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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

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Catteruccia F (2009) Transgenic technologies to induce sterility. in Malaria journal

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Dottorini T (2007) A genome-wide analysis in Anopheles gambiae mosquitoes reveals 46 male accessory gland genes, possible modulators of female behavior. in Proceedings of the National Academy of Sciences of the United States of America

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Rogers DW (2008) Molecular and cellular components of the mating machinery in Anopheles gambiae females. in Proceedings of the National Academy of Sciences of the United States of America

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Thailayil J (2011) Spermless males elicit large-scale female responses to mating in the malaria mosquito Anopheles gambiae. in Proceedings of the National Academy of Sciences of the United States of America

 
Description BBSRC research grant
Amount £600,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2011 
End 04/2014
 
Description EC FP7 (Malvecblok)
Amount £250,000 (GBP)
Funding ID 223601 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 01/2009 
End 06/2012
 
Description EC FP7 infrastructure funding
Amount £250,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 03/2009 
End 08/2013
 
Description ERC starting grant
Amount € 1,500,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 01/2011 
End 12/2015
 
Description Royal Society international Joint Projects
Amount £6,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 09/2009
 
Description Wellcome Trust Project grant
Amount £700,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 08/2014
 
Title Ab_Acps 
Description A number of polyclonal antibodies against male accessory gland genes 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact Studies on the fate and localization of male accessory gland proteins transferred to females during mating, in Anopheles gambiae and other species of the A. gambiae complex. 
 
Title Ab_Female reproductive tract 
Description We have generated antibodies against a number of proteins expressed exclusively in the female uterus and in the sperm storage organs 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact These antibodies are being used to unravel the function of female factors that are regulated by copulation. These studies are being completed and will be prepared for publication in the next few months. 
 
Title Field_mosquitoes 
Description We have collected mated couples from field swarms in Burkina Faso and have a collection of over 50 cDNAs of males and females isolated as virgins or at different time points after mating. Samples were collected from both M and S molecular forms, two incipient species of Anopheles gambiae 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact This material is invaluable for many other researchers who want to analyze field collected virgin and mated individuals for the expression of their genes of interest, and for researchers studying the processes of speciation in mosquitoes 
 
Title MA_mating 
Description Microarray data on gene expression in female mosquitoes before and after mating, at different time points 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact Identification of genes that are induced or repressed by mating and that play a role in female fertility, field studies in Burkina Faso, identification of female reproductive factors to target for future field intervention 
 
Title Male accessory gland samples 
Description Different male tissues (reproductive tissues, heads and rest of the body) collected at different time points before and after mating 
Type Of Material Biological samples 
Provided To Others? No  
Impact The identification of the changes occurring in male mosquitoes upon mating 
 
Title Mating_assay 
Description We developed an efficient method to isolate mosquito mating couples while still in copula. This method replaces previous ones where copulation was necessarily interrupted by the operator. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact Many other research groups now utilize this method for their mating experiments. Capturing mating couples allows to establish the exact time of mating, therefore permitting to perform studies (such as microarrays and mass spectrometry) where this information is necessary to the identification of factors regulated by mating. Furthermore it allows to assess with certainty the genotype of both male and female partners int eh couple, allowing genetic studies on the desired traits. 
 
Title Microarrays female tissues 
Description A number of female tissues (head, gut, ovaries, reproductive tract) collected before and after mating a t different time points 
Type Of Material Biological samples 
Provided To Others? No  
Impact The identification of the signalling cascades triggered in females by mating in different female tissues. This material has not been shared with other groups as yet but it will be made available upon request and it will be invaluable to researchers interested in the analysis of post-mating processes in females 
 
Title Virgin/mated spermathecae 
Description We have collected cDNAs from the spermathecae of virgin and mated females to perform microarray analyses on these reagents. 
Type Of Material Biological samples 
Provided To Others? No  
Impact The analysis is ongoing and will provide us with a number of candidate genes that may play an important role in mosquito fertility 
 
