Final Report Summary - INFRAVEC (Research capacity for the implementation of genetic control of mosquitoes)
Executive Summary:
Mosquitoes are known for transmitting a wide variety of infectious diseases that cause a tremendous burden to public health. Due to climate changes and to the increase in international trade and tourism the threats posed by mosquitoes are increasingly affecting large parts of Europe, causing understandable concerns among the populations of many Member States, particularly in the southern parts on the continent. Control methods, mainly based on the use of insecticides, are struggling to cope with the challenges posed by the biology and ecology of mosquito vectors. INFRAVEC aimed to bridge the gap between the recent advances in transgenic technology and its implementation as a novel powerful approach for vector control. To this aim, a large European Infrastructure has been put together, in which the coordination of efforts, expertise and facilities provided by the participating research groups and institutions contributed to expand the overall research capabilities of the research community. Four Infrastructure facilities have been integrated in the project: 1) the Mosquito Genetic Facility at Imperial College London; 2) the Mosquito Mass-rearing facility at the Centro Agricoltura ed Ambiente, CAA (which operates in close collaboration with the International Atomic Enargy Agency; 3) the Cambridge-based Bioinformatics facility of EMBL-EBI; and 4) the Mosquito Confined Release facility managed by UNIPG and offered a formidable research capability to external users that have applied for the services offered by the project and implemented five research projects aimed at utilizing basic knowledge of mosquito genetics and biology in an unprecedented effort to develop novel opportunities for mosquito control. INFRAVEC carried out a number of Networking, Joint Research, Transnational and Service activities, towards the objective of strenghthening research capability in Europe by sharing knowledge, resources and technology. These are: provision of access to the Infrastructure facilities; establishment of a mosquito line repository; development of mosquito high throughput sorting technologies; development of Ae. albopictus gene database; improvement of gene manipulation technologies; targeting mosquito vectorial capacity and EST sequencing; population structure analysis of mosquito species; validation of GM mosquito control measures under confined field conditions.
Project Context and Objectives:
INFRAVEC addresses the need of the scientific community to share facilities and integrate cuttingedge knowledge and technologies that are not readily accessible but nevertheless critical to exploit genetic and genomic information in the effort to control mosquito-borne diseases. Its objective is to provide laboratories that currently operate individually with limited coordination and little sharing of technologies, with the collective research capacity of the laboratories forming the core project infrastructure. INFRAVEC has provided resources to 31 institutions from European and African countries to enhance collaborative links, to execute joint research activity, and most importantly to enable individual researchers (from PhD students to established academics) to carry complex experimental activities by assigning research packages or ‘infrastructure access’ to be executed in the laboratory facilities and infrastructures of INFRAVEC. European laboratories have made crucial contributions in the field of mosquito molecular biology, genetics, biology, and epidemiology and have established a number of valuable facilities to perform insect mass rearing, mosquito genetic manipulation, single nucleotide polymorphism (SNP) population typing, and confined release experiments. These laboratories have so far mainly operated individually with limited coordination and little sharing of infrastructure and technologies. INFRAVEC has the objective to integrate these facilities into a coordinated infrastructure to promote scientific exchanges, facilitate the sharing of technological platforms, and enhance the research capacity of the scientific community. Its activities are articulated in networking, joint research projects, and promoting the use of the facility infrastructure by individual researchers. Networking activities have been aimed at improving and utilizing the facilities of the infrastructure as well as at strengthening and facilitating collaborative projects between institutions to improve, assess, and validate genetically manipulated mosquitoes for vector control. They include the development and maintenance of common facilities, such as the formation of a mosquito line repository facility by coordinating different laboratories to stock naturally occurring and genetically modified (GM) mosquito lines together with the formation of a database containing genetic and biological information of each line. INFRAVEC has supported four joint research and development activities aimed at (i) improving mosquito gene manipulation technologies, (ii) targeting the mosquito vectorial capacity, (iii) analyzing population structure and gene flow in mosquito species, and (iv) validating GM mosquitoes for vector control under confined field conditions. These projects were leveraged by access to the infrastructure with a range of complementary facilities to provide the research projects with a formidable capability to undertake complex experimental activities. The infrastructure of INFRAVEC consists of four laboratory facilities based in different European countries including (i) the insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna (Italy), (ii) the mosquito genetic manipulation laboratory at Imperial College London (UK), (iii) the mosquito confined release facility at the Biotechnology Centre of the University of Perugia (Italy), and (iv) the bioinformatics suite for data collection software development and information sharing at EMBL-EBI Cambridge (UK). Some of these facilities such as the bioinformatics suite at EMBL-EBI are widely utilized by the scientific community, whereas others offer their services sporadically and without coordination. INFRAVEC aims at integrating this infrastructure with networking activities and joint research projects to serve the mission of overcoming the existing capacity roadblock on the way to assess ethical, safety, efficacy, and feasibility issues of genetically manipulated mosquitoes for vector control. Simultaneously, INFRAVEC supports individual researchers to carry out research packages in the infrastructure facilities operated by the participants. The infrastructures are made available to external researchers or teams via a grant responsive mode. INFRAVEC publicizes the services offered by the infrastructure to external users and implements a peer review selection process to identify the projects that will be given support to utilize the infrastructure and contribute to the further development of the joint research activities.
Project Results:
INFRAVEC integrates in a multi-centre European Infrastructure a number of academic and industrial laboratories operating in the field of mosquito biology, genetics, epidemiology, genetic engineering, ecology and population biology. The project objectives have been to provide the participating laboratories and the European scientific community with a formidable leverage to progress in the understanding of mosquito biology and to develop new control measures against vector-borne diseases. A number of laboratory facilities have been integrated in a coherent manner within a single operating Infrastructure, which includes:
i) The insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna (Italy);
ii) The mosquito genetic manipulation laboratory at Imperial College London (UK);
iii) The mosquito confined release facility at the University of Perugia (Italy):
iv) The bioinformatics suite for data collection software development and information sharing at EMBLEBI Cambridge (UK).
The project has been articulated in networking and joint research activities specifically designed to improve the performance of the Infrastructure and to increase its capacity.
The networking activity included:
a) A multi-centre mosquito line repository facility to coordinate the implementation of standardized quality controls and the distribution to external users of both genetically manipulated and field-derived mosquitoes;
b) The development of a technology for the high-throughput sorting of mosquitoes on the basis of the expression of fluorescent markers in organs and tissues;
c) The sequencing and the annotation of the Ae. albopictus genome and tissue RNA sequencing to provide the scientific community with the knowledge on genome sequence, conservation and divergence between different mosquito species.
INFRAVEC also supported four collaborative research projects that aimed at strengthening the research capability and the scientific excellence towards the effort of expanding the basic knowledge of mosquito biology and of addressing the major bottlenecks in the development of vector control measures based on genetically modified (GM) mosquitoes. The following are the collaborative research projects implemented:
a) Improve gene manipulation technologies. The objective was to adapt the existing gene transfer technologies to mosquito species of emerging medical importance such as Ae. albopictus; and to develop a gene targeting technology in An. gambiae to selectively manipulate genes of interest. These advancements have significantly contributed to expanding the research capability of both the mosquito genetic manipulation laboratory at Imperial College London and the mosquito confined release facility at the University of Perugia;
b) Targeting the mosquito vector capacity. The aim was to generate a collection of An. gambiae mosquito lines in which genes essential for the mosquito’s ability to transmit disease have been identified and selectively disrupted. This work has provided the infrastructure and the participating laboratories with reference tools for addressing fundamental biological questions related to mosquito biology such as immunity and reproduction, and has enabled the identification of mosquito genes to be used in vector control measures;
c) Analyze population structure of mosquito species. The objective was to perform a systematic analysis of polymorphic loci in wild and laboratory Ae. albopictus populations. SNP frequencies and inheritance patterns will be used to monitor gene flow in field mosquitoes and to identify genetic bottlenecks when adapting mosquitoes to laboratory rearing conditions and mass production. This information will be invaluable for the correct functioning of the insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna;
d) Validation of GM mosquito control measures under confined field conditions. Large numbers of GM mosquitoes will be released in large semi-natural cages exposed to environmental conditions to assess the fitness of GM mosquitoes under confined “field” conditions and their efficacy in vector control methods. These experiments will be of paramount importance for assessing possible safety and environmental risks and for addressing regulatory requirements for subsequent field releases. The results of these experiments will provide guideline for future experiments and provide the rationale for adapting the structure of the facility for future tasks.
Work Package 2 Development of a mosquito line repository: A number of mosquito strains have been collected over years in remote locations in Asia and Africa and or genetically modified at the participating laboratories. They represent formidable models to study fundamental mechanisms of speciation, adaptation and evolution, whereas others exhibit unique properties resulting from natural diversity, tedious laboratory selection or transgenesis. A number of drawbacks prevents these invaluable resources from being utilized to their full power: (i) severe space limitations, especially in the laboratories producing GM mosquitoes; (ii) lack of information about the existing stocks; (iii) absence of efficient methods for molecular authentication; (iv) lack of standardized breeding procedures; (v) lack of control for persistent or cryptic pathogen infections (e.g. microsporidia infections). This WP promoted the formation of a mosquito stock repository facility by (i) coordinating efforts of the participating laboratories to stock mosquito lines in their insectaries; (ii) establishing and implementing standardized procedures for stock quality control; (iii) providing the community with centralized access to the lines/strains through a web-based database containing genetic and biological information for each line. The repository is designed to provide standardized material to individual laboratory across the world for studies aimed at investigating mosquito biology and at developing new control measure but most importantly dramatically enhances the research capability of the infrastructure facility, by overcoming limitations of breeding and maintaining mosquito lines due to logistics and resource constrains.
The mosquito stock census was completed and progress was made towards the implementation of standardized quality control processes and the addition of new lines. A dedicated informatics platform to implement the repository activities was developed and made available to the scientific community via the project website. A number of participating laboratories including IMPERIAL, UNIPG, UNIRM, CNRS, CNRFP, UNICAM, ICIPE, IP, KEELE provided information about the genetics, the biology and the geographic origin the Anopheles gambiae and Anopheles arabiensis mosquito lines that were kept in their laboratories. These include colonies from natural populations representing genetic diversity, mosquito colonies susceptible or refractory to Plasmodium development and mosquitoes showing different level of resistance to distinct classes of insecticides such as DDT, Deltamethrin, Lambda-cyhalothrin and Permethrin. The Anopheles gambiae PEST reference strain and M and S molecular forms were subjected to whole genome sequence to computationally extract a set of 1500 high-quality segregating polymorphic markers (SNPs) that were used to make a customized Illumina GoldenGate BeadArray genotyping chip. The chip was validated using a large collection of wild mosquitoes from Burkina Faso (IP). A sub-panel of 50 highly informative and robust markers has been derived based on the genotyping done to date. We have used this panel of markers as a genetic barcode for identifying mosquito strain on different genotyping platforms. The repository also includes lines derived from Aedes albopictus, an invasive mosquito species that has colonized vast regions of South Europe. This mosquito species has been responsible for the transmission of vector borne diseases in Italy Greece and Madeira. CAA has generated four Aedes albopictus lines using field samples collected in Italy and in Albania. One of these lines “Fellini” was subjected to a number of inbreeding crosses utilized to develop an “isofemale line” with limited genetic variability to serve as starting material for the “Aedes albopictus” genome-sequencing project (IMPERIAL).
Currently the repository has identified more than one hundred mosquito lines including Anopheles gambiae, Anopheles arabiaensis Aedes albopictus strains that are kept in the laboratories of the participating institutions. The information provided concerns the geographical origin, the biological characteristics and the phenotype of each individual line and stored in the database. Molecular probes have been distributed to the partners to carry out standardized genotyping assays in order to confirm the identity of the lines (UNIPG). Protocols have been implemented to standardize breeding conditions, genotyping and stock propagation. Procedures have been implemented to allow external users to access, request and deposit the lines of repository via a web interface.
Work Package 3 Development of mosquito high throughput sorting technologies: The objective of this work package was to develop an automated high throughput platform to screen mosquitoes for visible genetic markers. We have planned to develop a novel fully or semi-automated technologies for the screening of mosquitoes. This was achieved by two parallel approaches both based on adaptation of the "worm sorter" instrument COPAS (Complex Object Parametric Analyzers and Sorters), developed by Union Biometrica Inc. for Caenorhabditis elegans, to mosquito larvae. The development this technology dramatically expedites the identification of phenotypes under different experimental conditions. This has translated in a reduction of the workload and in enhanced research capacity of the infrastructure facilities particularly the mosquito genetic manipulation laboratory at Imperial College London and the mosquito confined release facility at the University of Perugia. Automated genetic screening reduces the time and effort to generate genetically manipulated mosquitoes and facilitates the analysis of their phenotype. This technology is also invaluable for its capability to selectively separate male and female mosquitoes on the basis of sex linked fluorescent markers for testing vector control measures under confined conditions. In this respect the sorting technology is critical to the successful implementation of different work packages including: (i) Networking Activities – WP2 ‘Development of a mosquito line repository’; (ii) Joint Research Activities – WP5 ’Improve GM technologies’, WP6 ‘Targeting vectorial capacity’, and WP8 ‘Validation of GM mosquito control measure’.
The instrument specification were set, reviewed and the prototype design approved in a number of meeting involving CNRS, UNIPG, IMPERIAL, ISRIM, CAA and Union Biometrica’s representatives in London, Strasbourg and Boston. Though the instrument still needs some improvement through modifications to the pressure and flow settings it is now largely fit for purpose for the medium throughput screening of certain transgenic backgrounds. The instrument was originally designed for use at the Laboratory of Biotechnologies of the University of Perugia, though it is IMPERIAL that initially took the responsibility for supervising the installation and initial use of the instrument in London. After the installation of the COPAS at the Imperial College, the instrument was validated for high throughput sorting of mosquito larvae of various stages based on the expression of fluorescent marker proteins in the eyes, ventral nerve cord or in the male reproductive organs. Upon delivery two major activities were carried out: 1) the validation of protocols for the breeding of large quantities of mosquitoes; and 2) the adaptation of the existing technology to achieve high-throughput, efficient sexing on the basis of fluorescent phenotypes. The team at CNRS closely collaborated to the tests that were performed at Imperial College.
Work Package 4: Development of Ae. albopictus gene database: In contrast to An. gambiae and Ae. aegypti, there are currently limited Ae. albopictus genome resources available with no large scale genomic sequencing and few cDNA sequences in the public nucleotide databanks. The aim of this WP has been to address this limitations and generate a database containing the genome sequence of Ae. albopictus together with a large number of Expressed Sequence Tags (ESTs) from different organs and perform suitable bioinformatics analyses to cluster into consensus sequences, predict the coding open reading frames (ORFs) and provide automated functional annotations. This database was expected to provide the first large scale gene discovery platform for this mosquito species which is of fundamental interest to the general mosquito research community. Knowledge of the Ae albopictus genome and transcriptome is instrumental in enhancing the research capability of the mosquito genetic manipulation laboratory and the mass rearing facilities by facilitating the development of genotyping tools for the identification of wild and reared mosquitoes. The genomic data available to the scientific community was expected to become more readily available with the development of a database at EMBL-EBI on the most invasive mosquito species.
The scope of this work package has expanded significantly because of the unexpected decrease in sequencing costs and the introduction of new technologies. It was decided therefore that there was value in creating a genome sequence from a newly established Ae. albopictus reference strain. This approach has the advantage of not relying on homology with Ae. aegypti in order to assemble a full genome sequence. DNA for sequencing was generated using an isofemale line generated at IMPERIAL starting from an Ae albopictus laboratory strain established at CAA. DNA material was shipped to the sequencing laboratory “GenePool” for the construction of genomic sequencing libraries. A small insert library (200 bp paired-end) has been constructed using the female DNA. After several attempts GenePool failed to construct a long insert mate-pair library. This task was transferred to UNIPG that were able to generate the mate-pair library Ae. albopictus genome has been sequenced at the University of Edinburgh's GenePool facility (200 bp paired-end) and University of Perugia (mate-pair library). The sequencing strategy assumes an estimated genome size similar to that of Ae. aegypti and therefore we used 20 lanes of Illumina sequencing given current production capabilities at the facility. The libraries were sequenced using the Illumina HiSeq 2000 platform in 2x101 nucleotide paired-end reads. Sequencing was performed using 2 lanes of the flow-cell, yielding ~220 million reads per cell. The data has been analyzed under a bioinformatic access project with INFRAVEC. Annotation of the genome has been achieved using automated procedures. The annotation process has been completed in three phases: 1) Identification and masking of repetitive sequence; 2) gene prediction; 3) prediction of gene function. The genome annotation pipelines included MAKER (http://gmod.org/wiki/MAKER). EMBL-EBI now uses MAKER as the default annotation pipeline to integrate the results from predictive algorithms into a single reference gene prediction set.
