Unravelling the role of the male accessory glands in the fertility of the malaria mosquito Anopheles gambiae

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

Abstract

Mosquitoes represent a major threat for human health, as they transmit a variety of infectious diseases that cause the death and suffering of millions of people each year. Among the most prominent infectious diseases is malaria, which is transmitted exclusively by mosquitoes of the Anopheles genus. Current strategies aimed at tackling this disease rely extensively on the control of vector populations in the field, mainly through the use of insecticide indoor residual sprays and insecticide-impregnated bednets. However, insurgence of resistance in mosquitoes and the lack of novel insecticidal compounds are major hurdles in the fight against malaria, and novel ideas and tools are urgently needed. This proposal focuses on the reproductive biology of the species of mosquito primarily responsible for the transmission of malaria in Africa, known as Anopheles gambiae. An. gambiae mosquitoes mate only once in their lifetime, which means that disrupting the reproductive process offers a good way of dramatically reducing natural populations in Africa. When they mate, the male mosquito transfers sperm to the female and then afterwards transfers a coagulated mass of proteins and seminal fluids known as a mating plug, which is formed in the male accessory glands (MAGs), the mosquito equivalent of the human prostate. The MAGs have been shown by a number of studies to play a very important role in An. gambiae fertility. Transfer of MAG secretions (mainly proteins and lipids) to female mosquitoes has been shown to reduce the female willingness to mate again. Furthermore, our previous studies have shown that transfer of the mating plug is essential for the correct storage of sperm in the female, and hence for female fertility. A deeper knowledge of factors and mechanisms shaping the function of the MAGs would undoubtedly provide novel tools and ideas for the control of mosquito populations in the field through the manipulation of their fertility. As an example, if molecular mechanisms ensuring male fertility were identified, then we could design chemicals to inhibit such mechanisms that could be delivered to field mosquitoes in order to limit their reproductive output. In this project we will perform a detailed analysis of the MAGs, unravelling their functions in regulating mosquito reproductive success. In particular, we will assess whether the MAGs are regulating male behaviour, and we will analyse the roles in fertility of small peptide hormones produced in these reproductive organs. The results obtained here will remarkably improve our knowledge of the reproductive processes occurring in Anopheles mosquitoes, and will provide us with tremendous opportunities to translate this knowledge into practical tools for vector control. To achieve our goals, we will validate the results obtained in the laboratory in field mosquitoes, in collaboration with Professor Dan Masiga, from the International Centre of Insect Physiology and Ecology (ICIPE), Kenya. Strong collaborative links are already established between our groups within the framework of EU projects and in the form of a joint PhD studentship. Our study will focus on An. gambiae mosquitoes, however it could subsequently be extended to other insect pests of medical and agricultural importance. We anticipate that this project will have a profound impact on malaria research, and will contribute to the training and scientific excellence of the next generation of scientists.

Technical Summary

Anopheles gambiae mosquitoes are the major vectors of malaria, a disease with devastating consequences for human health. Given the evolution of insecticide resistance in mosquitoes and the lack of new insecticidal compounds, novel methods for controlling the natural vector populations are urgently needed. A deeper understanding of the processes shaping the biology of these mosquitoes would undoubtedly help to develop tools aimed at limiting the spread of malaria. In this proposal, we will target mosquito reproduction, a major determinant of the An. gambiae capacity to transmit disease. We will focus our analyses on the male accessory gland (MAGs), a major player of mosquito reproduction, in both laboratory and field settings. We will then set the scene for translating this information into the identification of tools for the manipulation of fertility of field populations. The experimental activities will be divided in three objectives. In Objective 1, we will unravel the role of the MAGs in shaping mosquito fertility and behaviour, by performing a combination of transcriptional and functional studies that will reveal the multifaceted activities of these tissues. In Objective 2 we will instead focus on the assessment of the role of peptide hormones produced in the MAGs on the fertility and reproductive behaviour of mosquitoes. Results obtained in both objectives will then be validated in field mosquitoes in Objective 3. This study will reveal as yet unknown molecular mechanisms underlying reproductive success in mosquitoes, considerably increasing our knowledge beyond the state-of-the-art and critically contributing with innovative tools and ideas to the fight against malaria. Our results will also be instrumental for the study of reproduction in other insect pests of medical and agricultural importance.

Planned Impact

Expanding our knowledge beyond the state-of-the-art. This project will considerably expand our knowledge of the reproductive biology of Anopheles mosquitoes, major vectors of human malaria. Importantly, it is very likely that the extensive knowledge gained here will be directly applicable to many other insects, providing the means for applying our findings to other systems for the reduction of field populations, for instance through the generation of sterile males in Sterile Insect Technique programmes. As such this can be considered as a founding study. By expanding our general knowledge of reproductive processes crucial to the high reproductive rate of these mosquitoes, we will contribute towards the development of innovative ideas and novel tools for the reduction or eradication of natural mosquito populations. Training of staff and students This project will crucially contribute to the training of next generation of scientists, in the UK and in the collaborating disease endemic country (Kenya). The interdisciplinary approach utilized here will greatly enhance the scientific skills and knowledge of the postdoctoral fellow that will be hired on the project, providing them with an excellent opportunity to learn state-of-the-art techniques, to collaborate with international groups of the highest scientific caliber, to participate to international conferences and to perform field studies. Such trained postdocs are likely to benefit biotechnology and pharmaceutical industries, as well as academic Institutions based in the UK and abroad. Furthermore, our collaboration with ICIPE, Kenya, will provide young African scientists with an invaluable opportunity for training in their everyday scientific work, also through their participation at project meetings and international conferences. Dissemination of results The results of this project will be mainly disseminated by: 1) publication as research articles in a variety of specialised and high-impact journals; 2) scientific lectures/talks at international conferences; 3) review articles and book chapters; 4) websites, bloggers, podcasts interviews. Our group is actively disseminating results to scientific audiences and to the general public, as our results have been made available. We will also give talks to schools and societies. Social and economic impact Malaria constitutes a terrible burden for health services and economies in disease endemic countries, and it also represents an increasing health and economic problem for European countries. By elucidating the molecular mechanisms of vector reproduction that are so crucial to the mosquito capacity to transmit malaria, this project will impact global health issues. Moreover, it will provide a strong contribution to UK scientific competitiveness. This will be achieved through the development of highly innovative ideas and findings that in future studies will be translated into powerful and novel tools for insect control. As an example of possible applications, once identified in Objective 2, peptide hormones affecting mosquito fertility could be modified to block the events leading to successful fertilization. These would therefore act as a novel generation of insecticide-like compounds to reduce the size of field populations, with the possible advantage of a species-specific mode of action to target only transmission-competent species. Alternatively, genetic traits conferring a male sterility phenotype could be introduced into field populations by transgenic means, thereby preventing insemination. It is anticipate that the involvement of industrial partners will be sought at some stage for the development of these tools.

