SECOND RESEARCH CO-ORDINATION MEETING Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
Co-ordinated Research Programme on
Explore genetic molecular, mechanical and behavioral methods of sex separation in mosquitoes
Scientific Secretary: Jeremie Gilles & Kostas Bourtzis
Moscamed Brazil Juazeiro, Brazil 9 – 13 March 2015
1 Contents PROPOSAL FOR A COORDINATED RESEARCH PROJECT (CRP) ...... 3 Title ...... 3 Budget Cycle ...... 3 Project ...... 3 Division ...... 3 Responsible Project Officer ...... 3 Alternate Project Officer ...... 3 Summary ...... 3 Background Situation Analysis: ...... 4 To explore irradiation and classical genetic approaches for sex separation in mosquitoes - development of GSS based on irradiation and classical genetics ...... 5 To explore molecular approaches for sex separation in mosquitoes - Development of GSS based on molecular genetics ...... 7 To explore mechanical, behavioural, developmental and symbiont-based approaches for sex separation in mosquitoes ...... 9 Selected References ...... 11 Nuclear Component ...... 18 Explanation / Justification: ...... 18 Participation of Agency's laboratories (Yes/No) ...... 18 Assumptions:...... 18 Related TC Projects ...... 18 LOGICAL FRAMEWORK ...... 20 Narrative Summary ...... 20 Specific Objectives ...... 21 Outcomes ...... 22 Outputs ...... 22 FUTURE ACTIVITIES ...... 26 AGENDA ...... 41 PARTICIPANT ABSTRACTS ...... 44 LIST OF PARTICIPANTS ...... 73
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PROPOSAL FOR A COORDINATED RESEARCH PROJECT (CRP)
Title: Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes.
Budget Cycle: 2013-2018
Project: Project 2.1.4.4 (Development of the SIT for the control of disease transmitting mosquitoes) as part of Sub-programme 2.1.4 (Sustainable Control of Major Insect Pests).
Division: NAFA
Responsible Project Officer: Jeremie Gilles
Alternate Project Officer: Kostas Bourtzis
Summary: Requests from Member States for exploration of the potential of applying the Sterile Insect Technique (SIT) against mosquitoes in area-wide integrated vector management (AW-IVM) programmes continue to increase. However, because female mosquitoes, unlike male mosquitoes, can transmit disease, means to eliminate them from the mass production process are a critical pre-requisite. In addition, not releasing sterile females would increase SIT programme efficiency due to the fact that sterile males can then focus only on achieving matings with wild females. Thus mosquito SIT programme efficiency and safety would be considerably enhanced by the development of improved strains for mass-rearing and release. These include strains that: (a) produce only male insects for release and (b) carry easily identifiable markers to monitor released males in the field. Although also assessing mechanical, behavioural and developmental approaches, this CRP will primarily explore classical genetic and modern biotechnology techniques to accomplish female elimination in major mosquito vectors of disease. Whenever possible, these methods will be created with a view to application to a wide spectrum of mosquito species. Major beneficiaries will be operational AW-IVM programmes in Member States that plan to apply the SIT (classical, transgenic, and/or symbiont-based approaches) against mosquitoes. By the end of the CRP, methods for developing sexing strains will have advanced and some strains will be available for evaluation. The development and evaluation of such methods through this CRP will have the following tangible benefits for SIT mosquito control programmes: 1.) Safety of SIT mosquito programmes will be enhanced. Mosquito SIT implementation cannot include the release of even sexually-sterile biting females. Therefore mosquito SIT requires the exclusive release of sterile males, which is impossible on a large scale without sex separation methods.
3 2.) Male-only releases are several-fold more efficient than releases of both sexes. Consequently, when genetic sexing technology is available SIT programmes are significantly more efficient and therefore more cost-effective. 3.) As only the males are needed for the SIT, the production, handling and release costs can be reduced significantly if sexing strains are used and females are eliminated early in development.
Background Situation Analysis:
Among the major vectors of human diseases, mosquitoes are the most devastating ones. Urbanisation, globalisation and climate change have further accelerated the spread and outbreaks of new mosquito-borne diseases. In view of the problems associated with conventional mosquito control, such as resistance and health effects, major efforts are required to develop new or complementary control techniques, including the SIT, for major mosquito species.
The Sterile Insect Technique (SIT) is an increasingly important component of area-wide integrated vector management (AW-IVM) programmes for key insect vectors such as mosquitoes. With the increase in vector-borne diseases and their toll on human health and mortality, there have been recurring requests from Member States to develop tools and techniques for mosquito SIT, including the development of sexing strains (GC(56)/RES/19) to be able to apply the SIT to control mosquito vector populations (Resolution GC(52)/RES/13). The SIT has the ability to suppress or in special situations to eradicate existing vector populations and to prevent the establishment of new outbreaks.
Operational use of the SIT in insect agricultural pest species continues to reveal areas where new technologies could further improve efficiency and thus lead to more efficacious programmes. There are many aspects of the mosquito SIT package that require increased efficiency to be able to reach the operational level, e.g. improved mass rearing, release technology, quality control, field monitoring, etc. However, one critical area where important advances need to be made before any SIT application is possible concerns the development of genetic sexing strains (GSS). Unlike agricultural pests where the release of both sexes is primarily of economic concern, in mosquitoes, it is an essential prerequisite to release only males since females are blood feeders and transmit disease. Without male-only releases, SIT applications against mosquitoes are not possible.
In respect to symbiont-based control methods, the Incompatible Insect Technique (IIT) relies on the sustained, inundative releases of Wolbachia-infected incompatible males to sterilize the targeted female population. The success of IIT could be affected by the accidental release of females infected with the incompatible Wolbachia type. As the population is suppressed, there is the risk that accidental female release would permit the establishment of the incompatible Wolbachia, leading to population replacement instead of population elimination.
Currently, AW integrated pest management (IPM) programmes with an SIT component have been successfully implemented for several very important fruit fly and lepidopteran species where the development of improved strains, especially GSS (in the case of fruit flies), led to
4 major increases in applicability and efficiency of the SIT component. The experience gained from these programmes would prime the new CRP for the development of GSS in mosquitoes.
There are several mosquito species that are major vectors of different pathogens in different countries. Of these, based on the severity of the disease, requests from Member States and the availability of scientific tools and information, the consultants recommended that the initial efforts to develop mosquito GSS should focus on the following species: Anopheles arabiensis, Anopheles stephensi, and Anopheles gambiae (vectors of malaria), and Aedes albopictus and Ae. aegypti (vectors of dengue, chikungunya, zika and other viruses).
Mosquito GSS development can be achieved using different approaches, but they all rely on some form of stable genetic change introduced and maintained in the developed strain. Genetic change can be introduced either using irradiation and classical genetics (as in the case of the Mediterranean fruit fly GSS) or modern biotechnology, specifically genetic transformation. Both approaches have advantages and disadvantages relating to transferability of systems between species, stability in mass-rearing, regulatory approval, etc. In addition, existing alternative sex separation methods based on mechanical, behavioural developmental and symbiont-based approaches should also be considered. However, it should be noted that these alternative methods are based on species-specific traits and are currently appropriate only for small scale operations, highlighting the need to develop state of the art sexing strategies based on genetic and molecular approaches for large scale SIT and non-SIT operations.
Developing sex separation methods for mosquitoes is a prerequisite for the use of SIT in an AW-IVM approach. Four specific objectives should be addressed:
1. To explore classical genetic approaches including irradiation for sex separation in mosquitoes
2. To explore molecular approaches for sex separation in mosquitoes
3. To explore mechanical, behavioral, developmental and symbiont-based approaches for sex separation in mosquitoes
4. To continue encouraging and attracting participants to the CRP in the field of classical genetics
To explore irradiation and classical genetic approaches for sex separation in mosquitoes - development of GSS based on irradiation and classical genetics One example of how strain improvement can significantly enhance SIT applicability and efficiency has been the use of GSS in the Mediterranean fruit fly, Ceratitis capitata AW-IPM programmes, a technology developed through the Agency’s CRP programme with support from the FAO/IAEA Agriculture and Biotechnology Laboratories in Seibersdorf. Using irradiation and classical genetic approaches, a series of genetic sexing strains were developed for the Mediterranean fruit fly. Several of them were evaluated and are currently being used in all mass-rearing facilities of this pest for large scale SIT and AW-IPM programmes, including the VIENNA 8 strains. These achievements in the Mediterranean fruit fly field later resulted in the adoption of irradiation and classical genetic approaches for the development of
5 GSS in other major tephritid species like the Mexican fruit fly (Anastrepha ludens), the melon fly (Bactrocera cucurbitae) and the Oriental fruit fly (Bactrocera dorsalis).
To develop efficient sex separation methods, including genetic sexing strains for mosquito SIT programmes, basic genetic and molecular tools are required as well as any available mechanical and behavioural methods. This section provides an overview of what genetic and molecular tools are currently available in the toolbox and illustrates that despite recent advances in this field, there is a urgent need for further improvement.
The sequence of the genomes of some 20 species of mosquitoes are currently available and are invaluable resources for the development of GSS using transgenic and genome modification technologies. The first mosquito GSS were developed in ‘70s, using classical genetic approaches. However, these strains did not prove to be very useful for various reasons including instability of the genotypes produced in the lab and essential determinants of the effectiveness of the GSS. While none of the original strains are currently available, one of the original mosquito GSS was recreated based on dieldrin resistance in An. arabiensis. The use of insecticide resistance as a selectable marker is unlikely to be acceptable for large scale applications for a number of reasons including: (a) protection of the environment and human health; (b) accuracy and (c) potential contamination of the mass-rearing colony.
In view of the lack of highly efficient GSS there is a major demand for the development of new GSS for target mosquito species, which would be based on the successful strategy followed for Mediterranean fruit fly and other fruit fly species. An ideal GSS must have two features: (a) the ability to maintain desirable genotypes using selectable or morphological markers (b) a mechanism to ensure sex-specific selection. In addition, the development of GSS allowing early elimination of females in the mass production process (ideally at the embryonic stage), should be preferred to improve cost efficiency. Thus, the development of a mosquito GSS using conventional genetic technologies would demand the following: (a) isolation and mapping of genetic markers (preferably morphological markers) from natural populations and/or laboratory populations following mutagenesis; most of these markers will be useful for genetic studies, some of which may meet the criteria for a selectable marker; (b) irradiation-induced chromosomal rearrangements (i.e inversions, translocations) and their characterization; (c) isolation of conditional early lethal mutations (for example tsl mutation) and (d) combination of the above components into a stable GSS. Making the decision about the most suitable selectable marker is crucial and will ultimately result in the construction of a robust GSS. At this point, it should be emphasized that the isolation of genetic markers from natural populations or random mutagenesis is a rather challenging and labour-demanding serendipitous process. The achievement of this goal will require the recruitment of a dedicated and fully committed expert, as well as sufficient technical support, to join the mosquito group at the IPCL, Seibersdorf. Despite the fact that the principles of the classical genetic approach are easily applicable to most species, the actual tools that will be developed for a given species cannot be transferred to another mosquito species. On the other hand, the benefit of the classical genetic approach is that no legal restrictions or other regulations apply and strains can be directly used for SIT application.
Expected outputs
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1. Genetic markers from different sources including natural populations and / or mutagen-based screens. These markers will facilitate genetic studies as well as the isolation of suitable selectable markers for the development of mosquito GSS.
2. Chromosomal rearrangements in the target mosquito species. These chromosomal rearrangements will facilitate genetic studies as well as the development of stable mosquito GSS.
3. (Cyto)genetic data from the analysis of the tools developed above in [1.] and [2.]. Cytogenetic analysis will facilitate genetic studies and the evaluation of the most suitable GSS.
4. Morphological and selectable markers. These markers will allow the construction of GSS.
5. A classical GSS. The GSS will consist of at least: (a) a selectable marker; (b) an inversion and (c) a sex chromosome / autosome translocation.
6. Data from the evaluation of a classical GSS on a small scale using standard quality control parameters. This evaluation will provide data on the actual stability and the overall fitness of the developed GSS.
To explore molecular approaches for sex separation in mosquitoes - Development of GSS based on molecular genetics
Enhancing SIT through the development of molecular-based genetic sexing strains has been proven successful for fruit flies, including the tephritid species C. capitata and A. suspensa, for which 100% female lethality has been achieved through a well-understood apoptotic pathway combined with sex-specific splicing. The application of transgenic systems has the advantage of a faster development time and the ability to be transferred to other species. In addition, an approach to generate a male-only population by sex reversion of females to males has been tested in C. capitata with very encouraging results. Some of the molecular- based GSS have been successfully evaluated under semi-mass rearing and field cage conditions with support from the FAO/IAEA Agriculture and Biotechnology Laboratories in Seibersdorf. These achievements will help to explore different options for molecular-based GSS in mosquito species.
To explore the possibility of generating molecular-based GSS in mosquitoes using female- specific lethal genes, the community should transfer existing systems and/or identify endogenous candidate genes with sex-specific expression patterns. The isolation and in vitro validation of species-specific promoters and lethal effector genes that have been recently achieved in fruit flies can be of great help for the construction of similar conditional lethal systems in mosquitoes. Gene knockdown studies using germ-line transformation for the embryonic and sex-determination genes developed for fruit flies could be adapted to achieve sex-reversal in the target mosquito species. Similarly, cassettes consisting of fluorescent markers under the control of male-specific promoters such as the β2-tubulin or newly isolated endogenous ones have been developed. In this context, the recent generation of an Anopheles
7 gambiae strain with a Y-linked attP for site-specific recombination has demonstrated the possibility for reliable automated sex separation based on expression of fluorescent markers located on the Y chromosome. To this end, recent developments in FACS technology would allow high throughput sorting for mosquito mass rearing requirements. However, this technology still needs improvement. An alternative to automated sexing based on fluorescent markers are genetic sexing strains harbouring positive selection traits that could be easily introduced via the Y-linked attP site.
To improve the development and ecological safety of genetically engineered (GE) mosquitoes created for enhanced biological control programmes, site-specific systems or recombination-mediated cassette exchange (RMCE) have been developed and evaluated in fruit flies and could be explored for different mosquitoes. These systems address potential transgene instability, and new transposon vectors that allow post-integration immobilization could be applied in mosquitoes with a focus on the mentioned target species. In addition, the random nature of genomic insertions via transposons is problematic for GE strain development due to genomic position effects that can influence transgene expression, and insertional mutations that negatively affect host fitness and viability. Diminished transgene expression could result in the unintended survival of conditional lethal individuals, or the inability to identify them. A solution to this problem could be the phiC31-attP site-specific integration system, which has already been developed for An. gambiae, An. stephensi, Ae. aegypti, and Ae. albopictus. Another versatile option for genetic modification would be RMCE systems, recently established for the first tephritid, Anastrepha suspensa based on the Cre-loxP elements and D. melanogaster based on an attP system. Alternative options for site- specific modification are the Flp-FRT or site-specific nuclease systems. Recently, the first successful application of engineered endonucleases such as CRISPR and TALENs were reported in Ae. aegypti and An. gambiae genomes. A major concern for molecular-based SIT approaches is the evolution of resistance. It was recently reported that the use of independent food controllable lethality systems, for example tetracycline (TET) and quinic acid (Q) systems, would address the concern of resistance development and could be considered in large scale suppression programs. So far, the activity of this system has been proven functional in An. gambiae.
The identification and evaluation of landing sites for genomic insertions has worked very efficiently for the development of stable transgenic Mediterranean fruit fly strains and is currently being transferred to other tephritid species. The community has all the necessary tools and techniques to create, characterize, and identify several landing site strains for the proposed mosquito species. This step includes fitness tests to characterize the best strains for subsequent strain development and further modification. The fittest lines should then be used to test the site-specific recombination systems and the most effective recombination system for each individual mosquito should be identified and used to stabilize the transgene. These “optimal” lines (fit and stabilized) should then be distributed in the community as a toolset for comparative genetics and for creating the most efficient sexing strains in mosquitoes.
