Stable isotope-mass spectrometric determination of semen transfer in malaria mosquitoes

The potential use of stable isotopes to study mosquito mating was investigated by tracing the fate of labelled semen into spermathecae. [C-13] glucose was incorporated in the diet of the malaria mosquito Anopheles arabiensis. Treatments included labelling of either the larval water or adult sugar water, or a combination of both. After mating, 'spiked' spermathecae were analysed for isotope ratios using mass spectrometry. Results demonstrated that spermathecae positive for semen could successfully be distinguished from empty ones or controls (i.e. filled with unlabelled semen) using the raw delta C-13 values. Labelling during larval development and combined labelling of larvae and adults resulted in detectable values. The label persisted in spermathecae for up to 7 days after mating, and unlabelled sugar feeding of males labelled in the larval stage did not result in a detectable turnover of the semen label. There were no detrimental effects of the addition of labelled glucose on larval development and survival, adult size, male longevity and mating performance. We have proved that it is possible to label male mosquitoes and detect the semen label in females after insemination. This method offers great potential to study mating in mosquitoes and other insects and could prove useful in genetic control studies of medical or agricultural pest insects, with male mating success in the field as a critical verifiable indicator for a positive outcome of the intervention.