Description 3D structure transglutaminase 
Organisation University of Bath
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution The identification of the 3D crystal structure of the transglutaminase enzyme that is necessary to form the mating lug during copulation in Anopheles
Collaborator Contribution Analysis of 3D structure of the plug-forming transglutaminase (not published yet)
Impact Analysis of 3D structure of the plug-forming transglutaminase is helping our research on the function of this enzyme in mosquito reproduction. Multidisciplinary, involving chemistry and genetics
Start Year 2010
 
Description Field studies on Anopheles mating 
Organisation MURAZ Center
Country Burkina Faso 
Sector Academic/University 
PI Contribution We have isolated mating couples from natural swarms and have dissected reproductive tissues of male and female mosquitoes from thewe couples at different time points after copulation. We have then performed quantitative real time PCR on cDNAs from these tissues, for a large number of reproductive genes in order to assess their regulation upon mating.
Collaborator Contribution We have conducted a pilot study on the transcriptional response to mating in females caught in natural swarms in Burkina Faso and have confirmed our laboratory-based studies, therefore validating a number of mosquito candidate reproductive genes for further functional analysis. Our collaborators are experts in finding natural mosquito swarms in the field and without their help this study would have not been possible.
Impact Our data show that laboratory-based experiments aimed at studying the reproductive biology of Anopheles mosquitoes are trustworthy. Moreover we have confirmed that mating-induced changes are in some cases permanent. These data are currently being written for publication.
Start Year 2009
 
Description Interplay between mating and immunity in mosquitoes 
Organisation International Centre of Insect Physiology and Ecology (ICIPE)
Country Kenya 
Sector Charity/Non Profit 
PI Contribution Our studies on mosquito reproduction have attracted the attention of different groups working on the biology of the malaria mosquito and of the malaria parasite, resulting in this collaborative network comprising 3 African and 4 European groups. In particular, my research group is working at unraveling the interplay between reproduction and immunity with the view to find new tools for the control of field populations of Anopheles mosquitoes. We are also extending the studies on the reproductive biology of Anopheles gambiae to other mosquito species that are vectors of human malaria, with the view to identify factors and mechanisms important for fertility that are common to different vector species that could be targeted for field interventions
Collaborator Contribution Sharing of reagents and protocols, training of students/postdoctoral fellows in cell biology techniques. Discussions of novel ideas to tackle scientific questions related to our research.We are studying the rates of evolution of a number of reproductive genes in Anopheles gambiae mosquitoes.Based on this collaboration, we now share a PhD studentship to look at the role of mosquito reproductive genes in different anopheline vectors of human malaria.
Impact This collaboration has resulted in a publication, published in PLoS Biology in 2009 (Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito), and in a series of other studies that are currently being written up for publication.
Start Year 2009
 
Description Interplay between mating and immunity in mosquitoes 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Country France 
Sector Public 
PI Contribution Our studies on mosquito reproduction have attracted the attention of different groups working on the biology of the malaria mosquito and of the malaria parasite, resulting in this collaborative network comprising 3 African and 4 European groups. In particular, my research group is working at unraveling the interplay between reproduction and immunity with the view to find new tools for the control of field populations of Anopheles mosquitoes. We are also extending the studies on the reproductive biology of Anopheles gambiae to other mosquito species that are vectors of human malaria, with the view to identify factors and mechanisms important for fertility that are common to different vector species that could be targeted for field interventions
Collaborator Contribution Sharing of reagents and protocols, training of students/postdoctoral fellows in cell biology techniques. Discussions of novel ideas to tackle scientific questions related to our research.We are studying the rates of evolution of a number of reproductive genes in Anopheles gambiae mosquitoes.Based on this collaboration, we now share a PhD studentship to look at the role of mosquito reproductive genes in different anopheline vectors of human malaria.
Impact This collaboration has resulted in a publication, published in PLoS Biology in 2009 (Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito), and in a series of other studies that are currently being written up for publication.
Start Year 2009
 