Expected outputs
7. Construction and characterization of transgenic strains with site-specific landing sites and known performance characteristics. Currently transgenes integrate randomly into the genome. Adding site-specific landing sites to the
8 transgenes will allow for versatile subsequent modification of the loci. Beneficial strains can be selected and distributed to the community for further strain development.
8. Genetically modified strains from the use of ‘gene editing’ technologies (e.g. TALEN, CRISPR). Site-specific alteration of mosquito genomes by knock-in, knock-out of genes, or point mutations to introduce traits for sex separation.
9. Data on the structure, organization and patterns of gene expression of the sex chromosomes of mosquitoes. Understanding sex determination mechanisms will provide tools for developing genetic sexing strains.
10. Gene expression regulatory elements that confer sex-specificity. Regulatory elements could be promoter/enhancers or sex-specifically spliced sequences.
11. Effector molecules that can confer predictable phenotypes when expressed in mosquitoes. Effectors could be from the group of lethal genes, genes conferring phenotypes that allow sex separation, or sex conversion.
12. Strains of genetically modified mosquitoes that permit genetic sexing. Combinations of regulatory elements and effector molecules can be used to generate female-specific effects.
13. Quality control standards and parameters for evaluating genetically modified mosquito strains with known performance characteristics at small scale. This evaluation will provide data on the actual stability and the overall fitness of molecular-based GSS developed.
To explore mechanical, behavioural, developmental and symbiont-based approaches for sex separation in mosquitoes Mechanical, behavioural, and developmental tools for sexing need to be developed or existing ones to be refined to further improving the elimination of females at different developmental stages (ideally at the embryonic stage). The accessibility and integration of less efficient systems can provide a stop-gap measure that allows rapid start up with a minimum of investment, but are currently not considered long-term substitutes for true sexing systems. The following are being used and could be made more specific and efficient, or have not been explored in detail, but might be investigated by CRP participants. More than one method could be combined in order to attain better separation.
Mechanical Tools. Sex separation is possible at the pupal stage for some Aedes and Culex species due to size dimorphism between male and female pupae: females are usually larger. In other mosquito species, these differences are not significant enough to make sexing by size feasible. Many trials have been made in the past to assess pupal body size at different larval densities by measuring the dorsal width of the cephalothorax. While cephalothorax width was shown to be density dependent in Ae. albopictus, the results indicate that the size difference between males and females remains fairly constant across densities. Methods that have exploited this difference include: (i) sieving methods (e.g. standard sieves) for Ae. albopictus,
9 Ae. aegypti, and Cx. quinquefasciatus; (ii) use of the Fay-Morlan glass plate sex separation system (later modified by Focks) for numerous species; and (iii) use of the McCray adjustable opening separation system for Aedes sp.
Both female and male pupae are positively buoyant in pure water, but they may not have the same absolute density. Differences in buoyancy between male and female pupae could be explored using cold water (in which different rates of ascent might exist) or aqueous solutions whose density has been modified using e.g. salt or sugar solutions. If such differences are found, they could be exploited in mechanized sex-separation systems, so their utility likely overlaps that of pupa size methods.
Female adult mosquitoes are heavier and larger than males. These characteristics may be of value for separation of adults using a system similar to that which has been developed for Musca domestica in which a regulated air stream was used to separate anesthetized (CO2) adults. Such methods will be most efficient for species in which size difference between sexes is greatest.
Sexual dimorphism can be also used for automatic identification by artificial vision algorithms. Once both sexes have been identified, mechanical separation/removal of females controlled by computer can be used. One possible method to eliminate female pupae is to use high speed positioning and accuracy precision laser systems driven by a dual axis galvanometer optical scanner. These systems are widely used in industrial applications (ie. stereolithography rapid prototyping systems).
Behavioural Tools. For all blood-feeding mosquitoes, sex separation could occur at the adult stage by spiking blood with insecticides (malathion, dieldrin) or other mosquito toxins (ivermectin, Spinosad). Recent studies have shown that all female An. arabiensis could be eliminated by spiking blood meals with 7.5 ppm ivermectin within 4 days. For An. albimanus, using 0.5 % malathion in citrated bovine blood gave 95% effective elimination of females but resulted in significant (25%) male death due to inadvertent exposure. Improvements could involve toxicants with no vapour or contact toxicity.
Host-seeking behaviour by females could be exploited even if they do not actually consume any blood. By creating a mechanical system for capturing females that are drawn to host cues (heat, CO2, odours), females could be partitioned into separate chambers or killed upon resting on a metal screen (electrocution). If the electrocuting screen operated intermittently, females might rest on it more readily.
Males of some species (Culex and Anopheles particularly) begin swarming as young adults. Swarms contain very few females when they form in nature. This behaviour might be exploited for small-scale studies in which males could be collected from the swarms.
Developmental Tools. On average, male mosquitoes develop more rapidly than females (protandry). Mosquito larvae must achieve a critical weight to be able to pupate and this critical weight is higher for females leading to a longer developmental time. This feature has been exploited for Aedes particularly, but could be considered for other species where better sex-separation methods are not available. Protandry is known to be affected by several factors including rearing temperature, larval density and diet. Renewed efforts have been developed to standardize this process for at least three Aedes species, namely Ae. aegypti, Ae. albopictus, and Ae. polynesiensis.
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Ae. albopictus and Ae. polynesiensis harbour endosymbiotic microrganisms like Wolbachia which influence larval survival and adult fitness. Very little work has been done on the effect of this bacterium on pre-imaginal development comparing lines harbouring different Wolbachia strains (natural or artificially established) with their aposymbiotic counterparts. Exploring the role of bacterial endosymbionts (e.g. Wolbachia) in influencing the duration of pre-imaginal development, protandry, sex ratio and pupal dimorphism may allow the development of more effective sex separation methods using appropriate mosquito strains and mass rearing conditions. Recent evidence supports the need for further investigation of the Wolbachia properties on mosquito developmental biology. The dynamics of endosymbiont density in their host is also being investigated in view of its possible importance in SIT and/or IIT applications.
Expected outputs
14. Data that allows the use of Wolbachia symbiosis for sex separation. Wolbachia symbiosis may enhance protandry and could improve existing sex separation methods.
15. A set of parameters for using mechanical (e.g. size dimorphism), behavioural (e.g. phototaxis) and developmental (e.g. protandry) sex separation tools. These parameters will facilitate studies as well as the development of classical sex separation tools.
16. Data on the efficacy and reproducibility of mechanical, behavioural, and developmental tools under mid to large production scales using standard quality control parameters. This evaluation will provide data on the actual efficiency, reproducibility, and applicability of these tools.
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14 McMeniman CJ, Corfas RA, Matthews BJ, Ritchie SA, Vosshall LB. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. Cell 2014 Feb 27;156(5):1060-71
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15 Schliekelman P, Gould F (2000) Pest control by the release of insects carrying a female- killing allele on multiple loci. J Econ Entomol, 93:1566-1579.
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Yoshioka, M., J. Couret, F. Kim, J. McMillan, T. R. Burkot, E. M. Dotson, U. Kitron, and G. M. Vazquez-Prokopec. 2012. Diet and density dependent competition affect larval performance and oviposition site selection in the mosquito species Aedes albopictus (Diptera: Culicidae). Parasites & Vectors 5: 225.
17 Nuclear Component: All the activities in the CRP relate to the development and evaluation of mosquito strains for use in SIT programmes. The SIT relies on the use of ionizing radiation to sterilize large numbers of insects and currently there is no alternative that could replace radiation. However, there are developments taking place which intend to use molecular methods for generating lethality in field populations. These approaches are not included in this new CRP as their non-confined use would create significant concerns relating to biosafety and long-term effectiveness. Radiation-induced sterility provides a very high level of biosafety and can be used in combination with improved strains developed in this CRP. As radiation induces random dominant mutations, there is no possibility of resistance developing to this physical process, a possibility which cannot be excluded with molecular approaches that involve genomic insertions.
Explanation / Justification:
1. Publication of results. Activities and findings of the CRP will be published in a special issue of a peer-reviewed journal.
Participation of Agency's laboratories (Yes/No)
As few institutions are applying irradiation and classical genetics for the development of GSS in mosquitoes, the CRP needs therefore to be supported through adaptive research and development carried out at the IPCL, FAO/IAEA Agriculture and Biotechnology Laboratories, Seibersdorf as part of Project 2.1.4.4. This R&D will focus on the development of molecular markers using classical genetics, and the evaluation of marker strains and genetic sexing strains developed using modern biotechnology.
Assumptions:
Member States will continue to suffer major losses due to dengue, chikungunya, zika, and malaria outbreaks.
That IAEA Member States continue requesting the development of the SIT package for mosquitoes, and that enough scientists and institutions are willing to participate in the CRP and can be motivated to apply classical genetic approaches to develop sex separation systems in mosquitoes.
The demand for area-wide integrated insect pest management approaches, including SIT and augmentative biological control as non-polluting suppression/eradication components, continues to increase, mandating expansion and improvement in cost-effectiveness of these environment-friendly, sustainable approaches.
Related TC Projects
RAF5065: Promoting the sharing of expertise and physical infrastructure for mass rearing mosquitoes and integration of the sterile insect technique (SIT) with conventional methods for vector control, among countries of the region.
18 RAF5072: Exploring the use of sterile insect technique as a novel technique for control of vector mosquito for Chikungunya and dengue (Aedes albopictus) in the Indian Ocean Region (Phase I 2014-2015).
RAS5066: Promoting the sharing of expertise and infrastructure for dengue vector control surveillance towards integration of the sterile insect technique with conventional methods among South and South East Asian countries.
SUD5034: Supporting a Feasibility Study on the Suitability of the Sterile Insect Technique As a Strategy for the Integrated Control of Anopheles arabiensis.
SAF5013: Assessing the Sterile Insect Technique for Malaria Mosquitoes in a South African Setting.
MAR5019: Supporting a Feasibility Study Using the Sterile Insect Technique (SIT) for the Integrated Control of Mosquitoes.
SRL5044: Supporting a Feasibility Study Using the Sterile Insect Technique (SIT) for Integrated Control of Mosquitoes.
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LOGICAL FRAMEWORK:
Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
Overall Objective Requests by Member States in the area of mosquito control To explore genetic, molecular, N/A N/A using the SIT are increasing. To behavioural and mechanical make this nuclear technology methods of sex separation in available to Member States for mosquitoes several mosquito species, the development of male-only strains is either a requirement to reduce programme costs or an essential precondition. Given the large number of different target species and the lack of any efficient tools for mosquito control, strategies based on genetic, molecular, mechanical and behavioural techniques are amenable. Biological material is available. An expert on classical genetics and a technician in the mosquito group in Seibersdorf are recruited.
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Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
Specific Objectives
1. To explore irradiation and Classical GSS for at Reports and or Irradiation and classical classical genetic approaches least one mosquito published papers. genetics can be applied for the for sex-separation in species constructed. construction of mosquito GSS. mosquitoes
2. To explore molecular Molecular-based Reports and or Molecular approaches are approaches for sex separation GSS for at least one published papers. applicable to target mosquito in mosquitoes mosquito species species. constructed.
3. To explore mechanical, Mechanical, Reports and or Development and improvement behavioural, developmental behavioural and published papers. are possible. and symbiont-based developmental sex approaches for sex separation separation systems in mosquitoes for at least one mosquito species developed or improved.
Geneticists pursuing Agreements or Relevant scientists can be 4. To continue encouraging and a classical approach contracts issued and identified and encouraged to attracting participants to the attracted to the CRP. signed. join the CRP. CRP in the field of classical genetics
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Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
Outcomes
1. Classical genetic Irradiation protocols Data collected and Facilities and resources approaches for sex and classical genetic feasibility analysis available. separation in mosquitoes approaches assessed determined
2. Molecular approaches for Methods for Data collected and Facilities and resources sex separation in molecular-based sex feasibility analysis available. mosquitoes developed separation developed
3. Mechanical, behavioural, Protocols developed developmental and for improved sex Data collected and Facilities and resources symbiont-based separation methods feasibility analysis available. approaches for sex separation in mosquitoes improved and validated
Outputs
1. Genetic markers from Genetic markers Reports and or Biological material and different sources including isolated. published papers. methods available. natural populations and / or mutagen-based screens.
2. Chromosomal Chromosomal Reports and or Scientists working on classical rearrangements in the rearrangements published papers. genetics and methods are target mosquito species. selected. available.
3. (Cyto)genetic data from (Cyto)genetic Reports and or (Cyto)genetic methods, the analysis of the tools markers and published papers. chromosome maps and experts developed above in [1.] chromosomal are in place. and [2.]. rearrangements described.
4. Morphological and Morphological and Reports and or Morphological and selectable selectable markers. selectable markers published papers. markers are identifiable. confirmed.
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Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
5. A classical GSS. Classical GSS Reports and or Suitable genetic markers, constructed and published papers irradiation sources, characterized chromosomal rearrangements are available.
6. Data from the evaluation Classical GSS in Evaluation data Laboratory testing is feasible of a classical GSS on a laboratory testing identified and and quality control standards small scale using standard evaluated published are available. quality control parameters.
7. Construction and Strains with landing Reports and or Landing sites and quality characterization of sites constructed and published papers control standards are available. transgenic strains with site- performance specific landing sites and characteristics known performance assessed characteristics.
8. Genetically modified Accurate genome Reports and or Transgenic technologies, strains from the use of editing in target loci published papers genomic data are available and ‘gene editing’ technologies and species achieved endogenous repair mechanisms (e.g. TALEN, CRISPR). are functional.
9. Data on the structure, Sex determination Reports and or Tools and omics data for the organization and patterns pathways and sex published papers analysis of complex sex of gene expression of the specific expression determination pathways and sex chromosomes of in mosquitoes better mechanisms are available. mosquitoes. understood. Content of sex chromosomes studied.
10. Gene expression regulatory Regulatory elements Reports and or Regulatory elements can be elements that confer sex- tested published papers tested. specificity.
11. Effector molecules that can Effector molecules Reports and or Effector molecules are available confer predictable tested published papers and can be tested. phenotypes when expressed in mosquitoes.
12. Strains of genetically Molecular-based Reports and or Components for the application modified mosquitoes that sexing strains published papers of molecular-based permit genetic sexing. developed technologies are available
13. Quality control standards Standard quality Reports, protocols, Laboratory testing of GSS is and parameters for control protocols and or published feasible and decisions on evaluating genetically developed and papers standard testing parameters modified mosquito strains molecular-based have been made 23
Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
with known performance GSS at small scale characteristics at small evaluated scale.
14. Data that allows the use of Wolbachia influence Reports and or Wolbachia infection influences Wolbachia symbiosis for on sex separation published papers developmental parameters in a sex separation. exploited sex-specific manner
15. A set of parameters for Factors influencing Reports and or Tools for characterization are using mechanical (e.g. size the efficiency of sex published papers available. dimorphism), behavioural separation tools (e.g. phototaxis) and characterized developmental (e.g. protandry) sex separation tools.
16. Data on the efficacy and Efficacy and Reports and or Tools and standard quality reproducibility of reproducibility of published papers control protocols are in place mechanical, behavioural, tools assessed and developmental tools under mid to large production scales using standard quality control parameters.
17. Publication of results in a Papers drafted and Journal issue with Data for publication available. peer reviewed journal. submitted. published papers. Activities
1.Form a network of research Proposals evaluated Signed contracts and Suitable proposals submitted, collaborators and 6 Research agreements. funding available and approval Contracts, 11 of Contracts and Agreements Research by CCRA-NA committee. Agreements and 2 Technical Contracts awarded.
2. Organise 1st RCM to refine the 1st RCM held 2013. Participants’ activities Contracts and Agreements logical framework and plan the and logical framework signed by counterpart overall activities of the CRP. revised. organisations.
