Male-to-female transfer of 5-hydroxytryptophan glucoside during mating in Zygaena filipendulae (Lepidoptera) Mika Zagrobelny a, b, * , Mohammed Saddik Motawia a, b , Carl Erik Olsen a, b, c , Søren Bak a, b , Birger Lindberg Møller a, b, c a Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark b Villum Research Center “Plant Plasticity”, Denmark c Novo Nordisk Foundation Center for BioSustainability, Denmark article info Article history: Received 11 July 2013 Received in revised form 19 August 2013 Accepted 24 August 2013 Keywords: 5-Hydroxytryptophan glucoside Zygaena filipendulae Insect Mating Nuptial gift Spermatophore abstract Zygaena filipendulae accumulates the cyanogenic glucosides linamarin and lotaustralin by larval sequestration from the food plant or de novo biosynthesis. We have previously demonstrated that the Z. filipendulae male transfers linamarin and lotaustralin to the female in the course of mating. In this study we report the additional transfer of 5-hydroxytryptophan glucoside (5-(b-D-glucopyranosyloxy)-L- Tryptophan) from the Z. filipendulae male internal genitalia to the female spermatophore around 5 h into the mating process. 5-Hydroxytryptophan glucoside is present in the virgin male internal genitalia, and production continues during the early phase of mating. Following initiation of 5-hydroxytryptophan glucoside transfer to the female, the amount in male internal genitalia is drastically reduced until af- ter mating where it is slowly replenished. For unambiguous structural identification, 5- hydroxytryptophan glucoside was chemically synthesized and used as an authentic standard. The bio- logical function of 5-hydroxytryptophan glucoside remains to be established, although we have in- dications that it may be involved in inducing the female to stay in copula and delay egg-laying to prevent re-mating of the female. To our knowledge 5-hydroxytryptophan glucoside has not previously been reported present in animal tissues. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Zygaena filipendulae (Zygaenidae, Lepidoptera) are brightly colored day-flying moths, known for their high content of cyano- genic glucosides which they use as defense chemicals (Zagrobelny and Møller, 2011). Z. filipendulae contains the two cyanogenic glu- cosides linamarin and lotaustralin which they may sequester from their food plant Lotus corniculatus (Fabaceae) as well as de novo biosynthesize from the amino acids valine and isoleucine, respec- tively (Davis and Nahrstedt, 1982, 1987; Jensen et al., 2011). In addition to serving as defense compounds, the two cyanogenic glucosides have previously been shown to play an intimate role in the mating process of the moth, in the course of which the male transfers the two compounds to the female (Zagrobelny et al., 2007). In the field, adult Z. filipendulae females often perch on purple flowers, preferably Knautia arvensis (Dipsacaceae), waiting for males to approach them for mating. They emit pheromones which blend with volatile compounds emitted from the flowers to attract the male (Naumann et al., 1999). Known female pheromones extracted from species within Zygaenidae are unsaturated fatty acids esterified with a short chain chiral alcohol (Subchev et al., 2009; Priesner et al., 1984). Once a male has localized a putative female partner, the male begins courtship behavior, wafting plumes of volatiles from brushes on his abdomen (corremata) around her. Some males will not be accepted for mating and are therefore rejected by the female. Acceptance of the male is positively corre- lated to a high cyanogenic glucoside content in the male, presum- ably because it indicates increased fitness (Zagrobelny et al., 2007). When the female choose a male, often during the afternoon, mating will commence and last from approximately 16 h up to more than 20 h ((Fänger and Naumann, 1998; Hofmann and Kia-Hofmann, 2010) and personal observations). In the course of this time period, and often in the early evening, the male transfers a sper- matophore to the female, containing large amounts of proteins and * Corresponding author. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. Tel.: þ45 35331106. E-mail address: miz@life.ku.dk (M. Zagrobelny). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibmb.2013.08.007 Insect Biochemistry and Molecular Biology 43 (2013) 1037e1044