Description Interplay between mating and immunity in mosquitoes 
Organisation Sapienza University of Rome
Department Department of Public Health Sciences
Country Italy 
Sector Academic/University 
PI Contribution Our studies on mosquito reproduction have attracted the attention of different groups working on the biology of the malaria mosquito and of the malaria parasite, resulting in this collaborative network comprising 3 African and 4 European groups. In particular, my research group is working at unraveling the interplay between reproduction and immunity with the view to find new tools for the control of field populations of Anopheles mosquitoes. We are also extending the studies on the reproductive biology of Anopheles gambiae to other mosquito species that are vectors of human malaria, with the view to identify factors and mechanisms important for fertility that are common to different vector species that could be targeted for field interventions
Collaborator Contribution Sharing of reagents and protocols, training of students/postdoctoral fellows in cell biology techniques. Discussions of novel ideas to tackle scientific questions related to our research.We are studying the rates of evolution of a number of reproductive genes in Anopheles gambiae mosquitoes.Based on this collaboration, we now share a PhD studentship to look at the role of mosquito reproductive genes in different anopheline vectors of human malaria.
Impact This collaboration has resulted in a publication, published in PLoS Biology in 2009 (Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito), and in a series of other studies that are currently being written up for publication.
Start Year 2009
 
Description Royal Society International Joint Project 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Public 
PI Contribution We collaborated with CNRS-INSERM Strasbourg France at the analysis of the interaction between reproduction and immunity in mosquitoes
Collaborator Contribution PMID: 19036921
Impact PMID: 19036921
Start Year 2007
 
Description Semi-field Kenya 
Organisation International Centre of Insect Physiology and Ecology (ICIPE)
Country Kenya 
Sector Charity/Non Profit 
PI Contribution A PhD student from my research group is performing studies at the field station of Mbita aimed at functionally characterize a number of reproductive genes regulated by mating in Anopheles females. This research will identify whether these female genes are important for mosquito fertility and therefore whether they represent good targets for the manipulation of fertility in the field.
Collaborator Contribution we are testing the function of some of the reproductive factors identified in our laboratory analysis in field mosquitoes, taking advantage of the semi-field facilities available in the field station of Mbita.
Impact No direct benefits yet as research is still ongoing
Start Year 2011
 
Description Interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact A number of interviews with radio stations (from the UK, Australia, South Africa, Italy) and websites (BBC, others)

Contacted by academics to establish collaborations, and by members of public congratulating our research or asking for further information
Year(s) Of Engagement Activity 2009,2010
 
Description Interviews 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release on the paper entitled: Spermless males elicit large-scale female responses to mating in the malaria mosquito Anopheles gambiae. Video interview (live) by Al Jazeera television. Many live and recorded radio interviews conducted by BBC Radio 4, BBC Scotland, Italian Radio Channels, German broadcasters and many more. Front page of Nature News and BBC website and many others websites.

Large interest in the research performed by my group. Attention from general public. Invitation to give seminars.
Year(s) Of Engagement Activity 2011
 
Description Male Biology Meeting at IAEA 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 3-day meeting held by the International Atomic Energy Agency to discuss the biology of male mosquitoes with the view to develop novel vector control methods. The audience included scientists and health professionals from the US, Europe and Africa. My presentation of the data obtained in the MRC project have been perceived extremely favorably and a series of collaborations have been started with the IAEA and with members from Burkina Faso.

A series of collaborations have been started with the IAEA and with members from Burkina Faso that will lead to field work to confirm data obtained in laboratory conditions
Year(s) Of Engagement Activity 2008
 
Description Presentation to the Imperial College Rector 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Invited seminar to present my research activities to the Rector of Imperial College London, as a representative of young promising group leaders. Most prominent members of the Faculty of Life Sciences were also present. A 15 minute presentation, with very good feedback from the Rector himself and the audience.

Collaborations established as a consequence of the talk. Visibility of my research within the Faculty.
Year(s) Of Engagement Activity 2008
 
Description podcast interview at Imperial College 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A monthly podcast magazine held by Imperial College to highlight the most interesting research ongoing at College.

High interest in general public, resulting in a number of emails received on the subject and on additional interviews from journalists
Year(s) Of Engagement Activity 2009
URL http://www.imperial.ac.uk/media/podcasts