Short report

A ¹⁵N stable isotope semen label to detect mating in the malaria mosquito Anopheles arabiensis Patton

Michelle EH Helinski^1,2,3 , Rebecca C Hood¹ , Doris Gludovacz¹ , Leo Mayr¹ and Bart GJ Knols²

¹Agency's Laboratories Seibersdorf, International Atomic Energy Agency (IAEA), A-2444 Seibersdorf, Austria

²Laboratory of Entomology, Wageningen University and Research Centre, P.O. Box 8031, 6700 EH Wageningen, The Netherlands

³Department of Entomology, Ithaca campus, Cornell University. 3136 Comstock Hall, Ithaca, NY 14853-2601, USA

author email corresponding author email

Parasites & Vectors 2008, 1:19doi:10.1186/1756-3305-1-19

Published:	1 July 2008

Abstract

In previous studies it was determined that the stable isotope 13-carbon can be used as a semen label to detect mating events in the malaria mosquito Anopheles arabiensis. In this paper we describe the use of an additional stable isotope, 15-nitrogen (¹⁵N), for that same purpose. Both stable isotopes can be analysed simultaneously in a mass spectrometer, offering the possibility to detect both labels in one sample in order to study complex and difficult-to-detect mating events, such as multiple mating. ¹⁵N-glycine was added to larval rearing water and the target enrichment was 5 atom% ¹⁵N. Males from these trays were mated with unlabelled virgin females, and spiked spermathecae were analysed for isotopic composition after mating using mass spectrometry. Results showed that spermathecae positive for semen could be distinguished from uninseminated or control samples using the raw δ¹⁵N‰ values. The label persisted in spermathecae for up to 5 days after insemination, and males aged 10 days transferred similar amounts of label as males aged 4 days. There were no negative effects of the label on larval survival and male longevity. Enrichment of teneral mosquitoes after emergence was 4.85 ± 0.10 atom% ¹⁵N. A threshold value defined as 3 standard deviations above the mean of virgin (i.e. uninseminated spermathecae) samples was successful in classifying a large proportion of samples correctly (i.e. on average 95%). We conclude that alongside ¹³C, ¹⁵N can be used to detect mating in Anopheles and the suitability of both labels is briefly discussed.