3. Organise 2nd RCM to analyse 2nd RCM held 2015. Participants and RCM Progress satisfactory. progress in delivering research Progress Reports. outputs and plan the next phase of the project.
4. Organise 3rd RCM to analyse 3rd RCM held 2016. Participants and RCM Progress satisfactory and mid- 24
Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators
progress in delivering the Progress Reports. CRP evaluation approved by research outputs and plan the CCRA-NA committee. final phase of the project.
5. Organise final RCM to assess 4th RCM held 2018. Participants and RCM Final reports are submitted to the success of the CRP in reaching Final Reports the Agency. its objectives and review the final publication.
6. Publish the results of the CRP Scientific publication. Consensus can be found on in a special issue of an appropriate international international journal. journal and acceptance by journal obtained.
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FUTURE ACTIVITIES
Contract 17939: Margareth Capurro, University of São Paulo, Brazil (Collaborators: Jake Tu, Chun-Hong Chen)
Construction and characterization of GSS (Aedes aegypti and Aedes albopictus)
The goal of this project is to generate transgenic Aedes aegypti mosquitoes that will produce male-only progeny. The key components of this strategy are the embryonic zygotic genes (EZGs) promoters and RNAi that will knockdown the doublesex (Aedsx) pathway. The 3’ UTR of the transgene will include a SV40 polyadenylation signal to stabilize that transcript. This gene will be regulated by a Tet-system to produce laboratory colonies.
Aims for the next 18 months: Characterization of doublesex (Aedsx) expression profile in individual embryos of Aedes aegypti Evaluation of RNAi experiments to knockdown the female Ae. aegypti dsx pathway Microinjection of the construct designed and synthetized to transform Ae. aegypti and Aedes albopictus embryos Develop a transgenic line to knockout dsx by RNAi mechanism
Contract 17958: Flaminia Catteruccia, Harvard T. H. Chan School of Public Health, Boston, USA (Collaborators: Eric Marois)
Mosquito sex separation via TALEN-mediated female-specific lethality
Creation of an X-linked docking line in Anopheles gambiae: The transgenic line French Kiss (FK) was generated by piggyBac-mediated integration into the Ngousso laboratory line of Anopheles gambiae. Subsequent analysis revealed that this transgene was integrated on the X-chromosome within an intron of AGAP001069. This transgene contained transgenic cargo containing YFP, GFP and RFP fluorescent markers under different promoters of interest flanked by loxP sites. Additionally, the transgene contained an attP sequence outside of the loxP sequences, making FK a proto-docking line such that Cre-loxP recombination between the loxP sites would remove all transgenic cargo, leaving only the attP site for subsequent Phi-C31 transgene ‘docking’. Injections of Cre recombinase into FK embryos failed to yield the desired excision products, but crossing to a Cre-recombinase-expressing transgenic line yielded many larvae lacking fluorescent transgenic cargo. Although both FK and the derivative docking line (XK) both have normal Mendelian sex segregation, any attempts to insert a new transgene onto the XK docking line yields only transgenic females and no transgenic males. Specifically, in the G1 generation of transgenics (progeny of G0 which were injected as embryos, and outcrossed to wild type) 22 of 23 transgenic individuals were female, and the single transgenic male died during development. In the G2 generation - from G1 females outcrossed to wild type - 50% of daughters were transgenic and 50% wild type as expected, but all sons were wild type, with half as many sons as expected, thereby suggesting that transgenic males are inviable. This phenotype persisted despite transgenesis attempts with five different constructs, suggesting it is not a phenotype attributable to the transgenic cargo but due instead to the docking line itself. We hypothesize
26
that the insertion of novel transgenes onto the docking line disrupts the correct splicing of AGAP001069, despite the original FK and XK lines having no such phenotype. The protein encoded by AGAP001069 has over 93% homology to the Drosophila melanogaster protein Surf4, an essential yet haplosufficient transmembrane protein implicated in Golgi transport. Lethality due to disruption of the putatitve ortholog of surf4 is therefore not entirely unexpected. Unfortunately, this phenotype suggests that this X-linked docking line may be unsuitable for our originally proposed project. Additional experiments will determine this.
Male reproduction-specific promoter: We have recently identified a highly male-specific promoter that we believe may be of interest to others in the consortium. Among a variety of uses, such a male specific promoter could be used to facilitate a toxin-antitoxin system to enable selective survival of males. For this project we originally set out to develop a transgenic line with a fluorescent mating plug to enable identification of mated females based on plug fluorescence visible through their cuticle, in order to circumvent the need to identify mated females by tedious dissection and spermatheca analysis. Towards this end we decided to generate a transgenic line expressing the main plug structural protein Plugin (AGAP009368) fused to tdTomato under the control of the native Plugin promoter comprising the 2,688bp immediately upstream of the start codon. This transgenic line contains a 3xP3-YFP selectable marker inserted onto the X1 docking line, which harbors an attP site in a non-coding region on chromosome 2L between AGAP005126 and AGAP005127. Analysis of the resulting transgenic line reveals tdTomato fluorescence within the anterior segment of the Male Accessory Glands (MAGs) (Figure 1b) that is also easily visible through the adult male cuticle (Figure 1a). tdTomato expression is initially visible in the late male larval stage, consistent with the initial development of the MAGs. Upon dissection, tdTomato is not observed in any female adult or larval tissues, and initial results suggest that plugin-tdTomato mRNA transcripts are undetectable in adult females. Additionally, immediately following mating faint tdTomato fluorescence from the transferred mating plug is also visible through the female cuticle, although the fluorescence intensity is sub-optimal. In all, this preliminary data suggests that the plugin promoter is a male-specific reproductive promoter that has sufficient specificity to enable future transgenic sexing systems.
Aims for the next 18 months: Determine if the X-linked docking line we generated can be used successfully with gypsy insulators, which were present in the proto-docking line FK. During attempts to reintroduce new transgenes onto XK, we omitted to include insulators. We hypothesize that the inclusion of insulators in future transgenic constructs may be crucial for successful transgenesis in the XK line. If these experiments fail, we will attempt to generate and characterize a novel X- linked docking line by recommencing embryonic microinjections of piggyBac constructs into the An. gambiae G3 strain. Work towards identifying and characterizing a promoter expressed strongly during early embryonic development to facilitate TALEN expression.
Contract 17882: Andrea Crisanti. Imperial College of London (Collaborators: Philippos Papathanos, Mara Lawniczak, Federica Bernardini, Nikolai Windbichler).
Characterization of the YAttP-introgressed Anopheles arabiensis strain and its validation for the application of vector control strategies 27
We have previously characterized an Anopheles gambiae transgenic strain, named T4, with a transgenic construct inserted onto the Y chromosome. Expression of fluorescent maker genes in T4 males suggested that the transgene construct had inserted within a region of chromatin that allows transcriptional activity. The fact that the T4 locus was transcriptionally active made it an exciting target for genetic engineering. Using meganuclease-induced homologous repair a site-specific recombination signal, AttP, was introduced onto the Y chromosome and the resulting docking line, named YAttP, was proven to allow secondary integration. This transgenic strain allows automated sex separation and generation of male-exclusive genetic traits, therefore it represents a valuable tool for the improvement of vector control strategies already in use, such as sterile insect technique, and encourage the development of new approaches such as those based on endonuclease genes (Bernardini et al., 2014). A. arabiensis is another important vector of human malaria. The generation of a transgenic line in this species carrying a Y-linked AttP site is of great interest. Since active insertions onto the Y chromosome are extremely rare, we used a scheme based on crossing and selection to overcome F1 hybrid male sterility, predicted by Haldane’s rule, and we introgressed the engineered A. gambiae Y chromosome, YAttP, in the A. arabiensis genetic background. Fertility of YAttP-introgressed males, tested after up to 10 backcross generations, was comparable to fertility of wild-type A. arabiensis males. This observation makes the YAttP- introgressed strain a good candidate for the application of vector control strategies on A. arabiensis mosquitoes. During the next 18 months we will validate the value, in the context of vector control, of the YAttP-introgressed strain.
Aims for the next 18 months: Assess the genetic composition of the YAttP-introgressed males by deep DNA sequence analysis. Assess the reproductive fitness of YAttP-introgressed males (fertility, fecundity, mating competitiveness). Assess the possibility for site-specific integration in YAttP-introgressed males.
Contract 17962: Jaroslaw Krzywinski, The Pirbright Institute, UK (Collaborators: Francesca Scolari, Philippos Papathanos)
Creation of conditional female embryonic lethal strains of An. arabiensis
The objective of this research is to develop a transgenic sexing strategy in An. arabiensis to enable large-scale simultaneous removal of females to facilitate cost-effective production of males for SIT releases. The transgenic system will utilise female embryonic lethality caused by ectopic expression of the Y chromosome-linked gene involved in sex determination. Our experiments indicate that, unlike in females, the male development is not affected by ectopic delivery of the Y gene transcripts. During the next 18 months we will generate elements of the Gal4-UAS system and test the resulting transgenic strains of An. arabiensis for the expression of female embryonic lethal phenotype.
Aims for the next 18 months: Test the activity of the putative early promoters of selected genes in An. gambiae and An. arabiensis embryos. 28
Generate transgenic driver and responder lines of a bipartite Gal4-UAS system. Perform crosses between individual driver lines and the responder line and test the phenotype (sex ratio) of the progeny. If the above produce male-only generations, construct Tet-off vectors needed for generation of conditional female lethality.
Contract 17981: David O’Brochta, University of Maryland, College Park, USA (Collaborators: Jake Tu)
CRISPR/Cas9 mutagenesis of An. arabiensis and the creation of ΦC31 attP landing site lines
CRISPR/Cas9-based mutagenesis is an effective way to create mutations at specific sites within genomes. Efforts during the first 18 months resulted in a set of protocols that enable the efficient creation of CRISPR/Cas9-induced mutations in mosquitoes. piggyBac transposon-based vectors can be use to carry ΦC31attP sites into mosquito genomes and during the first 18 months we showed that piggyBac elements integrated into the genome of An. gambiae can be efficiently remobilized by chromosomal sources of piggyBac transposase.
Aims for the next 18 months: Create transgenic An. arabiensis lines that carry either 1) a stably integrated source of piggyBac transposase expressed in the germ-line (using promoter from vasa). 2) a single copy of a piggyBac elements with an ΦC31attP site and a reporter gene sensitive to ‘position effects’ Remobilize PB-attP in An. arabiensis to create a collection of lines with ΦC31attP landing sites on different chromosomes that are free from ‘position effects’. Use CRISPR/Cas9 to create visible mutations.
Contract 17952: Philippos Papathanos, University of Perugia, Italy (CRP collaborators: Andrea Crisanti, Jake Tu, Jaroslaw Krzywinski, Francesca Scolari; External collaborators: N. Windbichler, N. Besanszy, I. Sharakov)
Sex differentiation and determination in Anopheline mosquitoes
Dissecting the genetic circuitry that governs sexual dimorphism in mosquitoes, in which only females contribute to disease transmission, has the potential to enhance our capacity to generate sexing strains or improve population suppression systems. Over the last 18 months we have been heavily involved in the completion of two large genomics based efforts in anopheline mosquitoes: a) the 16 anopheles genomes project where we focused on the identification of sex biased genes and an analysis of their evolution in the genus and b) the characterization of the Y-chromosomes of the gambiae complex.
Aims for the next 18 months: investigating with RNA-seq-based transcriptome analysis of sexed adult and dissected germlines, the sex-biased transcriptional landscape of 4 of 17 anophelines, including two of the target species An. arabiensis, An. gambiae s.s. but also the 29
phylogenetically relevant An. minimus and An. albimanus. Extend the sex-biased gene expression analysis in these mosquitoes and document the evolutionary forces that are driving their genomic distribution and sequence and expression evolution. Functional analysis of sperm specific and Y specific genes using RNA interference. Initially we will test the system recently published by Whyard et al, where dsRNA is delivered by feeding developing mosquito larvae with bacterial strains engineered to express the dsRNA targeting mosquito sequences. This will help in the identification of genes that are important for maleness or male fertility which may be useful to the development of molecular tools for SIT or GSS. Development of a synthetic Y chromosome that increases its transmission based on the shredding of the X chromosome during spermatogenesis.
Contract 17896: Francesca Scolari, University of Pavia, Italy (Collaborators: Jake Tu, Romeo Bellini, Jaroslaw Krzywinski, Philippos Papathanos, Margareth Capurro)
Development of sex-specific markers in the tiger mosquito Ae. albopictus
A protocol that allows the unambiguous sexing of mature fourth instar larvae by microscope observation of developing gonads has been developed for Ae. albopictus (Rimini strain, CAA). This facilitated the characterization of male and female karyotypes using mitotic chromosomes obtained from larval imaginal discs. Both DAPI- and Giemsa C-banding stainings performed on such mitotic preparations consistently confirmed the presence of a male-specific banding pattern on Chromosome 1. Molecular analyses performed using a Representational Difference Analysis (RDA) approach permitted to obtain a number of potential male-specific/enriched sequences that could be used as targets for PCR assays for molecular sexing. In addition, the genome size of the Rimini strain was determined using a Real-time PCR-based method, and the annotation of the chemosensory gene repertoire of this species is ongoing (European Consortium). Antennal transcriptomes from male and female adult individuals have been separately developed. Aims for the next 18 months: The potential male-specific/biased location of the sequences derived using the Representational Difference Analysis (RDA) approach will be evaluated using RT- PCR and Fluorescence in situ hybridization (FISH) on the mitotic chromosomes spreads obtained from the imaginal discs of sexed fourth instar larvae. Potential variations in karyotype and genome size between Rimini and other strains and wild populations will be estimated. The chemosensory genes (Odorant Binding Protein, OBPs, and Odorant Receptors, OR) identified in the Ae. albopictus genome will be annotated. The set of OBP and OR genes identified from Ae. albopictus antennal transcriptome separately derived from male and female adult mosquitoes will be screened to identify any potential sex-specific expression profile. Protein expression, purification and binding to known active compounds will be initiated.
Contract 17855: Marc F. Schetelig, Irina Häcker, Justus-Liebig-University Giessen, Germany (Collaborators: David O’Brochta, Margareth Capurro, Jake Tu, Jeremie Gilles, Kostas Bourtzis)
30
Site-specific landing site systems in mosquitoes to improve transgenic insects for SIT
In the past 18 months we constructed landing site lines for loxN – lox2272 cassette exchange experiments in Ae. aegypti, and performed RMCE experiments in 3 of the 4 lines. The transformations didn’t yield a cassette exchange event, but instead we obtained donor plasmid integration either at the loxN or the lox2272 site, showing that lox-integration is functional in Ae. aegypti. Pursuing an alternative strategy, we cloned vectors needed for an attP-based RMCE system and created the corresponding landing site lines for attP cassette exchange experiments.
Aims for the next 18 months: Initiate the construction of vectors needed for the creation of landing site lines to perform loxN - loxP cassette exchange experiments. Create the landing site lines for loxN – loxP recombination in Aedes aegypti by germline transformation of early embryos with the above landing site vector and pBac helper. Perform loxN – loxP cassette exchange experiments by injecting homozygous embryos of the above landing site line(s) with the corresponding donor and Cre helper plasmids Construct the donor vector needed for attP cassette exchange, and perform cassette exchange experiments with the existing attP - attP landing site lines. Start lab scale fitness tests with the existing landing site lines (loxN-lox2272, attP- attP) and several of the lox-integration lines (loxN-integration, lox2272-integration) to identify the fittest line(s) with respect to viability, rearing efficiency and male competitiveness for downstream applications/further strain modifications.
Contract 17956: Zhijian Jake Tu, Virginia Tech/Biochemistry, USA (Collaborators: Jeremie Gilles, Kostas Bourtzis, Tony James, Marc Schetelig, Igor Sharakhov, Antony James)
Characterization and application of male-only transgenic lines in Anopheles mosquitoes
The first goal was to characterize currently available An. stephensi Guy1 transgenic lines: a) to determine whether female-killing or female conversion is responsible for the male-only phenotype. b) to determine the effect of ectopic Guy1 expression in the early embryos. a) We have shown that female-lethality, not sex-conversion, is responsible for the male-only phenotype. This is true for all lines and constructs tested. We also showed that the GUY1 protein was the cause of the lethality. We are pleased to report that all >6500 Guy1 transgenic individuals across different lines and multiple generations are males, making the Guy1 transgenic lines attractive for sexing and population suppression. b) We have shown that Guy1 transgenic females die prior to or shortly after hatching, suggesting that the phenotype is mostly embryonic lethal. We have also preliminary evidence suggesting that mis-regulation of dosage compensation may be the cause the female lethality. The second goal was to establish homozygous An. stephensi transgenic lines that carry a conditionally expressed Guy1 transgene. A Tet-off system should control transgene expression. The idea was to produce transgene-carrying females in the presence of tetracycline, so that homozygous individuals carrying the transgene may be obtained. In collaboration with Dr. Tony James at UC Irvine transgene should be integrated into pre- selected sites to ensure function of the transgene and minimize fitness cost associated with the insertion site. We have demonstrated that the Tet-off system will work in the early embryos in 31
An. stephensi. Embryonic injection demonstrated Tet dose-dependent suppression of reporter gene activity. Suppression of reporter gene was achieved when Tet was delivered to the embryo through feeding by adult females. Thus we now have all the components to homozygous An. stephensi transgenic lines that carry a conditionally expressed Guy1 transgene.
Aims for the next 18 months: Using the tested components to establish homozygous An. stephensi transgenic lines that carry a conditionally expressed Guy1 transgene. In addition to the Tet-off system, we also test the Tet-on system, which may have less toxicity. Evaluation of either the existing Guy1 lines or the homozygous Guy1 lines for suitability of mass rearing and competitiveness will be started in collaboration with Drs. Gilles, Bourtzis, and Lees at the IAEA (IPCL in Seibersdorf).
Contract 17959: Wimaladarma Abeyewickreme, Menaka Hapugoda. Nilmini Gunawardena Univ. of Kelaniya, Sri Lanka (Collaborators: Jeremie Gilles; Romeo Bellini; Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka; and Jeevanie Harischandra, Anti-malaria Campaign, Sri Lanka, Nayana Gunatilake, Industrial Technology Institute, Sri Lanka. (Collaborators: Jeremie Gilles; Romeo Bellini)
Mechanical sex separation in Aedes species
Our research group intends to explore mechanical and behavioral approaches for sex separation of Ae. albopictus. During the past 18 months we have been mainly working on establishment of laboratory colonies of Ae. aegypti and Ae. albopictus in two separate laboratories at the Anti Malaria Campaign and the Molecular Medicine Unit of the University of Kelaniya. Since it is important to standardize colonization following IAEA protocols to obtain maximum production with synchronization of development at different stages attention was given to have healthy colonies in both laboratories by exchanging ideas and sharing material between two laboratories. However we have made some experimental studies on separation of larvae/pupae separating Ae. albopictus males from females by hand after visual examination or using sieves of different mesh size. These preliminary studies show that mechanical separation is possible at pupal stage for Aedes albopictus. Attempts will be made to increase sex separation to near 100 % using several devices.
Aims for the next 18 months: Assess sex separation methods at the pupal stage using (i) standard sieves (ii) the Fay-Morlan glass plate sex separation systems with required modifications (Focks etc) separately or in combination. A modified adjustable opening separation systems (McCray) will also be tested Improve these methods taking into account the natural protandry of Aedes species and the collection of the male pupae only during the first 24h after the first pupae. Assess sex separation at the adult stage by feeding female mosquitoes with blood spiked with insecticides (malathion, dieldrin) and other potential mosquito toxins (ivermectin, spinosad).
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Explore seeking behaviour of males/females to create a mechanical separation system for capturing males/females that are drawn to host cues (heat, CO2, odours and other male attractants such as nectar, plant extracts, etc.). Partitioning of sexes into separate chambers and/or killing upon resting on different surfaces (eg. metal and electrocution) will also be investigated. Identification of markers for future collaborative molecular studies will be carried out during the larval rearing process. Attempt will be made to establish collaborative links with CRP partners particularly working on molecular aspects of Ae. albopictus for SIT.
Contract 17950: Hervé Bossin, Institut Louis Malardé, French Polynesia (Collaborators: Romeo Bellini, Maurizio Calvitti, Carlos Tur Lahiguera, Gustavo Salvador Herranz, Pablo Tortosa)
Developmental and mechanical sex separation in Aedes polynesiensis
The efficacy and sustainability of the Wolbachia incompatible insect technique (IIT) to control the disease vector Ae. polynesiensis is being evaluated through open release trials in French Polynesia. A proof of concept trial was conducted on the island of Raiatea and a ranging trial on the atoll of Tetiaroa, which support the pursuit of field evaluation. A suppression trial is under preparation, with the goal to control and possibly eliminate Ae. polynesiensis from an isolated islet (75 ha) on the atoll of Tetiaroa. This integrated strategy includes the release of incompatible males, which will start just before the onset of the rainy season when the size of the target population is lowest (Sept. 2015). Male releases will proceed for a period of 8 months encompassing the entire rainy season. Suppression will require the release of ca. 45,000 males each week. In the absence of a GSS strain for Ae. polynesiensis, sex separation currently relies on developmental (time to pupation) and mechanical (pupae size) differences between males and females.
Progress to date and main achievements: - The influence of rearing conditions (larval density, larval diet) on the stringency and reproducibility of developmental and mechanical sexing methods was further explored at ILM (standard rearing procedure) and in collaboration with CAA (mass-rearing SOP) through INFRAVEC support. Experiments conducted at a relatively small production scale indicate that protandry can be efficiently used for male/female pupae separation. A rearing procedure was developed in which ca. 50% of total Ae. polynesiensis males pupate on day 5 of development with no or little female contamination. L1 to pupae survival exceeds 90% and pupae to adult survival 98%. Further experiments were conducted at CAA using mass-rearing protocols with IAEA trays and IAEA diet to investigate the influence of larval density. Results support the use of larval densities comprised between 0.2 larvae/ml up to 1 larvae/ml without compromising rearing and sex separation parameters. Also the IAEA diet supplemented with Brewer’s yeast provided the best pupae productivity. Altogether, these and previous studies allowed the development of rearing conditions that deliver relatively high male yield with essentially no female contamination, adequate adult male size and survival, offering adequate developmental/mechanical sex separation in the context of the upcoming suppression trial. Testing of this sex separation approach under mass rearing conditions will now be conducted to ensure reproducibility.
Aims for the next 18 months: 33
Continue testing the influence of mass-rearing conditions (rearing tools, larval density, larval diet) on the stringency and reproducibility of developmental/mechanical sex separation. This work will be conducted at ILM using IAEA trays and diet formulation (collaboration with Romeo Bellini, CAA). Develop standard rearing and sorting procedures based on the optimal conditions defined above. Determine frequency size distribution curves of Ae. polynesiensis and Ae. aegypti pupae to assess the potential for laser-based sex separation (collaboration with Carlos Tur Lahiguera, TRAGSA, and Gustavo Salvador Herranz, Univ. Cardinal Herrera). Explore the potential for introgression of GSS traits under development for Ae. albopictus into the Ae. polynesiensis genetic background using interspecific crosses (collaboration with Pablo Tortosa, PIMIT). Compare the influence of Wolbachia strains on developmental parameters (e.g. protandry, time to pupation) of Ae. polynesiensis wild type (WolbA) and incompatible (WolbB) colonies toward their exploitation to improve sex separation. Monitor the dynamics of Wolbachia in Ae. polynesiensis wild type and incompatible strains and study the effects of rearing and sex-separation standard procedures on Wolbachia (collaboration with Maurizio Calvitti, ENEA).
Contract 17902: Maurizio Calvitti, R. Moretti, A. Desiderio, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Italy (Collaborators: Romeo Bellini, Arianna Puggioli, Pablo Tortosa, Jeremie Gilles, Herve Bossin)
Effect of Wolbachia infection and types (absence, native and artificial) in Aedes albopictus on pre-imaginal and sexual maturity
During the past 18 months, we started to evaluate the effect of a new Wolbachia infection (wPip strain) on the developmental biology of Ae. albopictus (ARwP strain). We considered protandry (male and female pupation times) and pupal size. Also the efficiency of mechanical separation of pupae, performed on the ARwP strain, was evaluated. Along with these specific studies, we conducted a preliminary characterization of the “wPip- Ae.albopictus” symbiosis with regard to the mean bacterial load of adult mosquitoes, its long- term changes, its response to the environmental pressures due to both small scale laboratory rearing and CAA rearing standard operational procedures (SOP). This knowledge is essential because most of the Wolbachia-influenced biological traits (Cytoplasmic Incompatibility, adult fitness), that make a mosquito strain suitable for use as genetic control tool, depend on Wolbachia density and on the long-term stability of the symbiosis. Effect of Wolbachia infection status (absence, native, artificial) in Aedes albopictus pre- imaginal and sexual maturity. Over the next 18 months, experimental activities will be carried out to confirm the preliminary findings that the wPip Wolbachia strain, established in Ae. albopictus (ARwP F80 strain) is able to influence the developmental biology of Ae. albopictus by determining an earlier development of pupae (pupation onset on average 5-6 hours earlier than that observed in the wild type strain Rimini F55) and an increased pupal size. A possible benefit of these effects is the observation of more efficient mechanical sex separation of the ARwP pupae with only 0.24% female contamination, compared to 1.59% obtained with the Rimini F55 wild type strain. Effects of rearing SOP on the “ wPip” Wolbachia titre. Concurrently with the experiments performed to assess the effects of Wolbachia on the developmental biology and the efficiency
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of mechanical separation we have developed a qPCR protocol in order to quantify the response of the wPip Wolbachia titer to the application of SOP developed for mass rearing and sex-separation at CAA. ARwP males produced using CAA SOP preserved their strong CI and showed a superior male mating competitiveness in semi-field cages, compared to the irradiated counterparts. Radiation biology of the ARwP strain in the perspective of developing a combined SIT- ITT. Considering the promising results obtained over the past 18 months in sex-separation efficiency, Wolbachia quantification in adults produced by SOP and male mating competitiveness assessed in large enclosure experiments, it is opportune to start investigating the radiation biology of the ARwP females in order to determine the lowest sterilizing dose in the perspective of developing a combined SIT-IIT strategy.
Aims for the next 18 months:
In order to find understand variations in the developmental biology of Ae. Albopictus, induced by the different Wolbachia strains, standard experiments on the developmental biology and sex separation will be repeated against the “AR-F91” aposymbiotic mosquito strain maintained at ENEA since 2009. This mosquito strain has the same genetic background as ARwP and Rimini strains. Concurrently, a permanent ARwP colony will be established at the CAA mass rearing facility to set up a series of experiments for monitoring Wolbachia density, adult fitness (females life-history parameters and male mating competitiveness) CI level after each 5 generations under CAA rearing SOP. Improvements recorded in the efficiency of mechanical separation of male and female pupae will be assessed in the long-term. Tests on the ARwP mosquito radiation biology will be started to find the lowest irradiation doses for female pupae to achieve female sterility. Combining the use of ARwP bidirectional CI with irradiation-induced female sterility (down to a low residual female fertility of 1 to 5%) would make this CI approach safer and widely applicable.
Contract 17925: R. Bellini, A. Puggioli, Centro Agricoltura Ambiente “G. Nicoli”. Italy (Collaborators: Maurizio Calvitti, Jeremie Gilles, Herve Bossin, Carlos Tur Lahiguera, Gustavo Salvador Herranz)
Exploiting sex dimorphism and protandry for Aedes albopictus sex separation
During the first 18 months of this CRP, we explored the possibility to increase the male productivity by enhancing the protandry and the sexual dimorphism of Aedes albopictus pupae. We checked if the natural sexual dimorphism is an inheritable character, which could be selected for the development of a strain with more marked differences in the size of the pupae as a function of sex. We crossed larger female pupae with smaller male pupae for four generations. After three generations of selection for small males and large females, we obtained a strain, which in comparison with the F0 had small adults of both sexes. From the data obtained in this exploratory experiment it seems that the pupal size is dominated by the male characteristics, with small males that bring down the size of females as well, therefore this approach seems to be unlikely in supporting a better sexing method. We analyzed the natural protandry of Ae. albopictus and verified the possibility to enhance it by artificial selection of a strain obtained by crossing the males that pupate earlier with the 35
females that pupate later on. After nine generations of selection we observed that the sex ratio, within 24 h from pupation onset, is significantly different compared to our control strain. We therefore decided to continue to work on this approach. Together with ENEA we evaluated the effect of a new Wolbachia infection (wPip strain), established in Ae. albopictus, on pre-imaginal development and sex separation. The male pupae productivity was similar between the two strains, while the percentage of residual females was significantly lower with ARwP strain. In cooperation with ILM we tested the effect of mass-rearing variables (larval density and larval diet) on the pre-imaginal development of Aedes polynesiensis, employing diet formulation and trays developed at the IAEA. For all the diets and larval densities tested, pupation occurred on the fifth day after hatching, with the highest percentage of pupation obtained using the IAEA-BY diet (at all densities), both on the first and the second day of pupation. The highest survival rate was obtained, at all densities, employing the BLP diet, also with the IAEA-BY diet a high survival rate was obtained, but only at the lower densities (1 and 2 larvae/ml), while with the IAEA diet the survival rate was lower compared to the other two diets at all the densities tested. The measurement of the wings is still in progress.
Aims for the next 18 months: Test different procedures to improve synchronous egg hatch and larval development. In our standard procedure, the Ae. albopictus egg hatch is stimulated with a nutrient broth culture overnight, therefore the newly hatched larvae collected in the morning may have 1-16 hours, which is a concern when coetaneous larvae are needed. We will test the effect of boiled water, shaking movement and combination of these methods including the use of nutrient broth, on the egg hatching in comparison with our standard procedure. Evaluate the possibility to have coetaneous larvae by means of brief overnight starvation upon hatching with our standard procedure using the nutrient broth; Based on the results obtained with this study, a protandry strain will be produced using more coetaneous larvae. The outcome will be regularly evaluated during the selection process by checking the following parameters: male and female pupae size, male pupae productivity, percentage of residual females present after the male pupae collection and sieving. The comparator will be the dataset obtained at the beginning of the selection process; Analyze the effect of pH on egg hatch and sex determination in Ae. albopictus; Continue the cooperation with ENEA establishing a permanent ARwP colony at the CAA mass rearing facility in order to monitor Wolbachia density, pre-imaginal development, efficiency of sex separation, adult fitness, and CI level, after each 5 generations of CAA rearing SOP; Collaborate with TRAGSA and the Universidad CEU Cardenal Herrera to analyze the frequency size distribution curves of pupae from different Ae. albopictus strains (including Rimini, Valencia and CAA protandry strains) following rearing SOP. TRAGSA will provide the image analysis software.
Contract 17945: Carlos Tur Lahiguera, TRAGSA, Gustavo Salvador Herranz, Universidad CEU Cardenal Herrera, Spain (Collaborators: Romeo Bellini, A. Puggioli, H. Bossin, P. Tortosa)
Aedes albopictus female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer
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During the past 18 months a hardware and software system was developed to identify and measure the size of Ae. albopictus pupae. Also a protocol to calculate the threshold between male and female size for each cohort has been developed and tested with positive results. Regarding the female removal hardware, all components have been selected and tested, including a CO2 laser, galvanometer scanner and a servo controller. Good progress was achieved in order to adjust the position of the pupae centroid coordinates and the laser beam path.
