Adámková M, Albrecht T, Šimek Z. Determination of testosterone and corticosterone in feathers using liquid chromatography-mass spectrometry. J. Chromatogr. A. 2019;1590:96–103. PubMed

R Core Team. R: A language and environment for statistical computing https://www.r-project.org (2019).

Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team. nlme: Linear and nonlinear mixed effects models https://cran.r-project.org/package=nlme (2019).

Wickham, H. ggplot2: Elegant graphics for data analysis https://ggplot2.tidyverse.org (2016).

StatSoft Inc. STATISTICA (data analysis software system) http://www.statsoft.com (2013).

Age-related mating rates among ecologically distinct lineages of bedbugs, Cimex lectularius