Transcription analysis has been carried on different tissues and organs. UNIPV in collaboration with the team at IMPERIAL Illumina RNA-seq libraries have been sequenced and analysed (EMBL-EBI) in order to identify transcripts involved in chemoreception. This information has also been used to support the annotation of the Ae. albopictus genome sequence, the development of an Ae. albopictus database resource and the identification of a large number of coding SNPs. Illumina libraries were prepared from the Ae. albopictus antennal RNA samples from four different geographic populations; Lampang Ban Rai (Thailand), Athens (Greece), Trento and Arco (North Italy). Poly-A mRNA in the total RNA samples was purified using Illumina poly-T oligo-attached magnetic beads. The poly-A RNA, the mRNA was also fragmented and primed with random hexamers for cDNA synthesis. The resulting libraries were validated using the Agilent 2100 bioanalyser to confirm the concentrations and size distribution and then normalized to 10nM concentration and sequenced using Illumina 2000 technology. The sequences have been assembled and annotated, transcription profiles were generated leading to the identification and characterization of genes expressed organ and tissues.
Work Package 5: Improve gene manipulation technologies: This work aimed to improve gene manipulation technology for An. gambiae and to adapt the existing technology to Ae. albopictus. We had planned on developing selective gene targeting methods to systematically disrupt mosquito genes involved in shaping the vectorial capacity i.e. those involved in sex determination, host-seeking behaviour, fertility and immunity. The full potential of genetic manipulation is well appreciated in studies aimed at investigating the biology of mouse and drosophila but has not been fully exploited in mosquitoes to investigate interactions with viruses and parasites and in developing environmentally safe GM insects for vector control. Important limitations are faced when studying the function of genes potentially involved in biological processes crucial for mosquito vectorial capacity such as fertility and sex differentiation, which cannot be efficiently targeted by transient RNA interference because of their temporal and spatial expression patterns. Furthermore, the use of transposons aimed to increase the transformation efficiency, raises safety and environmental issues that represent serious obstacles for the release of GM mosquitoes in the field as part of vector control measures. This relates to the stability of the genetic modifications, their unforeseen effect on other biological functions of the mosquito and their potential to spread to non-target insect species.
IMPERIAL, in collaboration with CNRS and KEELE, investigated the feasibility of developing a gene targeting technology based on the production of a recombinogenic linear molecule in the germline based on the concerted action of both the Isce1 endonuclease and the FLP recombinase on chromosomally-inserted transgenic targeting constructs. These efforts resulted in the successful validation of an alternative recombinase, Cre, that acts with high efficiency in the germline to excise Donor This approach yielded significantly more transgenic founders than the previous state-of-the-art, thereby translating in a significant improvement in efficiency and in a substantial cost reduction. Keele made major improvement to gene manipulation technology, particularly in relation to the utilization of phiC31 integrase to deliver DNA constructs at specific sites ‘docking sites’ (attP sequence). This revolved around the development of a piggyBac transformation construct, containing an attP sequence, a selectable marker, and a transcription unit encompassing the phiC31 integrase coding sequence (that directs the integration at the attP site) regulated by the germ-line specific nanos gene promoter. A series of attP strains were developed and characterized for their ability to uptake DNA sequences at specific sites. IMPERIAL in collaboration with CNRS developed additional lines that contain ‘docking sites’ dispersed at different genomic locations for future integrations of transgenes at pre-characterized genomic locations. Notably this technology has been used to edit the Y chromosome of A. gambiae swapping promoter and reporter genes at selected sites. Marked Y chromosomes were used to monitor the introgression of this chromosome in the related species A. arabiensis. CNRS and Imperial have also collaborated at the validation of the Cas-CRISP technology to selectively edit sequences in the anopheles gambiae genome. These studies relevealed that if CAS gene is provided as stable germ line integration the guide RNA techonology can be used to target specific sequences. In addition HEG technology proved to be a valuable and effective approach to selectively target gene sequences.
Work Package 6 Targeting mosquito vectorial capacity and EST sequencing: This work package aimed at developing a suite of novel tools based on fluorescent and luciferase reporters and on targeted gene disruption to address essential questions related to the mosquito biology and vectorial capacity. The availability of these lines were expected to dramatically increase the research capability of the infrastructure toward the objective of developing genetic vector control measures targeting malaria transmission. A number of diverse strategies have been put in place for the successful implementation of this workpackage objectives, targeting the different processes of mosquito biology that are relevant to disease transmission.
We have established a collection of An. gambiae transgenic fluorescence- or luciferase-based reporter lines in which promoter regions of immune response genes control the expression of fluorescent reporter markers. CNRS established additional transgenic reporter An. gambiae lines to follow activation of the immune system: one expressing GFP under the control of the TEP1 promoter, one expressing Luciferase and one expressing the red fluorescent gene tomato under the control of Rel1/2-inducible promoters, one expressing GFP under the control of the defensin promoter, and one expressing RFP under the control of the PPO6 promoter. These immune-responsive transgenic lines are designed to change color upon challenge with bacteria, viruses and parasites thereby allowing the performance of complex genetic screening as well as the analysis of parasite-vector interactions. These lines have been used to monitor natural bacterial infections, detected by the expression of the fluorescent reporter, providing a fast and reliable method to assess possible infections ongoing in insectary conditions thereby providing a formidable asset for the mosquito line repository developed in WP2. We have also mimicked in semi field condition the spead of a GFP asaia strains in large mosquito population thereby assessing for the first time the infection dynamics of a bacteria species in a target mosquito population. Furthermore wild type and reared mosquito population have been investigated to assess the microbiota profile to investigate how different bacteri species affect mosquito behaviour, ability to transmit malaria and longevity.
Work Package 7 Analyze population structure of mosquito species: Genetic methods of vector control will depend on a good knowledge of how novel genetic constructs will spread through a wild mosquito population, therefore baseline data of genetic diversity and population structure of target populations will be needed. This information has facilitated the design of experiments to be carried out at the confined field facility of INFRAVEC. Mosquito population structure has been analysed using high-throughput next-generation sequencing on a reduced portion of the genome using RADseq that allows many individual samples to be bar-coded and multiplexed on one Illumina lane with good coverage of each sequence, in effect doing SNP discovery and genotyping in one experiment. The data gathered allowed the investigation of genomic variation at over 100,000 separate sites, identifying over 5000 genome-wide single nucleotide polymorphisms (SNPs). Analysis of this data in the current reporting period identified very different population histories among different species (A. gambiae, A. arabiensis) at different locations and has been published (O’Loughlin et al 2014, MBE). The novel SNPs identified from RADseq have been made available on Dryad Digital Repository (doi:10.5061/dryad.hm6tt) and are in the process of being formatted for publication on the public data repository Vectorbase (www.vectorbase.org). Analysis is ongong to explore the use of RADseq for detecting recent population size changes. Simulated and real data suggest that it possible to detect a reduction in population size that occurred as recently as 100 generations ago using linkage disequilibrium analysis. This makes RADseq a potential tool for monitoring populations post-control interventions. As described in the previous report, we sequenced, assembled and annotated RNAseq libraries derived from antennal RNA from samples of four Ae. albopictus populations from Asia (Thailand) and Europe (Greece, and two North Italian populations in order to develop SNP markers related to chemoreception in collaboration with IMPERIAL and EMBL-EBI. Within the current time period we have continued our analyses of these data. Across the four libraries, considering all the transcripts, total of 958,216 high quality SNP loci were identified. Of these, 1947 were within the 78 Or transcripts and 463 in the 44 Obp transcripts. The predicted effects of the variants were determined manually using CLC Main Workbench 6 (http://www.clcbio.com). Considering only the SNPs in the Obps, over 98% of the SNP loci were bi-allelic. Almost 82% of the SNPs represented synonymous substitutions and only 13% of the SNPs resulted in amino acid substitions in the mature protein. The effects of the SNP variants in the Or transcripts are currently being assessed. These results will also benefit WP7, task 7 “Web-based portal to variation data in the context of available genome sequence data for the mosquito species”. This work resulted in the development of a data base of SSR fingerprints from nine geographic populations, two native Asian and seven adventive populations, which enabled us to 1) determine the degree of fluctuation of allele frequencies in space, and 2) to infer the dispersal patterns. SNP loci identified within transcripts related to chemoreception are in public domain.
Work Package 8 Validation of GM mosquito control measures under confined field conditions: GM technology has the potential to simplify the implementation of traditional pest control measures (e.g. SIT) by automating the hurdle of male and female separation through the expression of sex specific fluorescent markers or by using sex-specific toxic genes (as in WP6). The objective of this work package has been to leverage the formidable research capacity mobilized by INFRAVEC to validate in terms of cost, efficacy and safety GM mosquito technology for vector control. We have utilized laboratory reared mosquitoes to asses to key traits that are anticipated to predict the success of any genetic control measure: 1) the mating competitiveness defined as the ability of transgenic males to mate wild-type females (for ethical and safety reason only males are released as females contribute to diseases transmission); and 2) the overall fitness of the transgenic population defined as the ability of the transgenic mosquitoes to effectively compete with wild-type mosquitoes. Experiments were carried out in large confined enclosures to mimic more closely field conditions. These preliminary experiments have proved critical for the correct setting up of the confined field facility at UNIPG and at validating protocol to maximize its utilization.
The large cage containment facility located in Perugia has been fully developed into a functional venue for population simulations and performance measurement of promising genetic control technologies. As indicated by numerous expressions of interest and the access calls granted, there is widespread need for such a facility. All of the applicants thus far are expanding projects previously performed in small cages but which they expect to test more realistically in large cages. Many plan to include the large cage experiments in the first publication of the technology, indicating that such tests will become a routine part of technology rollout. The unique feature for this facility is that for the first time it has been incorporated the capacity to breed mosquito population, to sort them with automated high throughput technology and to test them for desired behavioural traits in semi-field confined condition. In this current reporting period we have assessed the competitiveness of GM male mosquitoes through testing in the large semi-field cage facility at UNIPG. Further work has been performed on the competiveness on GM mosquitoes carrying different transgenes at the same genetic locus in mating experiments in the large cage facility at UNIPG. Importantly, the full repertoire of courtship and mating behaviour can be replicated in this environment. It was established that GM mosquitoes had more reduced mating competitiveness in this environment than previously revealed in lab experiments.
Transnational access activities
Access to the consortium facilities has been offered to external researchers or research teams via a grant responsive mode. Information about the infrastructure facilities, the content of the units of access available and the modalities of participation in a call have been made available to potential users via the dedicated sections of the INFRAVEC website. In order to submit proposals, a registration phase and a dedicated selection procedure were put in place to ensure that potential applicants and their proposed research comply with the programme criteria.Since the project start, TNAccess calls have been launched on a rolling basis with monthly and bimonthly cut off dates for proposals collection and evaluation. INFRAVEC calls offered funding either through “activity packages”, that is coherent experimental work activities including one or more units of access to the selected facility, or through stand-alone access units. The majority of transnational access projects were implemented over the final part of the project. Upon receipt of the Information on the outcome of your INFRAVEC proposal letter, successful candidates were urged to contact the Facilities contact person to discuss the details of their projects. This passage proved to be critical in the TNA implementation and the construction of the report by the individual facilities, as it helped them to plan the necessary work and record the actual “quantity” of TNAccess activities offered to each user. In fact, the INFRAVEC registration and proposal submission tool was designed to enable applicants to select the type of unit/activity package requested, rather than the actual quantity (as in one or more units). Such calculation has been recorded at the facilities for each awarded proposal, after discussing the project’s technical details with the users.The Mosquito Mass Rearing and the Mosquito Confined Release Facilities met the expected level of TNA activities originally planned, whereas the Mosquito Genetic Facility at IMPERIAL recorded a significant deviation in the way the services have been requested and offered, as in the case of the higher number of subscribtions for HTP sequencing than expected. The Bio-informatics Facility at EMBL-EBI carried out as part of their TNA activities efforts the Mosquito Informatics Workshops for Vector Biologists (1st and 2nd Edition) in 2012 and 2014.
A total of 125 registrations were made out of which 84 proposals were submitted by individual researchers or research teams coming from different countries and research institutions or universities. 27 users attended the Bioinformatics Workshop for Vector Biologists at EMBL-EBI (1st and 2nd edition). 48 projects have been awarded and implemented TNA funding, whereas a few projects were withdrawn by the users after the award. The most popular unit was high-throughput sequencing, which was awarded both as a stand-alone unit and as part of an activity package that also included bioinformatics analisys. As for the activity packages, the two most subscribed were Development of genetically modified mosquitoes and Transcription profile analysis.
Potential Impact:
INFRAVEC has been crucial for consolidating European leadership in the field of vector biology, for progressing towards the development of novel genetic control measures and for assisting African laboratories in their effort to develop independent research profiles. This has been achieved by providing resources for implementing research projects, for establishing collaborative links, and for supporting individual researchers to carry out projects in the laboratories forming the project infrastructure facilities.
A number of important scientific advances have been achieved and published on more than 30 articles featuring on high impact scientific journals.
Crucial progresses have been made towards the objective of understanding at the genetic level the population structure of Anopheles gambiae mosquitoes culminating with the sequencing of and M and S genetic forms and the identification of previously unrecognized species. New sophisticated bioinformatics approaches have been developed to keep pace with unprecedented developments in sequencing technologies. New gene manipulation technologies have been developed that allow the selective targeting of mosquito genome sequences and the engineering of large mosquito populations. A confined release facility that can closely reproduce field environmental conditions in terms of light exposure, humidity, and temperature – including stochastic climate variability – has been utilized to assess the ecology of both field and genetically manipulated mosquitoes. Over 40 research projects have been granted access involving analysis of mosquito genetics, genome sequencing, bioinformatics, and gene manipulation to facilitate the sharing of technological platforms and enhance the research capacity of individual laboratory research. Access, measured in ‘units’ or in ‘activity packages’ (a collection of units), includes logistical, technological, and scientific support in the form of either service or specific training that is normally provided to external researchers using the given infrastructure. INFRAVEC has also promoted the formation of a mosquito line repository facility by coordinating different laboratories to stock, in their insectaries, naturally occurring and genetically modified mosquito lines together with the formation of a database containing genetic and biological information of each line. Coordination ensures that agreed standards have been implemented across different laboratories and that external users may have access to a unique collection of mosquito lines upon request through the INFRAVEC website. The consortium has also supported the development of a high-throughput technology for the automated sorting of large numbers of mosquitoes. A sorting instrument has been developed in collaboration with Union Biometrica Inc. to separate hundreds of thousands of mosquito larvae daily on the basis of the expression of fluorescent markers in tissues and organs. This technology has proven extremely useful to dramatically increase the yield of mass rearing and the throughput of genetic screens.
Three individual reports detailing the initial, interim and final dissemination plan have been issues to describe the consortium dissemination strategy and communication efforts. In particular, the spectrum of INFRAVEC’s dissemination activities and procedures included:
a) Organization of and participation at international events and conferences
INFRAVEC’s consortium members attended a number of international events, where both individual and cluster scientific results were presented. Non-exhaustive lists of conferences, project meetings, symposia and other international events attended by members of INFRAVEC were included in each dissemination report.
b) Scientific publications
Publishing high quality scientific articles in peer reviewed and internationally renowned journals was identified as a key dissemination element. Since the beginning of the projects a large number of publications were produced and listed in each reporting period. A comprehensive list of INFRAVEC-related publications features in the Links & Publications section of the project website.
c) Generic dissemination material such as brochures, dissemination on scientific media, fact sheets, newsletters
Additional dissemination activities carried out by the consortium members included links to and from the project website (from networks of excellence like EVIMALAR to innovative business services catalysers like the Genomics, Genetics and Biology Innovation Pole to scientific journals such as Pathogens and Global Health). Oral communications (posters and presentations) were held by INFRAVEC members at the national and international scientific events previously listed and on universities video walls and other high-tech dissemination tools.
d) The project website
The website represents the primary source of dissemination of news and information about INFRAVEC activities and it also features a common-restricted access platform for information exchange within the consortium. Since its creation, the INFRAVEC website has been regularly updated with new sections and additional dedicated web pages, as well as updates on the project documentation. The effectiveness of the project website as a dissemination tool is strongly dependant on its visibility through the main internet searching engines, (i.e. Google, Mozilla Firefox, Yahoo Search, Bing, etc.).Therefore, the public website has been properly designed and structured in order to ensure that it is displayed within the very first results as selected by such search engines. Selected key-words for the search engines relevant for the content of the project includes for instance the short name of the project, and terms like mosquito informatics (pointing to the workshops held by the European Bioinformatics Institute), access calls and vector control.
The Member Area of the website which can be accessed directly through the public website, allows members of the project to manage, store, download and share the whole project documentation: deliverables, presentations, relevant documents, templates, reports, results of technical reviews, etc.
e) Links and collaborations with other consortia
The dissemination activities implemented by INFRAVEC are intended on two levels: i) a level where each partner was responsible for presenting their activity and contribution to the project individually, within their own networks, at local or national level and ii) a more centralized level, which supports the visibility of the whole project and its results.
Most of the consortium efforts have been directed to disseminate INFRAVEC research findings as widely as possible; identify target audiences and encouraged participation in the different project activities. The Executive Committee is currently engaged in building awareness of the project, and the opportunities offered by the infrastructure to the scientific community among the different stakeholders to ultimately secure a commitment to the advancement of the INFRAVEC goals.
List of Websites:
The project website is www.infravec.eu. The Project Coordinator is Professor Andrea Crisanti, Professor of Molecular Parasitology, Faculty of Natural Sciences, Department of Life Sciences, Imperial College London. Project Management Office e-mail address: infravec@imperial.ac.uk.