Aims for the next 18 months: Finalize building of the sex sorting prototype by tuning the image acquisition system with the laser beam positioning device, and synchronizing both systems with the forward linear speed of pupae on the conveyor wheel. Develop a method to feed the system with new pupae, in order to drain and distribute them uniformly over the conveyor wheel, avoiding pupae physical contact. Perform testing of the sex sorting prototype with Ae. Albopictus pupae from Valencia strain. Collaborate with CAA and CRVOI to analyze the frequency size distribution curves of pupae from different Ae. Albopictus strains (including La Réunion, Rimini, Valencia and CAA protandry strains following CAA rearing SOP). Determine frequency size distribution curves of Ae. Polynesiensis and Ae. Aegypti to assess the potential for laser-based sex separation in collaboration with Hervé Bossin (Institut Louis Malardé, French Polynesia).
Contract 17926: Gulzamin Khan, Alam Zeb, Inamullah Khan, Nuclear Institute for Food and Agriculture (NIFA), Pakistan (Collaborators: C. Tur, R. Bellini, A. Puggioli, W. Abeyewickreme, P. Tortosa)
Exploring mechanical, behavioural and nutritional methods of sex separation in mosquitoes.
Mechanical, behavioural, nutritional and developmental tools for sexing may be needed when efficient systems for eliminating females in the embryo stage have not been developed or are not yet available. This project is proposed to develop a specific and efficient system for male female separation. Sex separation is possible at the pupal stage for both Aedes and Culex species due to size dimorphism between male and female pupae: females are usually larger. Based on the blood-seeking behaviour of female mosquitoes for egg development, females require a more substantial source of food than males to mature eggs, and so seek sources for blood feeding. It is hypothesized that the same behaviour of females consuming more substantial amounts of diets may exist at the larval stages. This could be exploited for separation of larvae, through the use of diets rich in protein and others rich in carbohydrate and different quantities fed to them during the early stages of development. Diets that are richer in protein might be preferentially used by female larvae and they may in turn produce larger female pupae which are more easily separable using sieving methods.
Aims for the next 18 months: Compare efficiency of Aedes albopictus pupae sex separation using sieving and/or Fay-Morlan separation methods (separately or in combination).
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Explore differences in buoyancy between male and female pupae for both Culex and Aedes using cold water in which different salts/ chemicals e.g. salt or sugar solutions. If such differences are found, they could be exploited further in mechanized sex- separation systems. Similarly, female adult mosquitoes are heavier and larger than males. These characteristics may be of value for separation of Culex in which size difference between sexes is the greatest. Assess sex separation at the adult stage by spiking blood with insecticides (malathion, dieldrin) or other mosquito toxins (ivermectin, imidaclropid, Spinosad and IGR [Pyriproxifen]). Test combination of diets with larval crowding for increased protandry, and difference in the pupal size. Explore protandry in Aedes albopictus and Culex quinquefasciatus for better sex- separation methods Effect of chilling time on sheath formation in female pupae Effect of time period in the mechanical sex separation due to different light stimuli
Contract 17954: René Gato Armas, Juan A. Bisset, Magdalena Rodríguez, Israel García, Domingo Montada, Aileen González, Misladys Rodríguez, Institute Pedro Kourí, Havana, Cuba
Conditional lethality by insecticide resistance for sex separation
During the coming 18 months, we plan to obtain a genetic sexing strain of Aedes aegypti based on recessive conditional lethality. We have developed a homozygote temephos-resistant strain under selective pressure, which is routinely maintained under pressure in the laboratory using the LC90 (90% lethal concentration) of temephos, using a susceptible strain recently colonized from specimens collected in the field as a control strain. Aims for the next 18 months: Resistant adult males will be X-ray irradiated to induce genetic disruption. Irradiated males will be crossed with virgin susceptible females and male progeny from individual F1 females will be backcrossed with susceptible females. Translocation of a gene conferring temephos resistance at the vicinity of the male locus will be tested by exposing the progeny to 90% lethal concentration of temephos and measuring the sex ratio. The strains of interest will carry a temephos-resistant gene linked to the M locus. In order to keep this strain free of heterozygous resistant females, GSS males will be continuously outcrossed with susceptible females and maintained under temephos selection.
Contract 17904: Lizette Koekemoer, Leyya Essop, Basil Brooke, Givemore Munhenga, Thabo R Mashatola, WITS Research Institute for Malaria and National Institute for Communicable Diseases, South Africa (Collaborators: Pablo Tortosa, Jeremie Gilles, Kostas Bourtzis).
Effect of irradiation on local South African strain Introgressed with a dieldrin resistant (ANO IPCL1) strain and the use of an alternative to dieldrin for production of «male only» adults.
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Our group has successfully introgressed the male-linked dieldrin resistance allele from material obtained from Seirbersdorf Laboratory to a local An. arabiensis population. For this, females collected from Kruger National Park (AMAL) were mated with GSS ANO IPCL1 males obtained from the Insect Pest Control Laboratory (IPCL) FAO/IAEA Agriculture and Biotechnology laboratories, Seibersdorf, Austria. This has enabled gender separation of newly laid eggs by exposing them to dieldrin, producing male larvae only. Stability of the introgressed strain was determined through rdl Taqman probe PCR (Bass et al, 2010) at each generation. Reproductive fitness parameters for the strain were investigated and optimal irradiation doses inducing male sterility while maintaining mating vigour have been determined. Our group is also evaluating the efficacy of other GABA receptor antagonists to replace dieldrin. This sexing system is facing a number of challenges prior implementation at a larger scale, motivating the need to develop an alternative cost effective and environmentally friendly sexing method. In this context, we are exploring the development of a sexing strain based on tsl.
Aims for the next 18 months. Finalize optimization of doses and times of exposure for five GABA receptor antagonists (fipronil; picrotoxin; alpha-endosulfan; lindane and isoxazole). We will use increasing doses of insecticide on L3-L4 larvae with both AMAL (SS) and GAMA (GSS=RS) strains. Once we have obtained probit curves, resistance tests will be carried out on eggs. In the absence of any morphological marker, we will first characterize temperature sensitivity of eggs, larvae and pupae, in order to determine the temperature killing 50% and 90% of the mosquitoes. For this, mosquitoes will be kept for 12 hours in climatic chambers with different temperature conditions, ranging from 33°C to 45°C (3°C increments). If the above task is achieved, develop homozygous temperature resistant and susceptible lines.
Contract 17953: Cyrille Ndo (OCEAC, Camerooon), Yacouba Poumachu (OCEAC, Cameroon), Parfait Awono-Ambene (OCEAC, Cameroon), Igor Sharakhov (University of Virginia Tech USA) (Collaborators: IPCL, Jeremie Gilles, Kostas Bourtzis)
Developing a genetic method for accurate sex separation and female elimination in An. arabiensis
The general aim of our project is to develop an An. arabiensis genetic sexing strain in which females are homozygous for a recessive temperature-sensitive lethal (TSL) leading to lethality under non permissive conditions (i.e. 40°C), thus allowing selection of heterozygous resistant males. Mosquito colonies have been established in our insectary for experiments (An. arabiensis from North Cameroon and An. gambiae from MR4). We performed mutagenesis of An. arabiensis male mosquitoes using 3 distinct EMS doses and screened isofamilies for the tsl phenotype as well as for the presence of morphological markers. We have identified 2 strains named ANA_S1 and ANA_S2 together with 3 other families expressing temperature sensitivity. So far, only 2 specimens with a white-larvae phenotype have been observed.
Aims for the next 18 months: Confirm the presence of tsl among the selected strains/families and determine whether temperature sensitivity is an inheritable character; 39
Continue screening both natural and laboratory reared populations in order to isolate morphological markers; alternatively, insecticide resistance conferring genes (kdr, rdl, ace1) maybe used as markers. If the 2 first tasks are achieved, create a Genetic Sexing Strain by irradiating wt males with the aim of translocating tsl+ on the Y chromosome.
Contract 17933: Pablo Tortosa, Célestine Michelle Atyame, Louis-Clément Gouagna Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Océan Indien, La Réunion, France (Collaborators: G. Munhenga and M. Calvitti)
Construction of an RdlR Genetic Sexing Strain in Aedes albopictus.
Our laboratory is currently developing 2 innovative vector control strategies, namely the Sterile Insect Technique and the Incompatible Insect Technique, targeting Aedes albopictus, a vector species of global medical concern. Although both strategies are showing promising results, they both require the availability of an efficient sexing strain allowing the mass production of sterile or incompatible males. In the frame of the CRP “Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes”, we will create an Aedes albopictus Genetic Sexing Strain (GSS) using the rdlR allele confering dieldrin resistance. We constructed homozygous rdlR and rdlS lines, and measured the levels of resistance to dieldrin conferred by these genotypes. Since dieldrin has been banned for decades and is thus not acceptable for mass production, we explored the possibility of using fipronil as an alternative insecticide. For this, we have measured the levels of fipronil resistance conferred by rdlR allele on Cx. quinquefasciatus reference strains (S-GABA and S-Lab, RR and SS, respectively) provided by Mylène Weill (ISEM, Montpellier, France).
Aims for the next 18 months: Measure fipronil resistance on homozygous (rdlR and rdlS) and heterozygous Ae. albopictus mosquito lines. Carry out irradiations on rdlR homozygous males Backcross irradiated rdlR homozygous males with SS females and identify sex ratio- biased families.
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SECOND RESEARCH CO-ORDINATION MEETING
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
“Explore genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes”
Moscamed Facility Juazeiro, Brazil 9 - 13 March 2015 Scientific Secretary: Jeremie Gilles & Kostas Bourtzis
AGENDA Monday, 9 March 2015
SESSION 1 (K. Bourtzis)
09.00-09.15 Opening and presentation of the participants 09.10-09.20 K. Bourtzis: Administrative details and introduction 09.20-09.30 T. C. Chao. Brazil and the IAEA Technical Cooperation. 09.30-10.00 K. Bourtzis, D. Zhang, Z. Xi, J. Gilles. Combining SIT and Wolbachia-based methods for the population control of Aedes albopictus. 10.00-10.30 M. L. Capurro. Construction and characterization of GSS (Aedes aegypti and Ae. albopictus).
10.30-10.45 COFFEE BREAK
10.45-11.00 D. O. Carvalho. Obtaining genetically sterile transgenic lines for Aedes aegypti male production 11.00-11.15 A. L.Costa-da-Silva, D. O. Carvalho, B. B. Kojin, M. L. Capurro. Promoting refractoriness in Aedes aegypti fed with Dengue virus infected blood. 11.15-11.30 H. R. Corrêa de Araújo. Using transgenic Aedes aegypti to study mate biology. 11.30-11.45 M. Pedrosa. Transgenic Aedes Project: Mass rearing and Quality Control. 11.45-12.00 L. Garziera. PAT - Transgenic Aedes Project – Release, Monitoring and Community Engagement.
12:00-13.30 LUNCH
SESSION 2 (Hervé Bossin)
13.30-14.00 I. Häcker, M. Schetelig. Site-specific landing site systems to improve transgenic insects for their use in SIT programs. 14.00-14.30 F. Criscione, Y. Qi, J. Biedler, B. Hall, I. Sharakhov, Z. J. Tu. Mosquito Y chromosome genes and sex-determination. 14.30-15.00 The Anopheles Y consortium: A. B. Hall, A. Sharma, C. Cheng, A. Cagnetti, et al, I. V. Sharakhov, Z. J. Tu, N. J. Besansky, P.A Papathanos. The Y chromosome of the Anopheles gambiae complex: cataloguing and characterizing content and putative functions determination in anopheline mosquitoes.
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15.00-15.30 COFFEE BREAK
15.30-16.00 F. Scolari, G. Savini, P. Gabrieli, M. Manni, L. M. Gomulski, G. Gasperi, A. R. Malacrida. Cytogenetic and molecular characterization of sex-specific markers in the tiger mosquito Aedes albopictus. 16.00-16.30 G. Rose, E. Krzywinska, J. Krzywinski. Sex determination and dosage compensation in Anopheles gambiae. 16:30-17.00 F. Bernardini, N. Windbichler, A. Crisanti. A functional study of the Y chromosome in the malaria vector A. gambiae. 19.00-22.00 IAEA reception.
Tuesday, 10 March 2015
SESSION 3 (Marc Schetelig)
09.00-09.30 A. Smidler, Robert W. Shaw, F. Catteruccia. Mosquito sex separation via TALEN-mediated female specific lethality. 09.30-10.00 W. Reid, D. A. O’Brochta, S. N. Surendran. CRISPR/Cas9 Gene Editing of Mosquito Genomes. 10.00-10.30 C.-H. Chen. Creating an insect synthetic population to fight dengue fever.
10.30-11.00 COFFEE BREAK
11.00-11.30 C. Ndo, Y. Poumachu, P. Awono-Ambene and J. Gilles. Developing genetic method for accurate sex separation and female elimination in the malaria vector Anopheles arabiensis. 11.30-12.00 P. Tortosa. Development of an Aedes albopictus genetic sexing strain based on dieldrin resistance: selection and identification of homozygous resistant and sensitive strains.
12.00-13.30 LUNCH
SESSION 4 (Pablo Tortosa)
13.30-14.00 G. Munhenga, L. Essop, L. Dandalo, B. Brooke, O. Wood, L. Robertson, L.L. Koekemoer. Effect of irradiation on local South African strain introgressed with dieldrin resistant (ANO IPCL1) strain and the use of an alternative to dieldrin for “male only” adults. 14.00-14.30 C. Tur Lahiguera, G. Salvador. Aedes albopictus female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer. 14.30-15.00 R. Bellini, A. Puggioli, R. Veronesi, M. Carrieri. Exploiting sex dimorphism and protandry for Aedes albopictus sex separation.
15.00-15.30 COFFEE BREAK
15.30-16.00 W. Abeyewickreme, M. Hapugoda, N. Gunawardena, J. Harischandra. Exploring Tools for Sex Separation of Dengue Vector Mosquitoes in Sri Lanka. 16.00-16.30 G. Z. Khan, A. Zeb. Exploring mechanical and nutritional methods of sex separation in Aedes albopictus mosquitoes. 16.30-17.00 R. G. Armas. Conditional lethality by insecticide resistance for sex separation 42
of Aedes aegypti. 17.00-17.30 H. C. Bossin, H. Petiti, M. A. Cheong Sang. The AeLIMIN+ suppression trial in French Polynesia: monitoring of Aedes polynesiensis rearing and sorting parameters under various rearing conditions. 17.30-18.00 M. Calvitti, R. Moretti, F. Marini, A. Desiderio. Exploring the role of a heterologous Wolbachia single-infection in Aedes albopictus.
Wednesday, 11 March 2015
08.30-10.00 Open discussion and composition of the working groups
10.00-10.30 COFFEE BREAK
10.30-12.00 Revision of the CRP documents (introduction, contracts) by groups
LUNCH 13.30-17.00 Revision of the CRP documents (introduction, contracts) by groups
19.00-22.00 Group dinner
Thursday, 12 March 2015
09.00-10.30 Revision of individual contracts and planning of the activities to carry out for the next 18 months
10.30-11.00 COFFEE BREAK
11.00-12.30 Revision of individual contracts and planning of the activities to carry out for the next 18 months
12.30-14.00 LUNCH
14.00-15.30 Development of the logical framework matrix (LFM)
15.30-16.00 COFFEE BREAK
16.00-17.30 Development of the logical framework matrix (LFM)
Friday, 13 March 2015
09.00-10.30 Presentation of the LFM to the entire group and drafting the final document
10.30-11.00 COFFEE BREAK
11.00-12.30 Drafting the final document/RCM report
12.30-14.00 LUNCH 43
14.00-16.00 Presentation of the RCM report
16.00-16.15 Closing
PARTICIPANT ABSTRACTS
Construction and characterization of GSS (Aedes aegypti and Ae. albopictus)
Margareth Lara Capurro Guimarães
Associate Professor, São Paulo University
When considering a mosquito release program, one of the first issues to decide is how to eliminate females. A greatest number of options might eventually be available for using transgenic mosquitoes, but the inherent characteristics of the target species itself also provide possibilities for interim measures until more efficient methods can be developed. Differences in intrinsic size, behaviour and development rate between females and males for sexing may be available. Ideal systems specifically kill female embryos using a treatment that can be manipulated during production. Such killing systems are far more efficient than using intrinsic sexual differences, but these systems require selectable genetic markers and sex- linkage created by rare random chromosomal rearrangements. While intrinsic sexual differences should not be considered long-term substitutes for more robust and efficient sexing systems, in the absence of these, the accessibility and integration of less efficient systems can provide a stop-gap measure that allows rapid start up with a minimum of investment. The goal of this project is to generate transgenic Aedes aegypti and Ae. albopictus mosquitoes that will produce males-only progeny. These mosquitoes will be of value to public health programs in charge of controlling dengue epidemics (regular SIT or sterile mosquitoes by transgenesis). The key components of this strategy are the embryonic zygotic genes (EZGs) promoters and RNAi that will knockdown the doublesex pathway. This gene will be regulated by Tet-On Tet-Off systems to produce laboratory colonies.
