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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 ( 15 N), 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.  15 N-glycine was added to larval rearing water and the target enrichment was 5 atom%  15 N. 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 δ 15 N‰ 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%  15 N. 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  13 C,  15 N can be used to detect mating in  Anopheles  and the suitability of both labels is briefly discussed.

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