Mosquitoes are known for transmitting a wide variety of infectious diseases that cause a tremendous burden to public health. Due to climate changes and to the increase in international trade and tourism the threats posed by mosquitoes are increasingly affecting large parts of Europe, causing understandable concerns among the populations of many Member States, particularly in the southern parts on the continent. Control methods, mainly based on the use of insecticides, are struggling to cope with the challenges posed by the biology and ecology of mosquito vectors. INFRAVEC aimed to bridge the gap between the recent advances in transgenic technology and its implementation as a novel powerful approach for vector control. To this aim, a large European Infrastructure has been put together, in which the coordination of efforts, expertise and facilities provided by the participating research groups and institutions contributed to expand the overall research capabilities of the research community. Four Infrastructure facilities have been integrated in the project: 1) the Mosquito Genetic Facility at Imperial College London; 2) the Mosquito Mass-rearing facility at the Centro Agricoltura ed Ambiente, CAA (which operates in close collaboration with the International Atomic Enargy Agency; 3) the Cambridge-based Bioinformatics facility of EMBL-EBI; and 4) the Mosquito Confined Release facility managed by UNIPG and offered a formidable research capability to external users that have applied for the services offered by the project and implemented five research projects aimed at utilizing basic knowledge of mosquito genetics and biology in an unprecedented effort to develop novel opportunities for mosquito control. INFRAVEC carried out a number of Networking, Joint Research, Transnational and Service activities, towards the objective of strenghthening research capability in Europe by sharing knowledge, resources and technology. These are: provision of access to the Infrastructure facilities; establishment of a mosquito line repository; development of mosquito high throughput sorting technologies; development of Ae. albopictus gene database; improvement of gene manipulation technologies; targeting mosquito vectorial capacity and EST sequencing; population structure analysis of mosquito species; validation of GM mosquito control measures under confined field conditions.
Project Context and Objectives:
INFRAVEC addresses the need of the scientific community to share facilities and integrate cuttingedge knowledge and technologies that are not readily accessible but nevertheless critical to exploit genetic and genomic information in the effort to control mosquito-borne diseases. Its objective is to provide laboratories that currently operate individually with limited coordination and little sharing of technologies, with the collective research capacity of the laboratories forming the core project infrastructure. INFRAVEC has provided resources to 31 institutions from European and African countries to enhance collaborative links, to execute joint research activity, and most importantly to enable individual researchers (from PhD students to established academics) to carry complex experimental activities by assigning research packages or ‘infrastructure access’ to be executed in the laboratory facilities and infrastructures of INFRAVEC. European laboratories have made crucial contributions in the field of mosquito molecular biology, genetics, biology, and epidemiology and have established a number of valuable facilities to perform insect mass rearing, mosquito genetic manipulation, single nucleotide polymorphism (SNP) population typing, and confined release experiments. These laboratories have so far mainly operated individually with limited coordination and little sharing of infrastructure and technologies. INFRAVEC has the objective to integrate these facilities into a coordinated infrastructure to promote scientific exchanges, facilitate the sharing of technological platforms, and enhance the research capacity of the scientific community. Its activities are articulated in networking, joint research projects, and promoting the use of the facility infrastructure by individual researchers. Networking activities have been aimed at improving and utilizing the facilities of the infrastructure as well as at strengthening and facilitating collaborative projects between institutions to improve, assess, and validate genetically manipulated mosquitoes for vector control. They include the development and maintenance of common facilities, such as the formation of a mosquito line repository facility by coordinating different laboratories to stock naturally occurring and genetically modified (GM) mosquito lines together with the formation of a database containing genetic and biological information of each line. INFRAVEC has supported four joint research and development activities aimed at (i) improving mosquito gene manipulation technologies, (ii) targeting the mosquito vectorial capacity, (iii) analyzing population structure and gene flow in mosquito species, and (iv) validating GM mosquitoes for vector control under confined field conditions. These projects were leveraged by access to the infrastructure with a range of complementary facilities to provide the research projects with a formidable capability to undertake complex experimental activities. The infrastructure of INFRAVEC consists of four laboratory facilities based in different European countries including (i) the insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna (Italy), (ii) the mosquito genetic manipulation laboratory at Imperial College London (UK), (iii) the mosquito confined release facility at the Biotechnology Centre of the University of Perugia (Italy), and (iv) the bioinformatics suite for data collection software development and information sharing at EMBL-EBI Cambridge (UK). Some of these facilities such as the bioinformatics suite at EMBL-EBI are widely utilized by the scientific community, whereas others offer their services sporadically and without coordination. INFRAVEC aims at integrating this infrastructure with networking activities and joint research projects to serve the mission of overcoming the existing capacity roadblock on the way to assess ethical, safety, efficacy, and feasibility issues of genetically manipulated mosquitoes for vector control. Simultaneously, INFRAVEC supports individual researchers to carry out research packages in the infrastructure facilities operated by the participants. The infrastructures are made available to external researchers or teams via a grant responsive mode. INFRAVEC publicizes the services offered by the infrastructure to external users and implements a peer review selection process to identify the projects that will be given support to utilize the infrastructure and contribute to the further development of the joint research activities.
Project Results:
INFRAVEC integrates in a multi-centre European Infrastructure a number of academic and industrial laboratories operating in the field of mosquito biology, genetics, epidemiology, genetic engineering, ecology and population biology. The project objectives have been to provide the participating laboratories and the European scientific community with a formidable leverage to progress in the understanding of mosquito biology and to develop new control measures against vector-borne diseases. A number of laboratory facilities have been integrated in a coherent manner within a single operating Infrastructure, which includes:
i) The insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna (Italy);
ii) The mosquito genetic manipulation laboratory at Imperial College London (UK);
iii) The mosquito confined release facility at the University of Perugia (Italy):
iv) The bioinformatics suite for data collection software development and information sharing at EMBLEBI Cambridge (UK).
The project has been articulated in networking and joint research activities specifically designed to improve the performance of the Infrastructure and to increase its capacity.
The networking activity included:
a) A multi-centre mosquito line repository facility to coordinate the implementation of standardized quality controls and the distribution to external users of both genetically manipulated and field-derived mosquitoes;
b) The development of a technology for the high-throughput sorting of mosquitoes on the basis of the expression of fluorescent markers in organs and tissues;
c) The sequencing and the annotation of the Ae. albopictus genome and tissue RNA sequencing to provide the scientific community with the knowledge on genome sequence, conservation and divergence between different mosquito species.
INFRAVEC also supported four collaborative research projects that aimed at strengthening the research capability and the scientific excellence towards the effort of expanding the basic knowledge of mosquito biology and of addressing the major bottlenecks in the development of vector control measures based on genetically modified (GM) mosquitoes. The following are the collaborative research projects implemented:
a) Improve gene manipulation technologies. The objective was to adapt the existing gene transfer technologies to mosquito species of emerging medical importance such as Ae. albopictus; and to develop a gene targeting technology in An. gambiae to selectively manipulate genes of interest. These advancements have significantly contributed to expanding the research capability of both the mosquito genetic manipulation laboratory at Imperial College London and the mosquito confined release facility at the University of Perugia;
b) Targeting the mosquito vector capacity. The aim was to generate a collection of An. gambiae mosquito lines in which genes essential for the mosquito’s ability to transmit disease have been identified and selectively disrupted. This work has provided the infrastructure and the participating laboratories with reference tools for addressing fundamental biological questions related to mosquito biology such as immunity and reproduction, and has enabled the identification of mosquito genes to be used in vector control measures;
c) Analyze population structure of mosquito species. The objective was to perform a systematic analysis of polymorphic loci in wild and laboratory Ae. albopictus populations. SNP frequencies and inheritance patterns will be used to monitor gene flow in field mosquitoes and to identify genetic bottlenecks when adapting mosquitoes to laboratory rearing conditions and mass production. This information will be invaluable for the correct functioning of the insect mass rearing facilities at the Centro Agricoltura Ambiente in Bologna;
d) Validation of GM mosquito control measures under confined field conditions. Large numbers of GM mosquitoes will be released in large semi-natural cages exposed to environmental conditions to assess the fitness of GM mosquitoes under confined “field” conditions and their efficacy in vector control methods. These experiments will be of paramount importance for assessing possible safety and environmental risks and for addressing regulatory requirements for subsequent field releases. The results of these experiments will provide guideline for future experiments and provide the rationale for adapting the structure of the facility for future tasks.