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Obtaining genetically sterile transgenic lines for Aedes aegypti male production Danilo de Oliveira Carvalho
Universidade de São Paulo
Aedes aegypti the major arbovirus vector is presented in all tropical and subtropical area of the world. New strategies are needed to control the disease and suppress the mosquito population. The use of SIT (Sterile Insect Technique) is one of the approaches to control the disease, and molecular tools providing genetically modified organisms are also a novel strategy to suppress mosquito population. This project aims to obtain new transgenic lines that produce an effective molecule to promote sterility. The antagonist of inhibitor apoptosis protein, michelob_x and the endonuclease, CviAII are the chosen molecules to promote sterility in testis during the sperm production through the promoter of ß2-tubulin and conditionally regulated by the tetracycline system to keep the line fertile for colony maintenance for egg production.
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Promoting refractoriness in Aedes aegypti fed with Dengue virus infected blood
André Luis da Costa-da-Silva, Danilo de Oliveira Carvalho, Bianca Burini Kojin and Margareth Lara Capurro
Laboratório de Mosquitos Geneticamente Modificados, Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP 05508-000, Brazil
Dengue fever ranks as the most important mosquito-borne viral disease in the world. There are 2.5 billion people living in endemic areas and recent estimates show as many as 390 million infections occur annually with alarming death numbers caused by serious manifestation of this disease. These circumstances emphasize the urgent need for alternative approaches to limit the transmission of dengue since Aedes aegypti control measures as integrated management are not sufficient and commercial vaccines for dengue are not available. The goal of this project is to produce Ae. aegypti transgenic lines exhibiting a dengue virus refractory phenotype after an infected blood meal, being not competents for transmitting dengue to humans. The key components of this strategy are the NS3 viral protease, a female-specific and blood- meal induced mosquito promoter, and an engineered anti-dengue gene (ADG). The ADG encodes a fusion protein with a transmembrane-endoplasmic reticulum (ER) localization signal from Sec 61 gamma, a cleavage site recognized by the viral protease and a complete open reading frame of michelob_x (mx), the mosquito orthologous gene of the reaper/grim- like inhibitor of apoptosis protein (IAP) antagonist genes that have a pivotal role in cell death regulation in Drosophila. We obtained 5 transgenic lines and RT-PCR showed that all of them are expressing ADG gene (A5.F, A5.X, A5.Y, A5.Z e A5.P4). A5.F females were fed with D2 infected blood and transgenic insects did not show a longevity reduction, suggesting the block of viral replication. We are performing D2 challenges, infecting these lineages by oral feeding or thoracic injection to test 2 possible assumptions based on phenotypes – triggering the apoptosis in D2 infected midgut cells can heal the infection in initial virus replication or generalized infection in mosquito body cells can kill the insect, both mechanisms driving to an incompatible phenotype of virus transmission.
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Using transgenic Aedes aegypti to study mate biology
Helena Rocha Corrêa de Araújo
Universidade de São Paulo
The technique of releasing modified mosquitoes for population suppression requires species to mate once, however there are studies that mention a second mate, which could compromise the success of the technique. Studies using techniques labeling mosquito sperm (spermatozoa and accessory gland fluids) shows that greatly facilitate the study of mating behavior. Those studies showing a second mate from females used techniques difficult to demonstrate properly the occurrence of a second mate. The aim of this project is to use a transgenic line to demonstrate more accurate the occurrence of second mate. For that, a virgin female was mated with a transgenic male and after the mating the male was changed for a wild-type male and spend 24 hours. A second group, the mating was interrupted and the transgenic male was changed for a wild-type male. As result, the females that had a complete mate had offspring only from the first male, and females with interrupted mate had offspring from the second male and in some matings the female presented mixed offspring. This project showed that without concerns that only females with interrupted mating had possibility to present mixed offspring or from the second male.
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Transgenic Aedes Project: Mass rearing and Quality Control
Michelle Pedrosa
Moscamed Brasil and Universidade de São Paulo
Moscamed Brazil has the biggest Aedes aegypti (OX513A – Oxitec Ltd) mass rearing in the world (UPAT – Transgenic Aedes production Unit). In a mass rearing program there are two sectors: Egg production and Males production for release. Both sectors need to be monitoring extensively for quality control, that is an additional sector completely independent from the previously sectors. Two independent protocols are applied for egg and male production. For egg production in our condition (female = 1.35 mm and male = 1. 16 mm) 1 adult cage (1,500 females plus 500 males in a 30 x 30 cylinder cages) produce approximately 105,000 eggs after two gonotrophic cycles. UPAT total egg production per week is 6 million. Around 20-30% of the total production is stored and eggs are maintained during three months. For Males production are assembled three batches per week, producing at least 335.000 males each one. The recovery of males is on average around 30% in the first sorting. Each batch is sorted and prepared in a day and sent on the next morning. The production of males is currently of 1 million males per week. Male production for release are been produced at UPAT and pupae are sent to LEMI (Laboratory of Emergence, Monitoring and Information) at Jacobina City, the release site, in which pupae are placed in release devices for emergence. There is several Quality Controls during the whole process: control of the strain (fluorescence/genetic marker and lethality without tetracycline), control of the size of the male pupae, control of female contamination and control of transport of pupae. This is the biggest production of Aedes aegypti OX513A of the world, however, it is necessary to optimize and improve the mass rearing system. Therefore, several experiments have been done and many others are planned for the near future.
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Transgenic Aedes Project – Release, Monitoring and Community Engagement
Luiza Garziera
Moscamed Brasil
The project is called Projeto Aedes Transgenico (PAT) (Transgenic Aedes Project). From the start an extensive community engagement campaign was implemented to insure full transparency regarding the project. The actions were planned to cover every habitants and employing as much as possible all media tool available to reach the target area. Overall PAT can be divided into two stages, and in both the methodologies and strategies used were similar: I –PAT Juazeiro (2010-2013): Field tests of transgenic line OX513A of Aedes aegypti for control of natural populations in localities of Juazeiro. This steps, the first year had successful transfer of transgenic mosquito strain from UK to Brazil with establishment and local optimization of scalable mass rearing methodologies. Field activities have concentrated on evaluating a range of behavioral, quality and mating competitiveness factors of RIDL Aedes males in the first experimental area of Itaberaba. This site proved ideal for initial evaluation of these factors due to its proximity to Moscamed (< 1 Km) and very high year round mosquito population. The relatively stable and very high wild mosquito population allowing evaluation of mating success over a range of release densities, similar to a dose response study one may conduct for a new insecticide. This has provided a substantially better understanding of release numbers required to achieve suppression for a given wild population. II-PAT Jacobina (2013-present): Implementation of design for population suppression of Aedes aegypti in the urban area of Jacobina, using the transgenic line OX513A. Community engagement - Use of genetically modified insects raises understandable concerns as with any new technology. From the outset the project partners have worked closely with the Brazilian regulatory system to obtained required permits for field activities. It has been a clear focus of PAT from the outset to adopt full transparency and a vigorous and proactive community engagement campaign. This cuts across all levels of the community from national, regional and local stakeholders, public health and vector control agencies, and the resident community. Engagement with media via radio, TV and press at local and national level has been vital for communication to as many people as possible, and is facilitated by full time journalist working on the project. In a country that suffers hugely from dengue, media interest remains high with continued, overwhelmingly positive support for the project from all 49
sectors. In addition to working with the national regulatory system, numerous meetings and seminars have been organized with public health and political leaders at all levels to provide information and opportunity for feedback. Community engagement needs to be tailored to the audience taking into account the cultural and social context. This is most significant when engaging with the local community at the field sites. A local scientist was engaged to provide expert advice on best communication strategy. This was built on existing experience from Moscamed, who brought considerable local experience from previous community engagement campaigns in the implementation of conventional fruit fly SIT programs, and from international experience from earlier RIDL SIT projects. It is important to note that community engagement began well in advance of any release. Implementation includes communication via local media (radio, TV and press), community meetings, printed information (posters and leaflets), school presentations, carnival parades and use of truck with loudspeakers. A jingle explaining the project was commissioned which accompanied local radio broadcasts and communication events. However, there is no substitute for face-to-face interaction on an individual basis, allowing specific questions to be addressed and for direct feedback and concerns to be aired. Intensive monitoring provides full opportunity for field staff to discuss the project with residents in their homes as they survey the local mosquito population. The relationship and trust established between field staff and community has been instrumental and began during baseline monitoring phase before any releases occurred. In addition, dedicated teams have conducted door-to-door visits of every residence to discuss the project. These combined efforts have resulted in visits to every house in the field sites, often on multiple occasions, ensuring the highest level of community engagement possible. In Jacobina, the activities are not door-to-door due to the large number of homes in the city, however besides the already mentioned activities, itinerant shares are performed in places where people congregate, with disclosure and explanation of the project, including demonstration of mosquitoes in cages. The results of reduction of a neighborhood and the release schedule of the other neighborhood are similarly disclosed and through the distribution of pamphlets by PAT technicians. Actions of the Community engagement must be continuous, being performed before, during and after the period of mosquitoes releases.
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Site-specific landing site systems to improve transgenic mosquitoes for their use in SIT programs
Häcker, I & Schetelig, MF
Justus-Liebig-University Gießen, Germany
A successful strategy to control pest insect populations is based on the Sterile Insect Technique (SIT), which uses the release of mass-reared, radiation-sterilized male insects to cause infertile matings and thus reduce the pest population level. Genetic modifications offer new possibilities in pest insect control, e.g. by developing conditional genetic sexing or sterility systems needed for SIT. Genetic modification is often achieved by random genomic insertion of a transgene construct via transposons. This is challenging for GMO strain development due to genomic position effects that could suppress transgene expression or by insertional mutations that negatively affect host fitness and viability. Recombinase-mediated cassette exchange (RMCE) allows for site-specific integration of transgene constructs into a well-characterized integration site of so- called landing site lines. RMCE has been successfully established in Drosophila and in Caribfly. There are different recombination systems available to establish RMCE in an insect species, like the lox, FRT or attP systems. Besides targeting transgene vectors to defined genomic insertion sites having minimal negative effects on gene expression and host fitness, RMCE would also allow for stabilization of the target site by removing transposable elements, which was successfully done in Medfly. This addresses a major concern for GM insect release programs as transgene loss or intra-genomic movement could result in loss of strain attributes, and may ultimately lead to inter-species movement resulting in ecological risks. We created several landing site lines for RMCE in Aedes aegypti. These landing site lines contain heterologous lox sites flanking a fluorescent marker. We inbred these lines to homozygosity and injected early embryos with the Cre recombinase and a donor plasmid containing the corresponding lox sites flanking a different fluorescent marker. A change of marker colour in the offspring (G1) of the transformed individuals (G0) would indicate a successful cassette exchange event. G1 individuals with the new marker could indeed be identified. However, positive individuals still showed the original marker in addition to the new colour, indicating integration instead of a recombination event, which was confirmed by molecular analysis. Further recombination experiments using different sites are planned.
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Mosquito Y chromosome genes and sex-determination
Frank Criscione 1*, Yumin Qi 1, Jim Biedler 1, Brantley Hall 1, Igor Sharakhov 2, and Zhijian Jake Tu 1
1. Department of Biochemistry, Virginia Tech, Blacksburg, VA 2. Department of Entomology, Virginia Tech, Blacksburg, VA * Current Address: Department of Entomology, University of Maryland.
Y chromosomes are responsible for the initiation of male development, male fertility, and other male-related functions in diverse species. We have recently discovered a few Y genes in Anopheles mosquitoes. In this presentation, I will discuss the function of one of the Y genes named Guy1 in An. stephensi. We have obtained a number of transgenic lines that express Guy1 on the autosomes and showed that all of the >6000 transgenic individuals are males. We have shown that this phenotype resulted from complete female lethality, not female to male conversion, and that the GUY1 protein is the cause of the lethality. We further demonstrated that the Guy1 transgenic males were more competitive than their non-transgenic male siblings under our laboratory conditions. We are generating conditional Guy1 expression lines to make homozygous transgenics, which will produce all males for genetic sexing and for population suppression.
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The Y chromosome of the Anopheles gambiae complex: cataloguing and characterizing content and putative functions
The Anopheles Y consortium: Andrew B. Hall, Atashi Sharma, Chengde Cheng, Alessia Cagnetti, et al, Igor V. Sharakhov, Zhijian Tu, Nora J. Besansky and Philippos-Aris Papathanos
Centre of Functional Genomics, Department of Experimental Medicine, University of Perugia
Y chromosomes have proven to be extraordinarily difficult to characterize, mostly due to their repeat-rich nature. Not much is known about the Y of anopheles mosquitoes. Based mainly on cytological evidence we know that they are mostly heterochromatic, heteromorphic and heterogeneous across different species and even within populations, and that in some anopheline species the Y harbors the primary signal of sex determination and sequences that influence mating behaviours. In collaboration with the laboratories of Jake Tu, Igor Sharakov, Andrea Crisanti and Nora Besansky, which together formed the Anopheles Y consortium last year, we have recently completed the first in depth analysis on the Y chromosome of species of the gambiae complex. We have documented content and its evolution, the expression landscape and its variation in natural populations. The discoveries we have made, represent a significant step in increasing our understanding of mosquito biology including hints at how sexual dimorphism is orchestrated and species barriers may operate. I will also present updates from our laboratory concerning the characterization of sex biased gene evolution in the anopheles genus, our recent sterp towards engineering of the Y chromosome and the progress in the development of invasive genetic drive constructs to control mosquito population by biasing their sex ratio.
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Cytogenetic and Molecular Characterization of of sex-specific markers in the tiger mosquito Aedes albopictus
Francesca Scolari, Grazia Savini, Paolo Gabrieli, Mose’ Manni, Ludvik M. Gomulski, Giuliano Gasperi, Anna R. Malacrida
Dept of Biology and Biotechnology ‘Lazzaro Spallanzani’ University of Pavia, Italy
The development of Genetic Sexing Strains (GSS) in the Asian tiger mosquito Aedes albopictus requires a deeper knowledge of the chromosomal repertoire of the species, with particular emphasis on the identification of the sex determining chromosomes. This is important as, unlike anophelines, the sex chromosomes of Ae. albopictus, like other culicine mosquitoes, are homomorphic (Rai 1963, Ann Entomol Soc Amer 56:160-170). To this aim, using the Ae. albopictus Rimini strain provided by Romeo Bellini Laboratory (CAA, Crevalcore), we developed a protocol that distinguishes the sex of mature fourth instar larvae by microscope observation of developing gonads. In particular, we based our discrimination on the presence of transverse pseudochambers, representing the spermatic tubes, to identify the testes. This protocol permits the unambiguous identification of male and female larvae and thus facilitates the characterization of their karyotypes using mitotic chromosomes obtained from larval imaginal discs. Both DAPI- and Giemsa C-banding stainings performed on such mitotic preparations consistently confirmed the presence of a male-specific banding pattern on Chromosome 1. We also performed molecular analyses using the Representational Difference Analysis (RDA) approach (Lisitsyn et al. 1993, Science, 259:946-951) to identify male-specific/enriched sequences to be used as targets for PCR assays for molecular sexing. From one RDA library (enzyme MseI), we isolated one particularly interesting sequence due to its blast hits to one Ae. albopictus microsatellite sequence. This is not unexpected, due to the available information on Aedes aegypti, where the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites, in addition to a higher rate of genome rearrangements than observed in autosomes 2 and 3. FISH and PCR-based assays on the mitotic chromosomes will confirm the physical location of this sequence on the sex- determining region of Ae. albopictus.
