Analysing the molecular basis of post-mating behaviours in Anopheles mosquitoes

Malaria is a major threat for human health. Malaria parasites are transmitted to humans exclusively by the bite of an infected Anopheles female mosquito. Anopheles females mostly mate a single time in their lifetime, after which they undergo a series of changes that dramatically modify their physiology and behaviour. For instance, they start laying their eggs and become refractory to further mating. The bases of similar post-mating behaviours have been extensively studied in another insect, the fruitfly Drosophila, and small proteins secreted by part of the male genital organs (namely the male accessory glands) and transferred to females during copulations, have been implicated as their triggers. However, very limited information is available in Anopheles mosquitoes. This proposal aims at understanding the causes of mating refractoriness in Anopheles, focusing on the analysis of the male accessory glands secretions and on how they exert their functions in females. This study will provide crucial information on how mosquito fertility can be manipulated with the aim to control field mosquito populations. If for instance the occurrence of mating could be efficiently simulated in virgin females in the field, mating refractoriness would be induced, eventually leading to population extinction and hence malaria control.

In many insect species, mating induces important behavioural and physiological changes in females. In Anopheles gambiae mosquitoes, the major human malaria vector, after the occurrence of insemination the phase of the female flight activity rhythm shifts from crepuscular to nocturnal, the rate of egg-laying increases, and females become mostly refractory to further sexual intercourses. The biological bases of post-mating behaviours have been largely studied in Drosophila, and male accessory gland (MAGs) secretions (in particular, accessory glands proteins Acps) have been implicated to play a major role. However, information on how male products influence behavioural changes upon mating in Anopheles mosquitoes is still very limited. A detailed understanding of the biological events and molecular species involved in these processes would expand the knowledge on crucial aspect of reproduction behaviour in Anopheles, providing important clues on how to manipulate mosquito fertility at the population level, a topic that has great medical and public health relevance.

This proposal aims at studying the genetic basis of post-mating behaviour in A. gambiae females, in particular focusing on the study of the role of male accessory gland secretions in inducing mating refractoriness and oviposition, and on the identification of the genetic changes triggered in females by male-derived molecules and cells. To this purpose, a combination of Bioinformatics, transgenic technologies, Mass spectrometry and Microarray analysis will be utilised with the following aims:

1) Characterize the Acps composition of A. gambiae MAGs by Bioinformatics and Mass Spectrometry analyses, and investigate their role in modulating female post-mating behaviours such as mating refractoriness and oviposition by developing transgenic male mosquitoes depleted of MAGs secretions and sperm function;

2) Evaluate the genetic changes triggered in A. gambiae females upon mating, with particular attention to the central nervous system (CNS). This will be achieved by performing a detailed Microarray analysis comparing the transcriptomes of virgin and mated females, and by following the fate and the effects of the injection of single synthetic peptides in the female hemocoel.

These efforts will provide a tremendous contribution to our understanding of the reproductive behaviour of A. gambiae mosquitoes and possibly to their control, and may be extended to other important vectors of human disease. Furthermore, the proposed project is anticipated to branch out into a series of in depth analyses that will be the subject of future studies.