Work Package 2 Development of a mosquito line repository: A number of mosquito strains have been collected over years in remote locations in Asia and Africa and or genetically modified at the participating laboratories. They represent formidable models to study fundamental mechanisms of speciation, adaptation and evolution, whereas others exhibit unique properties resulting from natural diversity, tedious laboratory selection or transgenesis. A number of drawbacks prevents these invaluable resources from being utilized to their full power: (i) severe space limitations, especially in the laboratories producing GM mosquitoes; (ii) lack of information about the existing stocks; (iii) absence of efficient methods for molecular authentication; (iv) lack of standardized breeding procedures; (v) lack of control for persistent or cryptic pathogen infections (e.g. microsporidia infections). This WP promoted the formation of a mosquito stock repository facility by (i) coordinating efforts of the participating laboratories to stock mosquito lines in their insectaries; (ii) establishing and implementing standardized procedures for stock quality control; (iii) providing the community with centralized access to the lines/strains through a web-based database containing genetic and biological information for each line. The repository is designed to provide standardized material to individual laboratory across the world for studies aimed at investigating mosquito biology and at developing new control measure but most importantly dramatically enhances the research capability of the infrastructure facility, by overcoming limitations of breeding and maintaining mosquito lines due to logistics and resource constrains.
The mosquito stock census was completed and progress was made towards the implementation of standardized quality control processes and the addition of new lines. A dedicated informatics platform to implement the repository activities was developed and made available to the scientific community via the project website. A number of participating laboratories including IMPERIAL, UNIPG, UNIRM, CNRS, CNRFP, UNICAM, ICIPE, IP, KEELE provided information about the genetics, the biology and the geographic origin the Anopheles gambiae and Anopheles arabiensis mosquito lines that were kept in their laboratories. These include colonies from natural populations representing genetic diversity, mosquito colonies susceptible or refractory to Plasmodium development and mosquitoes showing different level of resistance to distinct classes of insecticides such as DDT, Deltamethrin, Lambda-cyhalothrin and Permethrin. The Anopheles gambiae PEST reference strain and M and S molecular forms were subjected to whole genome sequence to computationally extract a set of 1500 high-quality segregating polymorphic markers (SNPs) that were used to make a customized Illumina GoldenGate BeadArray genotyping chip. The chip was validated using a large collection of wild mosquitoes from Burkina Faso (IP). A sub-panel of 50 highly informative and robust markers has been derived based on the genotyping done to date. We have used this panel of markers as a genetic barcode for identifying mosquito strain on different genotyping platforms. The repository also includes lines derived from Aedes albopictus, an invasive mosquito species that has colonized vast regions of South Europe. This mosquito species has been responsible for the transmission of vector borne diseases in Italy Greece and Madeira. CAA has generated four Aedes albopictus lines using field samples collected in Italy and in Albania. One of these lines “Fellini” was subjected to a number of inbreeding crosses utilized to develop an “isofemale line” with limited genetic variability to serve as starting material for the “Aedes albopictus” genome-sequencing project (IMPERIAL).
Currently the repository has identified more than one hundred mosquito lines including Anopheles gambiae, Anopheles arabiaensis Aedes albopictus strains that are kept in the laboratories of the participating institutions. The information provided concerns the geographical origin, the biological characteristics and the phenotype of each individual line and stored in the database. Molecular probes have been distributed to the partners to carry out standardized genotyping assays in order to confirm the identity of the lines (UNIPG). Protocols have been implemented to standardize breeding conditions, genotyping and stock propagation. Procedures have been implemented to allow external users to access, request and deposit the lines of repository via a web interface.
Work Package 3 Development of mosquito high throughput sorting technologies: The objective of this work package was to develop an automated high throughput platform to screen mosquitoes for visible genetic markers. We have planned to develop a novel fully or semi-automated technologies for the screening of mosquitoes. This was achieved by two parallel approaches both based on adaptation of the "worm sorter" instrument COPAS (Complex Object Parametric Analyzers and Sorters), developed by Union Biometrica Inc. for Caenorhabditis elegans, to mosquito larvae. The development this technology dramatically expedites the identification of phenotypes under different experimental conditions. This has translated in a reduction of the workload and in enhanced research capacity of the infrastructure facilities particularly the mosquito genetic manipulation laboratory at Imperial College London and the mosquito confined release facility at the University of Perugia. Automated genetic screening reduces the time and effort to generate genetically manipulated mosquitoes and facilitates the analysis of their phenotype. This technology is also invaluable for its capability to selectively separate male and female mosquitoes on the basis of sex linked fluorescent markers for testing vector control measures under confined conditions. In this respect the sorting technology is critical to the successful implementation of different work packages including: (i) Networking Activities – WP2 ‘Development of a mosquito line repository’; (ii) Joint Research Activities – WP5 ’Improve GM technologies’, WP6 ‘Targeting vectorial capacity’, and WP8 ‘Validation of GM mosquito control measure’.
The instrument specification were set, reviewed and the prototype design approved in a number of meeting involving CNRS, UNIPG, IMPERIAL, ISRIM, CAA and Union Biometrica’s representatives in London, Strasbourg and Boston. Though the instrument still needs some improvement through modifications to the pressure and flow settings it is now largely fit for purpose for the medium throughput screening of certain transgenic backgrounds. The instrument was originally designed for use at the Laboratory of Biotechnologies of the University of Perugia, though it is IMPERIAL that initially took the responsibility for supervising the installation and initial use of the instrument in London. After the installation of the COPAS at the Imperial College, the instrument was validated for high throughput sorting of mosquito larvae of various stages based on the expression of fluorescent marker proteins in the eyes, ventral nerve cord or in the male reproductive organs. Upon delivery two major activities were carried out: 1) the validation of protocols for the breeding of large quantities of mosquitoes; and 2) the adaptation of the existing technology to achieve high-throughput, efficient sexing on the basis of fluorescent phenotypes. The team at CNRS closely collaborated to the tests that were performed at Imperial College.
Work Package 4: Development of Ae. albopictus gene database: In contrast to An. gambiae and Ae. aegypti, there are currently limited Ae. albopictus genome resources available with no large scale genomic sequencing and few cDNA sequences in the public nucleotide databanks. The aim of this WP has been to address this limitations and generate a database containing the genome sequence of Ae. albopictus together with a large number of Expressed Sequence Tags (ESTs) from different organs and perform suitable bioinformatics analyses to cluster into consensus sequences, predict the coding open reading frames (ORFs) and provide automated functional annotations. This database was expected to provide the first large scale gene discovery platform for this mosquito species which is of fundamental interest to the general mosquito research community. Knowledge of the Ae albopictus genome and transcriptome is instrumental in enhancing the research capability of the mosquito genetic manipulation laboratory and the mass rearing facilities by facilitating the development of genotyping tools for the identification of wild and reared mosquitoes. The genomic data available to the scientific community was expected to become more readily available with the development of a database at EMBL-EBI on the most invasive mosquito species.
The scope of this work package has expanded significantly because of the unexpected decrease in sequencing costs and the introduction of new technologies. It was decided therefore that there was value in creating a genome sequence from a newly established Ae. albopictus reference strain. This approach has the advantage of not relying on homology with Ae. aegypti in order to assemble a full genome sequence. DNA for sequencing was generated using an isofemale line generated at IMPERIAL starting from an Ae albopictus laboratory strain established at CAA. DNA material was shipped to the sequencing laboratory “GenePool” for the construction of genomic sequencing libraries. A small insert library (200 bp paired-end) has been constructed using the female DNA. After several attempts GenePool failed to construct a long insert mate-pair library. This task was transferred to UNIPG that were able to generate the mate-pair library Ae. albopictus genome has been sequenced at the University of Edinburgh's GenePool facility (200 bp paired-end) and University of Perugia (mate-pair library). The sequencing strategy assumes an estimated genome size similar to that of Ae. aegypti and therefore we used 20 lanes of Illumina sequencing given current production capabilities at the facility. The libraries were sequenced using the Illumina HiSeq 2000 platform in 2x101 nucleotide paired-end reads. Sequencing was performed using 2 lanes of the flow-cell, yielding ~220 million reads per cell. The data has been analyzed under a bioinformatic access project with INFRAVEC. Annotation of the genome has been achieved using automated procedures. The annotation process has been completed in three phases: 1) Identification and masking of repetitive sequence; 2) gene prediction; 3) prediction of gene function. The genome annotation pipelines included MAKER (http://gmod.org/wiki/MAKER). EMBL-EBI now uses MAKER as the default annotation pipeline to integrate the results from predictive algorithms into a single reference gene prediction set.
Transcription analysis has been carried on different tissues and organs. UNIPV in collaboration with the team at IMPERIAL Illumina RNA-seq libraries have been sequenced and analysed (EMBL-EBI) in order to identify transcripts involved in chemoreception. This information has also been used to support the annotation of the Ae. albopictus genome sequence, the development of an Ae. albopictus database resource and the identification of a large number of coding SNPs. Illumina libraries were prepared from the Ae. albopictus antennal RNA samples from four different geographic populations; Lampang Ban Rai (Thailand), Athens (Greece), Trento and Arco (North Italy). Poly-A mRNA in the total RNA samples was purified using Illumina poly-T oligo-attached magnetic beads. The poly-A RNA, the mRNA was also fragmented and primed with random hexamers for cDNA synthesis. The resulting libraries were validated using the Agilent 2100 bioanalyser to confirm the concentrations and size distribution and then normalized to 10nM concentration and sequenced using Illumina 2000 technology. The sequences have been assembled and annotated, transcription profiles were generated leading to the identification and characterization of genes expressed organ and tissues.
Work Package 5: Improve gene manipulation technologies: This work aimed to improve gene manipulation technology for An. gambiae and to adapt the existing technology to Ae. albopictus. We had planned on developing selective gene targeting methods to systematically disrupt mosquito genes involved in shaping the vectorial capacity i.e. those involved in sex determination, host-seeking behaviour, fertility and immunity. The full potential of genetic manipulation is well appreciated in studies aimed at investigating the biology of mouse and drosophila but has not been fully exploited in mosquitoes to investigate interactions with viruses and parasites and in developing environmentally safe GM insects for vector control. Important limitations are faced when studying the function of genes potentially involved in biological processes crucial for mosquito vectorial capacity such as fertility and sex differentiation, which cannot be efficiently targeted by transient RNA interference because of their temporal and spatial expression patterns. Furthermore, the use of transposons aimed to increase the transformation efficiency, raises safety and environmental issues that represent serious obstacles for the release of GM mosquitoes in the field as part of vector control measures. This relates to the stability of the genetic modifications, their unforeseen effect on other biological functions of the mosquito and their potential to spread to non-target insect species.
IMPERIAL, in collaboration with CNRS and KEELE, investigated the feasibility of developing a gene targeting technology based on the production of a recombinogenic linear molecule in the germline based on the concerted action of both the Isce1 endonuclease and the FLP recombinase on chromosomally-inserted transgenic targeting constructs. These efforts resulted in the successful validation of an alternative recombinase, Cre, that acts with high efficiency in the germline to excise Donor This approach yielded significantly more transgenic founders than the previous state-of-the-art, thereby translating in a significant improvement in efficiency and in a substantial cost reduction. Keele made major improvement to gene manipulation technology, particularly in relation to the utilization of phiC31 integrase to deliver DNA constructs at specific sites ‘docking sites’ (attP sequence). This revolved around the development of a piggyBac transformation construct, containing an attP sequence, a selectable marker, and a transcription unit encompassing the phiC31 integrase coding sequence (that directs the integration at the attP site) regulated by the germ-line specific nanos gene promoter. A series of attP strains were developed and characterized for their ability to uptake DNA sequences at specific sites. IMPERIAL in collaboration with CNRS developed additional lines that contain ‘docking sites’ dispersed at different genomic locations for future integrations of transgenes at pre-characterized genomic locations. Notably this technology has been used to edit the Y chromosome of A. gambiae swapping promoter and reporter genes at selected sites. Marked Y chromosomes were used to monitor the introgression of this chromosome in the related species A. arabiensis. CNRS and Imperial have also collaborated at the validation of the Cas-CRISP technology to selectively edit sequences in the anopheles gambiae genome. These studies relevealed that if CAS gene is provided as stable germ line integration the guide RNA techonology can be used to target specific sequences. In addition HEG technology proved to be a valuable and effective approach to selectively target gene sequences.
Work Package 6 Targeting mosquito vectorial capacity and EST sequencing: This work package aimed at developing a suite of novel tools based on fluorescent and luciferase reporters and on targeted gene disruption to address essential questions related to the mosquito biology and vectorial capacity. The availability of these lines were expected to dramatically increase the research capability of the infrastructure toward the objective of developing genetic vector control measures targeting malaria transmission. A number of diverse strategies have been put in place for the successful implementation of this workpackage objectives, targeting the different processes of mosquito biology that are relevant to disease transmission.
We have established a collection of An. gambiae transgenic fluorescence- or luciferase-based reporter lines in which promoter regions of immune response genes control the expression of fluorescent reporter markers. CNRS established additional transgenic reporter An. gambiae lines to follow activation of the immune system: one expressing GFP under the control of the TEP1 promoter, one expressing Luciferase and one expressing the red fluorescent gene tomato under the control of Rel1/2-inducible promoters, one expressing GFP under the control of the defensin promoter, and one expressing RFP under the control of the PPO6 promoter. These immune-responsive transgenic lines are designed to change color upon challenge with bacteria, viruses and parasites thereby allowing the performance of complex genetic screening as well as the analysis of parasite-vector interactions. These lines have been used to monitor natural bacterial infections, detected by the expression of the fluorescent reporter, providing a fast and reliable method to assess possible infections ongoing in insectary conditions thereby providing a formidable asset for the mosquito line repository developed in WP2. We have also mimicked in semi field condition the spead of a GFP asaia strains in large mosquito population thereby assessing for the first time the infection dynamics of a bacteria species in a target mosquito population. Furthermore wild type and reared mosquito population have been investigated to assess the microbiota profile to investigate how different bacteri species affect mosquito behaviour, ability to transmit malaria and longevity.
Work Package 7 Analyze population structure of mosquito species: Genetic methods of vector control will depend on a good knowledge of how novel genetic constructs will spread through a wild mosquito population, therefore baseline data of genetic diversity and population structure of target populations will be needed. This information has facilitated the design of experiments to be carried out at the confined field facility of INFRAVEC. Mosquito population structure has been analysed using high-throughput next-generation sequencing on a reduced portion of the genome using RADseq that allows many individual samples to be bar-coded and multiplexed on one Illumina lane with good coverage of each sequence, in effect doing SNP discovery and genotyping in one experiment. The data gathered allowed the investigation of genomic variation at over 100,000 separate sites, identifying over 5000 genome-wide single nucleotide polymorphisms (SNPs). Analysis of this data in the current reporting period identified very different population histories among different species (A. gambiae, A. arabiensis) at different locations and has been published (O’Loughlin et al 2014, MBE). The novel SNPs identified from RADseq have been made available on Dryad Digital Repository (doi:10.5061/dryad.hm6tt) and are in the process of being formatted for publication on the public data repository Vectorbase (www.vectorbase.org). Analysis is ongong to explore the use of RADseq for detecting recent population size changes. Simulated and real data suggest that it possible to detect a reduction in population size that occurred as recently as 100 generations ago using linkage disequilibrium analysis. This makes RADseq a potential tool for monitoring populations post-control interventions. As described in the previous report, we sequenced, assembled and annotated RNAseq libraries derived from antennal RNA from samples of four Ae. albopictus populations from Asia (Thailand) and Europe (Greece, and two North Italian populations in order to develop SNP markers related to chemoreception in collaboration with IMPERIAL and EMBL-EBI. Within the current time period we have continued our analyses of these data. Across the four libraries, considering all the transcripts, total of 958,216 high quality SNP loci were identified. Of these, 1947 were within the 78 Or transcripts and 463 in the 44 Obp transcripts. The predicted effects of the variants were determined manually using CLC Main Workbench 6 (http://www.clcbio.com). Considering only the SNPs in the Obps, over 98% of the SNP loci were bi-allelic. Almost 82% of the SNPs represented synonymous substitutions and only 13% of the SNPs resulted in amino acid substitions in the mature protein. The effects of the SNP variants in the Or transcripts are currently being assessed. These results will also benefit WP7, task 7 “Web-based portal to variation data in the context of available genome sequence data for the mosquito species”. This work resulted in the development of a data base of SSR fingerprints from nine geographic populations, two native Asian and seven adventive populations, which enabled us to 1) determine the degree of fluctuation of allele frequencies in space, and 2) to infer the dispersal patterns. SNP loci identified within transcripts related to chemoreception are in public domain.
Work Package 8 Validation of GM mosquito control measures under confined field conditions: GM technology has the potential to simplify the implementation of traditional pest control measures (e.g. SIT) by automating the hurdle of male and female separation through the expression of sex specific fluorescent markers or by using sex-specific toxic genes (as in WP6). The objective of this work package has been to leverage the formidable research capacity mobilized by INFRAVEC to validate in terms of cost, efficacy and safety GM mosquito technology for vector control. We have utilized laboratory reared mosquitoes to asses to key traits that are anticipated to predict the success of any genetic control measure: 1) the mating competitiveness defined as the ability of transgenic males to mate wild-type females (for ethical and safety reason only males are released as females contribute to diseases transmission); and 2) the overall fitness of the transgenic population defined as the ability of the transgenic mosquitoes to effectively compete with wild-type mosquitoes. Experiments were carried out in large confined enclosures to mimic more closely field conditions. These preliminary experiments have proved critical for the correct setting up of the confined field facility at UNIPG and at validating protocol to maximize its utilization.
The large cage containment facility located in Perugia has been fully developed into a functional venue for population simulations and performance measurement of promising genetic control technologies. As indicated by numerous expressions of interest and the access calls granted, there is widespread need for such a facility. All of the applicants thus far are expanding projects previously performed in small cages but which they expect to test more realistically in large cages. Many plan to include the large cage experiments in the first publication of the technology, indicating that such tests will become a routine part of technology rollout. The unique feature for this facility is that for the first time it has been incorporated the capacity to breed mosquito population, to sort them with automated high throughput technology and to test them for desired behavioural traits in semi-field confined condition. In this current reporting period we have assessed the competitiveness of GM male mosquitoes through testing in the large semi-field cage facility at UNIPG. Further work has been performed on the competiveness on GM mosquitoes carrying different transgenes at the same genetic locus in mating experiments in the large cage facility at UNIPG. Importantly, the full repertoire of courtship and mating behaviour can be replicated in this environment. It was established that GM mosquitoes had more reduced mating competitiveness in this environment than previously revealed in lab experiments.
Transnational access activities
Access to the consortium facilities has been offered to external researchers or research teams via a grant responsive mode. Information about the infrastructure facilities, the content of the units of access available and the modalities of participation in a call have been made available to potential users via the dedicated sections of the INFRAVEC website. In order to submit proposals, a registration phase and a dedicated selection procedure were put in place to ensure that potential applicants and their proposed research comply with the programme criteria.Since the project start, TNAccess calls have been launched on a rolling basis with monthly and bimonthly cut off dates for proposals collection and evaluation. INFRAVEC calls offered funding either through “activity packages”, that is coherent experimental work activities including one or more units of access to the selected facility, or through stand-alone access units. The majority of transnational access projects were implemented over the final part of the project. Upon receipt of the Information on the outcome of your INFRAVEC proposal letter, successful candidates were urged to contact the Facilities contact person to discuss the details of their projects. This passage proved to be critical in the TNA implementation and the construction of the report by the individual facilities, as it helped them to plan the necessary work and record the actual “quantity” of TNAccess activities offered to each user. In fact, the INFRAVEC registration and proposal submission tool was designed to enable applicants to select the type of unit/activity package requested, rather than the actual quantity (as in one or more units). Such calculation has been recorded at the facilities for each awarded proposal, after discussing the project’s technical details with the users.The Mosquito Mass Rearing and the Mosquito Confined Release Facilities met the expected level of TNA activities originally planned, whereas the Mosquito Genetic Facility at IMPERIAL recorded a significant deviation in the way the services have been requested and offered, as in the case of the higher number of subscribtions for HTP sequencing than expected. The Bio-informatics Facility at EMBL-EBI carried out as part of their TNA activities efforts the Mosquito Informatics Workshops for Vector Biologists (1st and 2nd Edition) in 2012 and 2014.
A total of 125 registrations were made out of which 84 proposals were submitted by individual researchers or research teams coming from different countries and research institutions or universities. 27 users attended the Bioinformatics Workshop for Vector Biologists at EMBL-EBI (1st and 2nd edition). 48 projects have been awarded and implemented TNA funding, whereas a few projects were withdrawn by the users after the award. The most popular unit was high-throughput sequencing, which was awarded both as a stand-alone unit and as part of an activity package that also included bioinformatics analisys. As for the activity packages, the two most subscribed were Development of genetically modified mosquitoes and Transcription profile analysis.
Potential Impact:
INFRAVEC has been crucial for consolidating European leadership in the field of vector biology, for progressing towards the development of novel genetic control measures and for assisting African laboratories in their effort to develop independent research profiles. This has been achieved by providing resources for implementing research projects, for establishing collaborative links, and for supporting individual researchers to carry out projects in the laboratories forming the project infrastructure facilities.
A number of important scientific advances have been achieved and published on more than 30 articles featuring on high impact scientific journals.
Crucial progresses have been made towards the objective of understanding at the genetic level the population structure of Anopheles gambiae mosquitoes culminating with the sequencing of and M and S genetic forms and the identification of previously unrecognized species. New sophisticated bioinformatics approaches have been developed to keep pace with unprecedented developments in sequencing technologies. New gene manipulation technologies have been developed that allow the selective targeting of mosquito genome sequences and the engineering of large mosquito populations. A confined release facility that can closely reproduce field environmental conditions in terms of light exposure, humidity, and temperature – including stochastic climate variability – has been utilized to assess the ecology of both field and genetically manipulated mosquitoes. Over 40 research projects have been granted access involving analysis of mosquito genetics, genome sequencing, bioinformatics, and gene manipulation to facilitate the sharing of technological platforms and enhance the research capacity of individual laboratory research. Access, measured in ‘units’ or in ‘activity packages’ (a collection of units), includes logistical, technological, and scientific support in the form of either service or specific training that is normally provided to external researchers using the given infrastructure. INFRAVEC has also promoted the formation of a mosquito line repository facility by coordinating different laboratories to stock, in their insectaries, naturally occurring and genetically modified mosquito lines together with the formation of a database containing genetic and biological information of each line. Coordination ensures that agreed standards have been implemented across different laboratories and that external users may have access to a unique collection of mosquito lines upon request through the INFRAVEC website. The consortium has also supported the development of a high-throughput technology for the automated sorting of large numbers of mosquitoes. A sorting instrument has been developed in collaboration with Union Biometrica Inc. to separate hundreds of thousands of mosquito larvae daily on the basis of the expression of fluorescent markers in tissues and organs. This technology has proven extremely useful to dramatically increase the yield of mass rearing and the throughput of genetic screens.
Three individual reports detailing the initial, interim and final dissemination plan have been issues to describe the consortium dissemination strategy and communication efforts. In particular, the spectrum of INFRAVEC’s dissemination activities and procedures included:
a) Organization of and participation at international events and conferences
INFRAVEC’s consortium members attended a number of international events, where both individual and cluster scientific results were presented. Non-exhaustive lists of conferences, project meetings, symposia and other international events attended by members of INFRAVEC were included in each dissemination report.
b) Scientific publications
Publishing high quality scientific articles in peer reviewed and internationally renowned journals was identified as a key dissemination element. Since the beginning of the projects a large number of publications were produced and listed in each reporting period. A comprehensive list of INFRAVEC-related publications features in the Links & Publications section of the project website.
c) Generic dissemination material such as brochures, dissemination on scientific media, fact sheets, newsletters
Additional dissemination activities carried out by the consortium members included links to and from the project website (from networks of excellence like EVIMALAR to innovative business services catalysers like the Genomics, Genetics and Biology Innovation Pole to scientific journals such as Pathogens and Global Health). Oral communications (posters and presentations) were held by INFRAVEC members at the national and international scientific events previously listed and on universities video walls and other high-tech dissemination tools.
d) The project website
The website represents the primary source of dissemination of news and information about INFRAVEC activities and it also features a common-restricted access platform for information exchange within the consortium. Since its creation, the INFRAVEC website has been regularly updated with new sections and additional dedicated web pages, as well as updates on the project documentation. The effectiveness of the project website as a dissemination tool is strongly dependant on its visibility through the main internet searching engines, (i.e. Google, Mozilla Firefox, Yahoo Search, Bing, etc.).Therefore, the public website has been properly designed and structured in order to ensure that it is displayed within the very first results as selected by such search engines. Selected key-words for the search engines relevant for the content of the project includes for instance the short name of the project, and terms like mosquito informatics (pointing to the workshops held by the European Bioinformatics Institute), access calls and vector control.
The Member Area of the website which can be accessed directly through the public website, allows members of the project to manage, store, download and share the whole project documentation: deliverables, presentations, relevant documents, templates, reports, results of technical reviews, etc.
e) Links and collaborations with other consortia
The dissemination activities implemented by INFRAVEC are intended on two levels: i) a level where each partner was responsible for presenting their activity and contribution to the project individually, within their own networks, at local or national level and ii) a more centralized level, which supports the visibility of the whole project and its results.
Most of the consortium efforts have been directed to disseminate INFRAVEC research findings as widely as possible; identify target audiences and encouraged participation in the different project activities. The Executive Committee is currently engaged in building awareness of the project, and the opportunities offered by the infrastructure to the scientific community among the different stakeholders to ultimately secure a commitment to the advancement of the INFRAVEC goals.
List of Websites:
The project website is www.infravec.eu. The Project Coordinator is Professor Andrea Crisanti, Professor of Molecular Parasitology, Faculty of Natural Sciences, Department of Life Sciences, Imperial College London. Project Management Office e-mail address: infravec@imperial.ac.uk.