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Sex determination and dosage compensation in Anopheles gambiae
Graham Rose, Elzbieta Krzywinska, Jaroslaw Krzywinski
The Pirbright Institute, UK
In insects sex is determined by a short cascade of genes. A key gene (primary signal) at the top controls a few subordinate genes that are differentially spliced in males and females. In Anopheles mosquitoes the primary signal comes from a dominant maleness factor linked to the Y chromosome. The only known element of the cascade in mosquitoes is the last step, the doublesex gene, whose sex-specific transcripts encode male and female forms of the DSX protein, which modulate downstream sexual differentiation processes. Earlier, we identified a Y chromosome gene in An. gambiae that has characteristics of the primary signal, including early embryo onset of expression and female killing effect, which may be due to disturbance of dosage compensation. Here we performed in vivo tests, which indicate the identity of the Y gene with the maleness factor. There is rather scant evidence for dosage compensation in An. gambiae. Therefore, to address the issue of likely female killing mechanism mentioned above, we analyzed global expression levels in the An. gambiae fourth instar larvae and pupae using high-coverage RNA-seq data. In both sexes, the median expression ratios of X-linked to autosomal genes were close to 1.0, and within or above the ranges of expression ratios between the autosomal pairs, consistent with complete compensation. Confirmation of the existence of dosage compensation in Anopheles opens a challenge for identification of the components of dosage compensation machinery and for deciphering their interaction with the sex determination pathway genes.
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A functional study of the Y chromosome in the malaria vector A. gambiae
F. Bernardini, N. Windbichler, A. Crisanti
Imperial College of London
Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. We previously characterized a transgenic strain of this mosquito species, obtained by transposase-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair a site-specific recombination signal was introduced onto the Y chromosome and the resulting docking line, named YAttP, was proven to allow secondary integration. To demonstrate its utility, the activity of a germline-specific promoter when located on the Y chromosome was studied. Anopheles arabiensis is another important vector of human malaria. Since active insertions onto the Y chromosome are extremely rare, a scheme based on crossing and selection was used to overcome F1 hybrid male sterility and introgress the modified Anopheles gambiae Y chromosome in the Anopheles arabiensis genetic background. Fertility of Y-introgressed males, tested after up to 10 backcross generations, was comparable to fertility of wild-type Anopheles arabiensis males. The molecular manipulation of the Y chromosome in Anopheles gambiae, opens up a number of ways to explore one of the most fascinating of evolution’s upshots and its introgression in the Anopheles arabiensis genetic background may answer important questions on the similarity and differences in Y chromosome biology of closely related species. The Y-linked fluorescent transgenes allow automated sex separation of these important vector species, providing the means to generate large single-sex populations. Furthermore, the possibility of introducing genes of interest specifically onto the Y chromosome make these strains a valuable tool for vector control strategies.
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Mosquito sex separation via TALEN-mediated female specific lethality
Andrea Smidler, Robert W. Shaw, Flaminia Catteruccia
Harvard T.H. Chan School of Public Health
In mosquitoes effective sex-separation is critical for the development and implementation of Sterile Insect Technique to suppress wild mosquito populations. We proposed to develop a system in which two TAL endonucleases (TALENs) targeting an essential gene are transgenically inserted onto the X-chromosome, such that trans-heterozygous females are inviable and males are viable. Towards this end, we proposed to develop an X-linked PhiC31 docking line in Anopheles gambiae with the help of our collaborator Dr. Eric Marois. Dr Marois generated the transgenic line “French Kiss”, by PiggyBac-mediated integration in which the transgene inserted onto the X chromosome within an intron of AGAP001069. This transgene was designed such that successful Cre-Lox recombination would excise the entirety of the transgenic cargo except for an AttP site, thereby resulting in an X-linked docking line. Injection of Cre-recombinase into this line was unsuccessful, but crossing this line to a Cre- expressing transgenic (carried out by Dr. Eric Marois) resulted in successful excision of the transgenic cargo and the creation of an X-linked docking line. Unfortunately, reintroduction of transgenes into this docking line has many undesirable phenotypic effects, and initial results suggest that this line is not likely suitable for its originally intended purpose. In the future, we will continue efforts to generate X-linked docking lines with more desirable phenotypes. Male-only populations could in principle be generated by the development of a toxin-antidote system where males are rescued via the male-specific expression of an antidote. To this end, although not included in our initial proposal, we have identified a highly specific male reproductive promoter that we expect may be useful for other participants of this consortium. This promoter drives the expression of the male accessory gland (MAG) protein Plugin (AGAP009368), the major constituent of the mating plug in Anopheles gambiae. We set out to generate a transgenic line where Plugin is fused to tdTomato and under the expression of the native Plugin promoter. Preliminary data shows it is highly and specifically expressed in the anterior compartment of the MAGs and shows undetectable levels of expression in females. Moreover, expression of the Plugin-tdTomato fusion protein in the MAGs generates a fluorescent mating plug that can be visualized in mated females without the need for
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dissection. Based on the highly sex-specific nature of this promoter, we expect that it could be harnessed to facilitate a toxin-antitoxin system that permits the survival of only males.
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CRISPR/Cas9 Gene Editing of Mosquito Genomes
William Reid1, David A. O’Brochta1, Sinnathamby Noble Surendran2
1University of Maryland College Park, 2University of Jaffna
Gene editing technologies refer to a small but diverse collection of systems that allow users to create site-specific DNA endonucleases capable of recognizing almost any sequence in a genome and thereby triggering the cells endogenous DNA repair machinery that can result in imperfect repair of the site, resulting in small deletions. Under some conditions the cells’ endogenous repair machinery can be forced to copy any DNA sequence into the double stranded gap created by the editing system allowing users to create custom alterations of a genes sequence. As part of an effort to develop proficiency in working with the CRISPR/Cas9 gene-editing system in mosquitoes, we attempted to create null mutations in the Ligase IV gene of Aedes aegypti which is expected to debilitate part of the insect’s DNA repair system used to join the broken ends associated with double-strand breaks in the DNA and to bias repair toward homology dependent DNA repair (so-called ‘knock-ins’. Aedes aegypti Ligase IV mutagenesis efforts with CRISPR/Cas9 were successful and we will report on what approaches we have found to be effective in using this technology in mosquitoes, compare those to what others are finding and describe how we are now ready to use this technology to build Anopheles arabiensis genotypes that will facilitate the creation of sex separation technology in this species. Programming the Cas9 DNA endonuclease to recognize specific sequences requires the design and synthesis of a short guide RNA. Guide RNA design is critically important and greatly affects the efficacy of the editing process. Criteria for finding sequences that are optimal for use as guide RNAs are evolving and we will discuss what we have found to be effective as well as procedures for identifying mutagenized genomes and creating lines homozygous for the resulting mutations.
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Creating an Insect Synthetic Population to Fight Dengue Fever
Chun-Hong Chen
Institute of Molecular and Genomic Medicine, National Health Research Institutes Taiwan
The worldwide annual incidence of dengue infection has been estimated by the World Health Organization to be 50 million cases. Compared with other vector-borne diseases, only malaria, which causes over one million deaths annually, has an incidence higher than that of dengue infection. During the past decade, many researchers have focused on the development of transgenic mosquitoes for alternative vector control strategies aimed at either reducing mosquito abundance or preventing the transmission of pathogens. Transgenic methodologies have created new opportunities to prevent infectious diseases through the manipulation of the genomes of insect vector populations. By integrating anti-pathogen effector genes in the genome of Aedes aegypti, the principal vector of dengue worldwide, my research team at the National Health Research Institute (NHRI) in Taiwan has developed strains of transgenic mosquitoes in which the replication of DENV-1, -2, -3, and -4 is prevented through an RNA interference-based mechanism. We will further optimize the anti- pathogen effector genes to target genomic sequences in these DENV serotypes and the chikungunya virus (CHIKV). The overall goal of our research is to prevent dengue and chikungunya fever through the replacement of wild, disease-susceptible mosquito populations with genetically engineered, disease-refractory mosquitoes. An important step toward the implementation of our population-replacement strategy has been the pioneering development and adaptation of the Medea selfish genetic element, which drives the inheritance of the anti- pathogen gene, sustaining a frequency of the disease-refractory phenotype of 100% in the replacement mosquito population. The outcome of our proposed research will reduce the threat of vector-borne diseases in threaten countries worldwide, and positively impact public health on a global scale.
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Developing genetic method for accurate sex separation and female elimination in the malaria vector Anopheles arabiensis
Cyrille Ndo1, Yacouba Poumachu1, Parfait Awono-Ambene1 and Jeremie Gilles2
1-Organisation de Coordination pour la Lutte contre les Endémies en Afrique Centrale (OCEAC), Yaoundé-Cameroon 2- Insect Pest Control Laboratory, International Atomic Energy Agency
To produce only male adults to be released in a future Sterile Insect Technique (SIT) control program targeting the major African malaria vector Anopheles arabiensis, we are investigating the use of temperature-sensitive lethals (TSL) for sex separation and female elimination. Wild type males of An. arabiensis from North Cameroun were mutagenized using 3 doses of Ethyl metanelsulfonate (EMS) and mated with virgin females for 3 consecutive days. The fecundity and eggs fertility of each female were assessed and L1 larvae of each dose were screened for mortality after heat shock at 40°C for 30 min. Feeding of mosquitoes with sucrose solution containing EMS for 48h resulted in high sterility at all doses. Therefore only 24h treated mosquitoes were used in experiments.The mean number of eggs laid by females mated with EMS-treated males as well as egg hatch rate per dose didn’t varied significantly. Laval mortality recorded after heat shock at 40°C for 30 min suggested that the dose of 0.005M was the most effective. At this dose 53.55% of families tested showed up to 25% mortality suggesting that they may contain a TSL. Among these families, 24 showing up to 80% mortality over generations have been selected and maintained at 26±2°C. Experiments are underway to confirm the presence of the TSL and to determine its inheritance pattern.
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Development of an Aedes albopictus Genetic Sexing Strain based on dieldrin resistance: selection and identification of homozygous resistant and sensitive strains
Pablo Tortosa
CRVOI-Université de La Réunion
In the frame of the present CRP, we propose to use an allele confering dieldrin resistance for the construction of an Aedes albopictus Genetic Sexing Strain (GSS). In the first year of research activities, we carried out insecticide selection and molecular genotyping in order to construct 2 homozygous lines for the rdl allele (sensitive and resistant lines). For this, we first sampled Ae. albopictus eggs by using ovitraps at a sampling site located at the edge of St Denis, the main capital of the island where we previously reported a high resistance to Dieldrin. Eggs were then immersed in water and a sub-sample of larvae was genotyped to measure the actual frequency of rdlR allele in the field (f0 generation). Third and 4th instar larvae were submitted to increasing concentrations of dieldrin through 3 generations, and an RdlR isofemale line, named R-Run was obtained. Similarly, an isofemale homozygous RdlS strain, named PDP, was obtained by genotyping mosquitoes sampled in an elevated portion of the island (Plaine des palmistes), where the levels of resistance are notoriously low. In the next step, R-Run and PDP strains will be submitted to irradiation and translocations conferring insecticide-dependent sex ratio biased towards males will be identified following 2 screens that will be presented.
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Effect of irradiation on local South African strain Introgressed with Dieldrin resistant (ANO IPCL1) strain and the use of an alternative to dieldrin for production of « male only » adults.
Givemore Munhenga1, 2, Leyya Essop1,2, Leonard Dandalo1,2, Basil Brooke1,2, Oliver Wood1,2, Leanne Robertson1,2 and Lizette L Koekemoer1,2
1 Centre for Opportunistic, Tropical and Hospital Infections, National Institute for Communicable Diseases, Private Bag X4, Sandringham, Johannesburg 2131, South Africa 2 Wits Research Institute for Malaria, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
In the context of eliminating malaria transmission by 2018 the South African government is investigating use of the sterile insect technique (SIT) as an additional tool for vector control intervention. In a mosquito SIT programme it is important to have a sexing system in place to assist in exclusively obtaining male offspring for sterilization. Using classical genetic approach we developed a GSS strain by transferring the male dieldrin resistance on a local An. arabiensis population. Although this approach can effectively eliminate 99.9% of females there are concerns on its efficient application under mass rearing conditions because of the inherently high sterility levels. In addition dieldrin is known to bioaccumulate and biomagnify along food chains, this has led to problems on its use in SIT programmes which are technically considered as environmentally friendly. These concerns have given rise to the need to search for alternative sexing methods. In our presentation we will describe work carried out to evaluate efficacy of other GABA receptor antagonist on a newly developed GSS strain (GAMA). We will also present work on investigations of the presence of temperatures sensitive lethal (tsl) in a newly established Anopheles arabiensis strain (AMAL).
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Aedes albopictus female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer
Carlos Tur Lahiguera, Gustavo Salvador
Centro de control biológico de Plagas (TRAGSA), Universidad CEU Cardenal Herrera
The purpose of this work is to develop mechanical, behavioral, and developmental tools for sexing Aedes albopictus pupae. This subject is included in the frame of the Co-ordinated Research Programme on “Explore mechanical, molecular, behavioral or genetic methods of sex separation in mosquitoes”. The proposal method is based on identification of both sexes by artificial vision algorithms due to the existing pupae sexual dimorphism. Previous data analysis have shown substantial difference in size between male and female pupae when analysis is performed within the same cohort. This enables an artificial vision system to separate up to 70 percent of males with a negligible risk of female presence. The percentage of sorted males can be also increased due to protandry. First, Watershed algorithm is used to separate pupae that are in contact and measure their individual size. Then, the frequency curve of pupae size is obtained with two differentiated peaks. Each peak represents the highest range of size for both sexes. Size threshold between males and females is obtained using an Expectation-Magnification algorithm. Both the frequency curve and the threshold are updated in real time with any new pupae analysis, which allows the continuous analysis of large amounts of pupae in a very short time. Once the threshold size has been calculated, a second picture of the pupae is obtained to calculate the female centroid coordinates and direct a laser beam using a system driven by a dual axis galvanometer optical scanner. The laser beam will kill all female pupae and only male pupae will survive. During the first 18 months a hardware and software system has been developed to identify and measure the size of Aedes albopictus pupae. Also a protocol to calculate the threshold between male and female size for each cohort has been developed and tested with positive results. Regarding the female removal hardware, all components have been selected and tested, including a CO2 laser, galvanometer scanner, servo controller and electronic components. Good progresses have been achieved in order to adjust the position of the pupae centroid coordinates and the laser beam path.
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Exploiting sex dimorphism and protandry for Aedes albopictus sex separation
Romeo Bellini, Arianna Puggioli, Rodolfo Veronesi, Marco Carrieri
Centro Agricoltura Ambiente “G. Nicoli”
Currently our system of sexing Aedes albopictus for SIT studies is based on the sieving of pupae in the water. Pupae are collected once per production cycle at a fixed time (approximately 24 hours from the beginning of pupation to better exploit protandry). On average the male pupae productivity with this method is in the range 25-30% (calculated on the total number of reared males), with an acceptable residual presence of females around 1%. These values are not satisfactory in case of mass rearing and a better performing system should be developed in order to increase the male pupae production and at the same time keep the percentage of female presence as low as possible. We explored the possibility to increase the male productivity by enhancing the protandry and the sexual dimorphism of the pupae. We checked if the natural sexual dimorphism in Ae. albopictus is an inheritable character which could be selected for the production of a strain with more marked differences in the size of the pupae in function of sex. Therefore we crossed larger female pupae with smaller male pupae for four generations. From the data obtained, it seems that the character of pupal dimension is dominated by the male, with small males that bring down the dimension of female pupae as well. We also analyzed the natural protandry of Ae. albopictus and verified the possibility to enhance it by artificial selection of a strain obtained by crossing the males that pupate earlier with the females that pupate later on. After nine generations of selection we observed that the sex ratio, within 24 h from pupation onset, is significantly different compared to our control strain. Together with ENEA we also evaluated the effect of a new Wolbachia infection (wPip strain), established in Ae. Albopictus, on pre-imaginal development and sex separation. In cooperation with ILM we tested the effect of mass-rearing variables (larval density and larval diet) on the pre-imaginal development of Aedes polynesiensis, employing diet formulation and trays developed at the IAEA.
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Sex separation of Aedes albopictus in Sri Lanka using Behavioural and Mechanical methods
W.Abeyewickreme and Jeevanie Harischandra
Faculty of Medicine, University of Kelaniya and the Anti Malaria Campaign
The activities were carried out in two institutions which have been involved in conducting studies in relation to SIT for dengue vectors in Sri Lanka , Aedes aegypti and Aedes albopictus, namely the Anti Malaria Campaign (AMC) and the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya (UOK). Anti Malaria Campaign has been working on project activities initiated through IAEA TC project SRL 5044 “ Supporting a feasibility study using Sterile Insect Technique for integrated control of mosquitoes in Sri Lanka.”
With the trainings obtained through the TC project the Entomologist and two technical staff members were able to learn he basic techniques in relation to laboratory colonization of Aedes mosquitoes and other related techniques for the use of SIT for dengue vectors. As a result AMC was able to establish colonies of Aedes aegypti and Aedes albopictus in their newly established insectaries for which most of the equipment were received through the TC project. AMC staff have been able to master the techniques in colonization to keep healthy colonies of mosquitoes following standard protocols blood feeding , hatching of eggs, correct larval feeding to get syncronous pupation and emergence. In addition, the AMC staff tried a method to separate larvae and pupae of Ae. Albopictus using mechanical sieves which were received by AMC under the project SRL 5044. The mesh size of the sieve used was 1.12 mm which was the smallest mesh size in the set. When the mixture of larvae and pupae was poured on to the sieve which was kept on a tray with water up to 1 cm and a light source from a torch is given 90% of pupae can be separated from larvae. The limitation here is the time taken for separation is around thirty minutes, thus this method has to be modified to increase the separation up to 100% and to reduce the time taken. Separation of sexes in the pupa stage was not successful using the sieve with 1.12 mm mesh. This has to be tried again with modifications and other methods. For Aedes albopictus the sex separation during the pupa stage can be done using the size difference of the sexes female being larger and males smaller. Visual hand-picking separation experiments showed that the smaller sized pupae separated by handpicking emerged as 100
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% adult males. However the lager sized puape left emerged as 98% females with 2% males mixed with them. Mechanical sieving (Bellini, 2007) will be further tested for Ae. albopictus and refined. Other new methods using differences in behavior and size of male and female pupae are being tested at the laboratories of the UOK under the project. These methods will include use of Hox’s separation method, Use of simple chamber devices for pupal separation, make use of fast movement of males compared to female pupae .Further experiments on sex separation of Aedes albopictus pupae are in progress.
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Exploring Mechanical and Nutritional Methods of Sex Separation in Aedes albopictus Mosquitoes
Gul Zamin Khan and Alamzeb
Nuclear Institute for Food & Agriculture (NIFA) Peshawar, Pakistan
Vector borne diseases are emerging threats in Pakistan and thus require particular attention. In Pakistan the first documented report of dengue DENV-2 was written in 1985 followed by donfirmed outbreak of Dengue Hemorrhagic Fever (DHF) reports during 1994. Later on, three dengue serotypes; DENV-1, 2 and 3 were also reported from different localities of Pakistan. Two Aedes species are important vectors for dengue spread in Pakistan; Aedes aegypti are the main vectors in the southern urban & peri-urban areas and Aedes albopictus is more important vector in the rural areas of central and northern areas of Pakistan. Different diet ingredients such as wheat, Rice, Maize, Chickpea, bean peanut and yeast were tested at 1% and 2% in single and combined diet experiments. The results showed that the diet comprising mainly of carbohydrates enhanced the male size and percentage of males ratio. Similarly, the protein based diets favoured females both in terms of size female ratio. Developmental period was also prolonged when single diet was used. Under behavioral studies different light intensities were tested. Males/females were exposed to different light intensities for the possible separation of both sexes. Among the different light intensities tested 100 watt of Inflorescent and 1000 watt of Incadescent light were found effective in separating the sexes. Under Mechanical separation, different sieves of mesh sizes were tested for the mechanical separation in repeated trials. The results showed that mesh size (12) was relatively more effective in sex separation. Recently, we have received sieve set of different mesh sizes from IAEA, we will try to standarlize its use for mechanical separation after the development of appropriate diet formula.
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Conditional lethality by insecticide resistance for sex separation of Aedes aegypti
René Gato Armas
Institute of Tropical Medicine Pedro Kourí, Havana, Cuba
Irradiation with Cobalt 60 was used to induce translocations in a temephos resistant strain of Ae. aegypti. Both late pupae and young virgin male adults were treated to Co60. Pupae were irradiated shortly before emergence, and adults ≈ 48 hrs post emergence. Doses applied ranged between 20 and 80 Gy. Insects were individually restricted in wells of 96-well PCR plate to achieve dose uniformity. The exposed males were crossed to Rockefeller susceptible females individually. The resulting eggs were counted and hatch rate was recorded. The resulting offspring were exposed to LC90 of temephos for 24 hours on larval stage. The survivors were counted by sex. Males from families showing few females were selected for subsequent cycles of crossing/ insecticide-exposition. Insect irradiation on pupa stage had no negative effect on the emergence of adults. Mosquitoes irradiated at 60 and 80 Gy showed high degree of sterility, so the number of descendants was not enough to maintain the lines. A reasonable number of lines derived from males irradiated at 20 and 40 Gy were selected as promising candidates for GSS. Several candidate lines were exposed to temephos following the same protocol used for press the parental resistant strain. Most of candidates did not survive, even at relatively low doses of temephos. The line 40 A-2 was considered the most promising candidate, since that showed high degree of temephos resistance and relatively good efficiency in female elimination. There was an evidence of the presence of heterozygous in the lines and the overall efficiency of the rearing-female separation process was not satisfactory. Probably the cause of the fails is in relation with the mechanism of resistance to temephos in Ae. aegypti, that has been associated with elevated insecticide detoxification catalyzed by actetylcholinesterase. Recently, deltamethrin- resistant strain was collected in Havana and appears be a good candidate for developing a GSS.
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The AeLIMIN+ suppression trial in French Polynesia: monitoring of Aedes polynesiensis rearing and sorting parameters under various rearing conditions
Hervé C. BOSSIN, Hereiti PETIT, Michel A. CHEONG SANG
Institut Louis Malardé, Papeete, Tahiti, French Polynesia
The emergence and expansion of dengue and other arboviruses (e.g., chikungunya, zika) have become major international public health concerns. The situation is particularly critical in several (European) overseas territories and island countries in the Caribbean and the Pacific where Aedes aegypti, the most important vector worldwide, and other Aedes vectors such as Ae. albopictus and Ae. polynesiensis are present. French Polynesia and other Pacific island countries were severely affected by three, nearly concomitant arboviral outbreaks (dengue, zika, chikungunya) in the past couple years. Besides the public health burden, Aedes mosquitoes and vector-borne diseases also greatly impact tourism, an essential economic resource of most (European) overseas territories and many other island countries. In view of the epidemic and entomological context, the development of innovative research to implement alternative or supplemental vector control methods that are efficient and sustainable at a cost affordable for Pacific islands has become essential. For Ae. polynesiensis, a suppression trial (AeLIMIN+) is underway on an isolated islet (75 ha) on the atoll of Tetiaroa, French Polynesia. This integrated control approach will rely in part on the release of Wolbachia incompatible males mosquitoes to seek, court and mate wild females, thereby reducing their reproductive capacity. Entomological and environmental data are being collected at the field site to characterize and monitor the dynamics of the Ae. polynesiensis mosquito before, during, and after integrated IIT intervention. Control actions scheduled in 2015 consist of classical control measures (mostly removal of breeding containers) and the deployment of gravid Aedes traps (GAT) for initial suppression and Wolbachia male releases to complete the suppression. Ca. 40,000 incompatible males will be released weekly starting Sept. 2015 for a period encompassing the tropical rainy season. QC (rearing/sorting parameters, male mating competitiveness, lab and field Wolbachia molecular monitoring) will be undertaken throughout the project. The scale of the operation requires a progressive transition from classical laboratory rearing (standard larval trays and diet) toward (semi-)industrial mosquito mass production and sex separation systems better suited for male- only mosquito releases. In the absence of a genetic sexing strain fro Ae. polynesiensis,
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male/female sorting relies upon developmental (protandry) and mechanical sex separation techniques. Current efforts are focused on the determination and reproducibility over time of rearing and sorting indicators (survival rate, time to pupation, male:female pupae ratio, male survival) under different rearing regimen. Preliminary results indicate the need to pursue optimization of the rearing conditions and sorting methodology. Testing of Aedes polynesiensis mass-rearing parameters using diet formulation and trays developed at the IAEA was also initiated at Centro Agricoltura Ambiente “G. Nicoli” (Italy) in the framework of the FP7 EU project “INFRAVEC”. These experiments will need to be pursued at ILM Tahiti following procurement of IAEA trays and diet.
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Exploring the role of a heterologous Wolbachia single-infection in Aedes albopictus
Maurizio Calvitti, Riccardo Moretti, Francesca Marini & Angiola Desiderio
ENEA
I. Effect of mass-rearing and mechanical sexing methods on Wolbachia titre and Cytoplasmic Incompatibility in the “ARwP” Aedes albopictus strain
As part of the CRP on “Exploring Genetic Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”, our main scientific objective is to investigate whether a heterologue Wolbachia infection (wPip strain taken from Culex pipiens molestus), that has replaced the native double infection (wAlbA+wAlbB strains), is able to change some Aedes albopictus developmental parameters (i.e male and female pupation times, protandry, pupal size) leading to a more efficient mechanical pupal sorting (i.e., to a lower percentage of female contamination among adults to be released). In this respect, preliminary experiments have been carried out in cooperation with the Centro Agricoltura Ambiente G. Nicoli (CAA, Bologna). Results will be shown and discussed in the Arianna Puggioli’s (CAA) presentation. In our laboratory at the ENEA Casaccia Research Center (Rome), we concurrently assessed by qPCR the impact of mass rearing conditions and mechanical sexing methods on Wolbachia titre in the same transinfected Ae. albopictus strain, named ARwP. In fact, mass rearing and sexing methods rely on environmental conditions (larval overcrowding and exposition of the pupae to high temperatures) that could affect the Wolbachia density. This risk could be higher in case of artificial and recently established symbiosis. Eventual lessening of Wolbachia (as ascertained in some cases in literature) could results in a weakened Cytoplasmic Incompatibility and even in the emergence of aposymbiotic adults. These unwanted events, could compromise the efficacy of the ARwP strain in genetic control projects. Herein, we present preliminary and unpublished results of Wolbachia molecular quantification in ARwP adult mosquitoes subjected to standard mass-rearing and mechanical pupal sorting methods at the CAA, without a preliminary selection.
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LIST OF PARTICIPANTS
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Brazil Germany Ms CAPURRO-GUIMARAES Margareth Ms Irina Haecker Universidade de Sao Paulo Fraunhofer Institute for Molecular Biology and Instituto de Ciencias Biomedicas Applied Ecology (IME) Av. Prof. Lineu Prestes, 2415 Project Group "Bioresources" Butantan Winchesterstrasse 2 05508-000 SAO PAULO 35394 GIESSEN BRAZIL GERMANY Email: [email protected] E-Mail: [email protected]
Cameroon Italy Mr NDO Cyrille Ms SCOLARI Francesca Organisation de Coordination pour la lutte contre Dipartimento di Biologia E Biotecnologie les endémies en Afrique Centrale (OCEAC) Universita degli Studi di Pavia 2.0 Rue du Centre Pasteur Via Ferrata 9 P.O.Box 288 27100 PAVIA (PV) YAOUNDÉ ITALY CAMEROON E-Mail: [email protected] E-Mail: [email protected]
France Mr CALVITTI Maurizio Mr TORTOSA Pablo BIOTEC/AGRO Institut de recherche pour le développement (IRD) Italian National Agency for New Technologies, Representation de La Reunion, CS41095 Energy and Sustainable Economic Development Parc Technologique Universitaire (ENEA) 2 rue Joseph Wetzell Lungotevere Thaon di Revel, 76 97495 SAINTE CLOTILDE CEDEX 00196 ROMA FRANCE ITALY E-Mail: [email protected] E-Mail: [email protected]
French Polynesia Mr PAPATHANOS Philippos Mr BOSSIN Hervé Polo d'Innovazione di Genomica, Genetica e Institut Louis Malarde(ILM) Biologia Laboratoire de recherche en entomologie médicale Edificio D 3 Piano B.P. 30 Via Gambuli 98716 TAHITI 06132 PERUGIA FRENCH POLYNESIA ITALY E-Mail: [email protected] E-mail: [email protected]
Italy United Kingdom Ms PUGGIOLI Arianna Ms BERNARDINI Federica Centro Agricoltura Ambiente Imperial College London Via Roma, 46 Crisanti Lab, 5th Floor 40052 BARICELLA Sir Alexander Fleming Building ITALY SW7 2AZ LONDON E-Mail: [email protected] UNITED KINGDOM E-Mail: [email protected]
Pakistan Mr KRZYWINSKI Jaroslaw Mr ZEB Alam The Pirbright Institute
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Nuclear Institute for Food and Agriculture (NIFA) Ash Road, Pirbright G.T. Road GU24 0NF WOKING Tarnab UNITED KINGDOM 25000 PESHAWAR E-Mail: [email protected] PAKISTAN E-Mail: [email protected]
South Africa Mr MUNHENGA Givemore Vector Control Reference Laboratory National Institute for Communicable Diseases Centre for Tropical, Opportunistic and Hospital Infections National Health Laboratory Service Private Bag X4, Sandringham JOHANNESBURG 2131 SOUTH AFRICA E-Mail: [email protected]
Spain Mr TUR LAHIGUERA Carlos Empresa de Transformacion Agraria, SA (TRAGSA) Instituto Valenciano de Investigaciones Agrarias - IVIA Carretera Moncada a Naqueda Km 4,5 46113 VALENCIA SPAIN E-Mail: [email protected]
Sri Lanka Mr ABEYEWICKREME Wimaladharma University of Kelaniya, Faculty of Medicine Thalagolla Road P.O. Box 06 RAGAMA, GAMPAHA SRI LANKA E-Mail: [email protected] Ms PEDROSA Michelle Biofábrica Moscamed Brasil Quadra D-13, Lote 15 Distrito Industrial do São Francisco JUAZEIRO-BA. 48.908-000 BRAZIL Email: [email protected]
Ms GARZIERA Luiza Biofábrica Moscamed Brasil Quadra D-13, Lote 15, Distrito Industrial do São Francisco
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Juazeiro-BA. 48.908-000 BRAZIL Email: [email protected]
Mr Tsu Chia Chao National Nuclear Energy Commission (CNEN) Rua General Severiano 90, Botafogo 22294-900 RIO DE JANEIRO, RJ BRAZIL Email: [email protected]
Scientific Secretary
IAEA Mr BOURTZIS Konstantinos Insect Pest Control Laboratory FAO/IAEA Agriculture & Biotechnology Laboratoies 2444 Seibersdorf, Austria [email protected]
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