Publications


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Gabrieli P (2014) Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae. in Proceedings of the National Academy of Sciences of the United States of America
Shaw WR (2014) Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae. in Proceedings of the National Academy of Sciences of the United States of America
 
Description Objective 1. Assessing the role of the MAGs in Anopheles reproduction

The first objective of this project is to unravel the role of the male accessory glands (MAGs) in ensuring reproductive success of Anopheles gambiae mosquitoes. With the view to identify genes important for reproduction, we have performed microarray analysis of the MAGs at three time points (3 hours, 12 hours and 24 hours) after mating. Mated tissues were compared to those from age-matched virgin males in four biological replicates using single channel Agilent 4x44K microarrays. This analysis has identified a large number of genes that are regulated by mating in the male mosquito. Surprisingly, a total of 4,319 genes were found to be differentially expressed after mating (P<0.05 FDR corrected), of which 2,296 were up-regulated and 2,023 were down-regulated. Maximum differential expression was observed at 12 (1,658 genes) and at 24 hours post mating (1,487 genes). In particular, genes that are important for the synthesis of steroid hormones were upregulated after mating, suggesting that males need steroid hormones for mating-related behaviours. While the microarray analysis is completed now, functional analyses of the hormonal pathway are underway (see aim 2) and will be incorporated into a manuscript describing both microarray data and follow up functional studies.



We have also studied the function of the Paired transcription factor in MAG development and gene expression. Paired was silenced by RNA interference (RNAi) injections in larvae: the initial hypothesis was that Paired silencing during larval development would generate adult males with no MAGs, as shown in Drosophila melanogaster. MAG-less males would then be used in mating experiments to assess the role of MAG secretions in modulating the post-mating physiology of females. Our results show that gene expression is reduced in Paired-silenced MAGs. However no obvious phenotype in terms of female post-mating behaviour was detected in matings between silenced males and virgin females: females were still capable of laying eggs and were normally refractory to further copulation. In order to determine the function of seminal secretions in triggering these post-mating responses, an in vivo system to identify matings defective in the transfer of MAG secretions was therefore developed. This system is based on previous work from the laboratory showing that coagulation of secretions into forming a mating plug can be prevented by silencing a MAG-specific transglutaminase enzyme. Further work has allowed us to use this system to selectively identify females that receive no mating plug during mating without the need to dissect them. Our work is demonstrating that transfer of coagulated secretions is necessary to trigger oviposition and refractoriness to further copulation, allowing us to circumvent the problems associated with developing MAG-less males. These experiments are being finalised at present and it is anticipated that a manuscript will be submitted before summer 2013.



Objective 2. Analysis of the role of small MAG-derived peptide hormones in fertility and female behaviour.

In this objective we proposed to identify small peptide hormones produced by the MAGs and assess their function. To this aim, we have targeted by dsRNA injections the male genes (glutaminyl peptide cyclotransferase, PHM and PAM) putatively involved in the formation of peptide hormones. We have generated antibodies against the three genes and determined their profile of expression. We have also assessed that RNAi injections are effective at silencing these genes by qRT-PCR and by immunoblotting. Phenotypic assay will be used to understand the role of these peptide hormones in male and female reproductive biology. Assays will include the analysis of sperm movement, mating receptivity, egg laying, fecundity and fertility of the mated females. At the same time, a pathway important for the synthesis of steroid hormone has been identified following the microarray analysis in Objective 1. Functional analyses are underway to determine whether these hormones affect female post-mating physiology. A manuscript containing these study and the microarray data will be submitted before the end of 2013.



Objective 3. Validation of candidates in field mosquitoes.



We have laid the ground for performing functional assays in semi-field conditions. To this end, we have established and validated mating protocols in semi-field cages (malariasphere, large outdoor cages exposed to environmental conditions and containing resting, breeding and swarming sites) at the Mbita Point field station of ICIPE, Kenya. We have observed occurrence of swarming and mating the first day following a release of males and female mosquitoes from laboratory cages into the semi-field facility. We have captured mating couples and assessed occurrence of insemination. The results obtained indicate that only 50% of the captured matings produced insemination. Our explanation for this observation is that different energy requirements imposed by flying in large containers relative to small laboratory cages prevent males from transferring a full complement of seminal fluid. Experiments are underway to determine whether improvements in the male diet can induce an increase in insemination rate.
Exploitation Route These results have the potential to be translated into tools for the control of mosquito populations transmitting malaria in Africa.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology