FIRST RESEARCH CO-ORDINATION MEETING Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture

Co-ordinated Research Programme on

Explore mechanical, molecular, behavioural or genetic methods of sex separation in mosquitoes

Scientific Secretary: Jeremie Gilles & Kostas Bourtzis

IAEA Headquarters Vienna, Austria 30 September -4 October 2013

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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 and developmental approaches for sex separation in mosquitoes ...... 9 Selected References...... 11 Nuclear Component...... 15 Explanation / Justification: ...... 15 Participation of Agency's laboratories (Yes/No) ...... 15 Assumptions: ...... 15 Related TC Projects ...... 16 LOGICAL FRAMEWORK ...... 17 Narrative Summary ...... 17 Specific Objectives ...... 18 Outcomes ...... 18 Outputs ...... 20 FUTURE ACTIVITIES ...... 23 AGENDA ...... 34 PARTICIPANT ABSTRACTS ...... 37 LIST OF PARTICIPANTS ...... 51

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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 in view of 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. To the extent that it is 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.

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.

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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 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.

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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 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 physical, mechanical, and behavioural 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, behavioural and developmental 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 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 ).

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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 huge potential for further improvement.

Several mosquito genomes are already available while many more are currently being sequenced. Genomic information will be of great value in terms of identifying common genetic elements that can be used for the development of GSS for many mosquito species.

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 stability issues. None of them are available anymore. More recently a revival of one of these GSS was established based on dieldrin resistance for An. arabiensis , although using such markers may not be acceptable for large scale application for obvious reasons regarding: (a) protection of the environment and human health; (b) accuracy and (c) potential contamination of the mass- rearing colony. In addition, the earlier the elimination of females is achieved, the better the cost efficiency of the GSS.

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 consist of two principal components: (a) a selectable marker and (b) a sex chromosome / autosome translocation, which is required for sexing purposes. Thus, the development of a mosquito GSS would demand the following: (a) isolation and mapping of genetic markers (preferably morphological ones) either from natural populations and/or EMS-induced genetic screens; 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 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 which 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

1. Isolation and mapping of genetic markers from different sources including natural populations and / or EMS-based screens. These markers will facilitate genetic studies as well as the isolation of suitable selectable markers for the development of mosquito GSS.

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2. Development and characterization of irradiation-induced chromosomal rearrangements in the target mosquito species. These chromosomal rearrangements will facilitate genetic studies as well as the development of stable mosquito GSS.

3. Cytogenetic analysis of the tools developed in [1] and [2]. Cytogenetic analysis will facilitate genetic studies and the evaluation of the most suitable GSS.

4. Isolation and characterization of morphological and selectable markers. These markers will allow the construction of GSS.

5. Construction and characterization of 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. 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 Another option for enhancing SIT in mosquitoes has been the development of transgenic sexing strains (TSS) in fruit flies. These systems have the advantage of a faster development time and the ability to transfer to other species. Recently, two early TSS have been developed for the tephritid fruit fly species C. capitata and A. suspensa , both with 100% female lethality. They use endogenous components of the fruit fly genome to induce lethality through a well-understood apoptotic pathway in flies. 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 TSS strains 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 TSS in mosquito species.

To explore the possibility of generating mosquito GSS 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 to be extended to 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 could be developed. To this end, recent developments in FACS technology would allow high through-put for mosquito mass rearing requirements.

To improve the development and ecological safety of genetically engineered (GE) mosquitoes created for enhanced biological control programmes, site-specific systems or

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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. Alternative options for site-specific modification are the Flp-FRT or site- specific nuclease systems.

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. 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 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. Unravelling the molecular mechanisms of sex determination and sex-specific expression in mosquitoes. Understanding sex determination mechanisms will provide tools for developing genetic sexing strains.

9. Isolation and testing of regulatory elements to achieve sex-specificity. Regulatory elements could be promoter/enhancers or sex-specifically spliced sequences.

10. Isolation and testing of effector molecules. Effectors could be from the group of lethal genes, genes conferring phenotypes that allow sex separation, or sex conversion.

11. Construction of transgenic sexing strains in mosquitoes. Combinations of regulatory elements and effector molecules can be used to generate female- specific effects.

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12. Evaluation of transgenic strains with known performance characteristics evaluated at a small scale using standard quality control parameters. This evaluation will provide data on the actual stability and the overall fitness of the transgenic strains developed.

To explore mechanical, behavioural and developmental approaches for sex separation in mosquitoes Mechanical, behavioural, and developmental tools for sexing may be needed since efficient systems for eliminating females in the embryo stage have not been developed or are not yet available. The latter are far more efficient. 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 not currently 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 , 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 (CO 2) adults. Such methods will be most efficient for species in which size difference between sexes is greatest.

This 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).

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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). Feeding An. arabiensis females with blood spiked with 5ppm ivermectin and Spinosad solution resulted in complete female elimination between 48h and 72h after the first blood feeding. 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, CO 2, 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 .

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.

Expected outputs

13. Characterization of parameters influencing the efficiency of mechanical, behavioural, and developmental sex separation tools. These parameters will facilitate studies as well as the development of classical sex separation tools.

14. Evaluation of 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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Union Biometrica (2004) Sex-specific GFP-expression in Drosophila embryos and sorting by COPAS flow cytometry technique.

Windbichler N, Papathanos PA, Catteruccia F, Ranson H, Burt A, Crisanti A (2007) Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos. Nucl Acids Res , 35:5922-5933.

Windbichler N, Papathanos PA, Crisanti A (2008) Targeting the X chromosome during spermatogenesis induces Y chromosome transmission ratio distortion and early dominant embryo lethality in Anopheles gambiae . Plos Genetics , 4.

Yamada H, Benedict MQ, Malcolm CA, Oliva CF, Soliban SM, Gilles JRL (2012) Genetic sex separation of the malaria vector, Anopheles arabiensis , by exposing eggs to dieldrin. Malaria J, 11:208

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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 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.

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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.

SUD5032: Investigating the Use of the Sterile Insect Technique for Controlling Mosquitoes in Northern Sudan.

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.

PAK5049: Support for Capacity Building in Baseline Data Collection for Mosquito Dengue Vector Management in Pakistan.

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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 required 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 published Irradiation and classical classical genetic approaches least one mosquito papers. genetics can be applied for the for sex-separation in species constructed. creation of mosquito GSS. mosquitoes

2. To explore molecular Transgenic GSS for Reports and published Transgenic approaches are approaches for sex separation at least one papers. applicable to target mosquito in mosquitoes mosquito species species. constructed.

3. To explore mechanical, Mechanical, Reports and or Development and improvement behavioural and behavioural and published papers. is possible. developmental approaches for developmental sex sex separation in mosquitoes separation systems for at least one mosquito species developed or improved.

4. To continue encouraging and Geneticists pursuing Agreements or Relevant scientists can be attracting participants to the a classical approach contracts issued and identified and encouraged to CRP in the field of classical attracted to the CRP. signed. join the CRP. genetics

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 feasibility analysis available. mosquitoes developed molecular-based sex separation developed

3. Mechanical, behavioural Protocols developed Data collected and Facilities and resources 18

Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators

and developmental for improved sex feasibility analysis available. approaches for sex separation methods separation in mosquitoes improved and validated

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Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators

Outputs

1. Isolation and mapping of Genetic markers Reports and or Biological material and genetic markers from isolated. published papers. methods available. different sources including natural populations and / or EMS-based screens.

2. Development and Chromosomal Reports and or Scientists working on classical characterization of rearrangements published papers. genetics and methods are irradiation-induced selected. available. chromosomal rearrangements in the target mosquito species.

3. Cytogenetic analysis of the Genetic markers and Reports and or Cytogenetic methods, tools developed in [1] and chromosomal published papers. chromosome maps and experts [2]. rearrangements are in place. described.

4. Isolation and characterization Morphological and Reports and or Morphological and selectable of morphological and selectable markers published papers. markers are isolated and selectable markers. confirmed. evaluated.

5. Construction and Classical GSS Reports and or Suitable genetic markers, characterization of a classical constructed and published papers irradiation sources, GSS characterized chromosomal rearrangements are available.

6. Evaluation of classical GSS Classical GSS in Evaluation data Laboratory testing is feasible. in small scale using standard laboratory testing identified and quality control parameters evaluated published

7. Transgenic strains with site- Strains with landing Reports and published Landing sites and performance specific landing sites and sites and papers protocols are available. known performance performance characteristics characteristics assessed

8. Unravelling the molecular Sex determination Reports and or Tools for the analysis of mechanisms of sex pathways and sex published papers complex sex determination determination and sex specific expression pathways and mechanisms are specific expression in in mosquitoes better available. mosquitoes understood

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Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators

9. Isolation and testing of Regulatory elements Reports and published Regulatory elements are regulatory elements to tested papers identifiable. achieve sex specificity

10. Isolation and testing of Effector molecules Reports and or Effector molecules are effector molecules tested published papers available.

11. Construction of transgenic Transgenic sexing Reports and published Components for the application sexing strains in mosquitoes strains developed papers of transgenic technologies are available Transgenic GSS in Reports and published Laboratory testing is feasible 12. Evaluation of transgenic laboratory testing papers strains with known evaluated performance characteristics at small scale using standard quality control parameters.

Factors influencing Reports and or Tools for evaluation are 13. Characterization of factors the efficiency of sex published papers available. influencing the efficiency of separation tools mechanical, behavioural and characterized developmental sex separation tools Efficacy and Reports and or Tools and QC protocols are in 14. Evaluation of the efficacy and reproducibility of published papers place reproducibility of mechanical, tools assessed behavioural and developmental tools at small to large scale using standard quality control parameters

Papers drafted and Journal issue with Data for publication available. 15. Publication of results in a submitted. published scientific peer reviewed journal. 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 1 st 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. 21

Objective Verifiable Means of Verification Important Assumptions Narrative Summary Indicators

3. Organise 2 nd 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 3 rd RCM to analyse 3rd RCM held 2016. Participants and RCM Progress satisfactory and mid- progress in delivering the research Progress Reports. CRP evaluation approved by outputs and plan the final phase of CCRA-NA committee. 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 Sao Paolo, Brazil (Collaborator: Jake Tu)

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 • Characterization of EZGs expression profiles in Ae. aegypti • Evaluation of RNAi experiments to knockdown the female Ae. aegypti dsx pathway • Design and synthesis of the construct for germ line transformation of Ae. aegypti embryos

Contract 17958: Flaminia Catteruccia, Harvard School of Public Health, Boston, USA (Collaborators: Eric Marois)

Mosquito sex separation via TALEN-mediated female-specific lethality

We will insert attP sequences linked to a fluorescent marker into the An. gambiae genome by PiggyBac -mediated transgenesis. We will then identify those individuals carrying the attP- containing transgenes on the X chromosome and establish stable attP-carrying docking lines from these individuals. This will be achieved in collaboration with Eric Marois from the Centre National de la Recherche Scientifique in Strasbourg. We will then characterize expression of a fluorescent marker inserted at that site to guarantee that there are distinct differences between the fluorescent intensities of hemizygous males and homozygous females. We will perform tests to assess the reproductive fitness of these lines by analyzing the progeny of females (fertility, fecundity, mating competitiveness) mated to mixtures of males.

Aims for the next 18 months: • Insert attP sequences linked to a fluorescent marker into the mosquito genome by piggyBac -mediated transgenesis. • Identify those individuals carrying the attP-containing transgenes on the X chromosome and establish stable attP-carrying docking lines from these individuals. • Characterize expression of a fluorescent marker inserted at that site to guarantee that there are distinct differences between the fluorescence intensities of hemizygous males and homozygous females. • Perform tests to assess the reproductive fitness of these lines by analyzing the progeny of females (fertility, fecundity, mating competitiveness) mated to mixtures of males. 23

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. We will capitalize on our recent discovery that ectopic delivery of the Y chromosome-linked putative primary sex determination gene Yg in embryos leads to embryonic lethality of the An. arabiensis females, whilst leaving males unaffected. During the next 18 months we will generate proof of concept transgenic strains of An. arabiensis with a female embryonic lethal phenotype.

Aims for the next 18 months: • Identify early zygotic genes that would donate promoters to drive ectopic expression of the Yg . • Identify and isolate promoter sequences of selected early zygotic genes. • Test the activity of the early promoters using an in vivo reporter system. • Generate transgenic strains with embryonic female lethal phenotype driven by the Yg expression using a bipartite Gal4-UAS system.

Contract 17981: David O’Brochta, University of Maryland, College Park, USA (Collaborators: Jake Tu)

Identification and isolation of gene and regulatory sequences from An. stephensi and the creation of An. arabiensis with visible mutant phenotypes

Genes and regulatory sequences with sex-, tissue- or temporal-specific expression will enable the construction of a wide variety of transgenes of potential use in the sex separation of mosquitoes.

Aims for the next 18 months: • Conduct enhancer-trap and gene-trap screens in An. stephensi , the only mosquito species in which these technologies are currently available, to identify enhancers and genes with temporal and spatial patterns of expression of use to this CRP. • Physical isolation of regulatory sequences of the most immediate use to this CRP will be initiated. • Site specific endonucleases (TALENS and/or CRISPRs) will be used to mutagenize selected genes to produce visible phenotypes.

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Contract 17952: Philippos Papathanos, University of Perugia, Italy (Collaborators: Andrea Crisanti, Jake Tu, Jaroslaw Krzywinski)

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. With the imminent completion of the 16 Anopheles genomes project there is a unique opportunity to conduct a comparative analysis on the evolutionary history of the sex-biased transcriptome and describe the conservation and de novo invention of mosquito sex determination systems.

Aims for the next 18 months: • Investigate using RNA-seq-based transcriptome analysis of sexed adult and dissected germlines, the sex-biased transcriptional landscape of 5 of these 17 anophelines, including two of the target species An. arabiensis, An. gambiae s.s. but also the phylogenetically relevant An. minimus, An. dirus and An. albimanus. • Catalogue sex and tissue-biased gene expression in these mosquitoes and document the evolutionary forces that are driving their genomic distribution and sequence and expression evolution. • Using this data, we aim to initially validate the sex-specific expression of a number of these genes, of which some may be directly involved in sex determination while others may be downstream resulting in sexual differentiation and probe gene function using transient RNAi experiments. • The regulatory regions of genes that display useful expression patterns, in relation to sex and developmental time (e.g. embryos), will be tested for their ability to drive expression of transgenes in transgenic strains. In the future, combinations of effectors and regulatory elements will be brought together to generate novel GSS.

Contract 17896: Francesca Scolari, University of Pavia, Italy (Collaborators: Jake Tu, Romeo Bellini, Jaroslaw Krzywinski, Philippos Papathanos)

Development of sex-specific markers in the tiger mosquito Ae. albopictus

Aims for the next 18 months: • Identification and characterization of DNA sequences specific for the male sex- determining chromosome in Ae. albopictus . • Apply the Representational Difference Analysis (RDA) approach already applied successfully in our laboratory in Bactrocera oleae , and which is currently giving promising results also in B. dorsalis . The RDA data will provide male-specific sequences which will allow the development of male/female-specific primers to be used for unambiguous molecular sexing of early developmental stages. • To confirm the location of the identified sequences on the male sex-determining chromosome, we plan to optimize Fluorescent in situ hybridization (FISH) on the mitotic chromosomes of Ae. albopictus , which we are currently cytogenetically characterizing. This will also be of key importance for the physical mapping of genes and contigs soon available thanks to the oncoming Ae. albopictus genome. • Screen our newly identified set of Odorant Binding Protein (OBP) genes from Ae. albopictus antennal transcriptome to determine whether sex-specific expression at

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the larval/adult level may be potentially useful for behavioural sex-separation approaches.

Contract 17855: Marc F. Schetelig, Justus-Liebig-University Giessen, Germany (Collaborators: David O’Brochta, Margareth Capurro, Jake Tu, Jeremie Gilles)

Site-specific landing site systems in mosquitoes to improve transgenic insects for SIT

Aims for the next 18 months: • Initiate the construction of vectors needed for creation of landing site/ cassette exchange systems. First vectors would include a transposon system (e.g. pBac) combined with fluorescent markers (e.g. EGFP, DsRed) and site-specific systems like the phiC31/ attP or CRE/l oxP • Create first landing site lines in Aedes aegypti by germline transformation . In this context we will test the phiC31/ attP or CRE/l oxP systems and the combination of different lox sites to find the best recombination system for Ae . aegypti . • Set up evaluation parameters to test the fitness and performance of transgenic strains in collaboration with the IPCL in Seibersdorf. We will start applying these protocols to the created lines in a comparative lab-scale testing to select the best (fittest) lines for subsequent 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

Aims for the next 18 months: • Characterize the currently available An. stephensi Guy1 transgenic lines: a) determine whether female-killing or female conversion is responsible for the male- only phenotype. The Tu lab will use independent Y chromosome markers to determine whether XX males are present. If such individuals are found, the Sharakhov lab will verify the XX karyotype by performing chromosome in situ hybridization using X-specific probes; b) determine the effect of ectopic Guy1 expression in the early embryos. • Establish homozygous An. stephensi transgenic lines that carry a conditionally expressed Guy1 transgene. A Tet-off system will be used to control transgene expression. The idea is to produce transgene-carrying females in the presence of tetracycline, so that homozygous individuals carrying the transgene may be obtained. We will collaborate with Dr. Tony James at UC Irvine to integrate the transgene into pre-selected sites to ensure function of the transgene and minimize fitness cost associated with the insertion site. We will try to establish one such line in the next 18 months, but it could take us longer.

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Contract 17959: Wimaladarma Abeyewickreme, Univ. of Kelaniya, Sri Lanka (Collaborators: Jeremie Gilles; Romeo Bellini; Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka; Anti-malaria Campaign, Sri Lanka)

Mechanical sex separation in Aedes species

Our research group intends to explore mechanical and behavioural approaches for sex separation of Ae. albopictus . Since mechanical separation has been found possible at pupal stage for Aedes spp. attempts will be made 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) and (iii ) adjustable opening separation systems (McCray). • 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) or IGR (Pyriproxifen) and other potential mosquito toxins (ivermectin, Spinosad). • Explore seeking behaviour of males/females to create a mechanical separation system for capturing males/females that are drawn to host cues (heat, CO 2, odours and other male attractants such as nectar). 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 (Collaborat: Jeremie Gilles, Romeo Bellini)

Mechanical sex separation in Aedes polynesiensis

The feasibility and efficiency of Wolbachia -based (IIT) strategy to control the disease vector Ae. polynesiensis are being evaluated through open release trials in French Polynesia. Two trials have been conducted so far on islets of increasing sizes on the island of Raiatea (O'Connor, 2012) and more recently on the atoll of Tetiaroa. IIT relies on the sustained, inundative release of 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. The process of sex separation for non-classical SIT strategies like the IIT must therefore be particularly stringent. In the absence of GSS strains, methods currently available for Aedes sex separation exploit mechanical, behavioural or developmental differences between males and females.

To that end, the effects of rearing conditions on several parameters key for Ae. polynesiensis SIT were recently studied (Hapairai, 2013). Larvae were reared to adulthood in the laboratory 27

under a range of temperatures and larval densities. We studied the effect of these variables on several life table parameters of relevance to male-only releases including: larval survivorship, developmental time to pupation, male to female ratio, male pupae yield, adult male size and survival. The range of tested rearing temperatures (20, 25, 27, and 30°C) and larval densities (50, 100, 200, and 400 larvae/L) was selected to be within the range of conditions allowing larval growth and survival. Larval survivorship was the highest when larvae were reared at a density of 200 larvae/L and at all temperatures tested except at 20°C. Male to female ratios were male biased at all temperatures and densities. Time to pupation decreased with increasing temperatures. Larval density and temperature influenced the proportion of males pupating on first day of pupation, with 43% to 47% of total male pupae produced on the first day when reared at 25°C. No significant difference in mean wing length was observed between male mosquitoes reared in the laboratory (except at 20 and 30°C for some densities) and field collected males. Altogether, the study allowed the identification of rearing conditions that deliver high male yield with essentially no female contamination, adequate adult male size and survival. Ae. polynesiensis thus appears particularly amenable to developmental and mechanical sex separation offering good prospects for Ae. polynesiensis population suppression trials that rely on the production and release of large numbers of incompatible or sterile males. Further testing of the accuracy and reproducibility of developmental and mechanical sexing method under mass production conditions is required.

Aims for the next 18 months: • Test the effects of mass-rearing variables (larval density, larval diet) on the stringency and reproducibility of developmental (differential time to pupation) and mechanical (pupal size) sexing methods as a means for separating Ae. polynesiensis males from females. This work will be conducted in collaboration with CAA (Romeo Bellini). IAEA diet formulation and trays will be used for these tests. • Develop standard rearing and sorting procedures based on the optimal conditions defined above. • Determine how the use of modern molecular tools (genome sequencing, RNAseq) might sustain the development of a GSS for Ae. polynesiensis and develop a grant proposal to support this effort.

Contract 17902: Maurizio Calvitti, R. Moretti, F. Marini, A. Desiderio, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Italy (Collaborators: Pablo Tortosa, Jeremie Gilles, Romeo Bellini)

Effect of Wolbachia infection status (absence, native, artificial) in Aedes albopictus pre- imaginal and sexual maturity.

Over the next 18 months, experimental activities aimed at assessing the feasibility of IIT and/ or to combine Wolbachia with nuclear components for Aedes albopictus control will be carried out (in cooperation with CRVOI-Reunion Island).

Aims for the next 18 months: • Complete the preparation of the Ae. albopictus colonies that will be studied: AR (Wolbachia free), SR (wild superinfected) and AR wP (wPip transinfected). In order to prevent confounding effects due to possible bottlenecks we are completing the phase of out-crossing with wild mosquitoes collected from the field (treated with antibiotics, to be outcrossed with the AR strain). 28

• Develop RT-PCR protocols for each of the Wolbachia strains involved in the research, since Wolbachia density will be monitored to associate any effect of mass rearing conditions (life history traits) on the bacterial load. • In cooperation with the CAA (IAEA collaborating Center), outline the specific rearing conditions (temperature and larval density) that will be applied with the goal of providing evidence for eventual influences of Wolbachia infection type or absence on those developmental parameters useful for mechanical separation of male and female pupae. Tests will be conducted both in ENEA and at the CAA facilities. • At the end of the first 18 months, we expect to conclude the comparative work (collection of all planned data) on the wild type and Wolbachia free lines.

Contract 17925: R. Bellini, A. Puggioli, Centro Agricoltura Ambiente “G. Nicoli”. Italy (Collaborators: Maurizio Calvitti, Jeremie Gilles, Herve Bossin)

Improve sex separation methods for Aedes albopictus

The main activities we plan to conduct during the investigation are: (mass-)rearing Aedes albopictus colonies in highly standardized conditions and separation of male from female pupae by mechanical sieving (24 hours from onset of pupation); acquiring data on the effect of specific components of the larval diet and larval density on the size of the pupae produced in order to explore the possibility to increase the size difference between male and female pupae and possibly enhancing the protandry; analyzing the protandry of Ae. albopictus and exploring the possibility of enhancing time-to-development differences between males and females.

Aims for the next 18 months: • Develop an Ae. albopictus strain by crossing small males with big females. Small males will be selected by a metal sieve ≤1,300-1,350 µm, while big females will be selected by a metal sieve ≥ 1,450-1,500 µm. Progressive adjustments of the sieve mesh will be adopted using evidence from each new selection. At each generation at least 250 males and 250 females will be employed. • Test the possibility of creating an Ae. albopictus strain by crossing first pupating males with last pupating females. Precocious males as well as late females will be collected every hour manually from the larval rearing tray. At each generation at least 250 males and 250 females will be collected and put into the cage (40 x 40 x 40 cm) for egg production. • Investigate protandry in the egg hatching process. Using the currently available standard egg hatching procedures we intend to check the possible influence of selection according to the timing of egg hatching performed in previous generations. Egg batches will be conditioned and pools of 300-400 eggs will be put into a series of hatching containers. At different time intervals (e.g. 2, 4, 6, 8, 10 h) the containers will be opened, the egg paper withdrawn and the water with newly hatched larvae put into a tray for larval rearing and successive sex screening. • The following parameters will be checked: male pupal size, male pupal productivity at 24 h from beginning of pupation, percentage of residual females present after the male pupae are collected and sieved, at the first generation and in the following generations. A period of around 2 months is expected to be necessary for each generation.

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• Together with ENEA collaborators, evaluate the effect of Wolbachia infection and types in Ae. albopictus pre-imaginal development. • Provide support to ILM by testing the effects of mass-rearing variables (larval density, larval diet) on the stringency and reproducibility of developmental (differential time to pupation) and mechanical (pupal size) sexing methods as a means to separate Ae. polynesiensis males from females. IAEA diet formulation and trays will be used for these tests.

Contract 17945: Carlos Tur Lahiguera, Gustavo Salvador Herranz, Universidad CEU Cardenal Herrera, Spain (Collaborators: Jake Tu, Jeremie Gilles, Romeo Bellini)

Aedes albopictus female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer

Our proposed method of sex separation for Aedes albopictus is based on the size dimorphism between male and female pupae. Normally females are larger than males. When analyzing the size of pupae by artificial vision and image analysis, the difference in number of pixels may be sufficient to differentiate males and females automatically. The overall objective is to eliminate female pupae using high speed positioning and accuracy precision laser systems driven by a dual axis galvanometer optical scanner.

Aims for the next 18 months: • Develop a hardware and software system for image acquisition and individual recognition, capable of segmenting pupal images for different individuals. As pupae can cluster, the software must be able to identify individuals even though they may be touching. • Create frequency distribution curves of size for males and females, and establish a threshold percentage of males to prevent female contamination and a second for the purpose of colony maintenance. • Optimise the system for automatic discrimination between male and female pupae (depending on the selected threshold and the occupied area in an orthographic projection), the number of males and females and the geometric centres of each individual, in order to generate the path to be followed by the laser beam to remove females. • Create an optimum rearing (diet optimization to enhance protandry) to exploit developmental features and reduce to a minimum the initial presence of females. • Perform testing of the software for sex differentiation with optimized rearing. • After testing the software with our Aedes albopictus colony, we will also test the software in optimized rearing conditions at the Centro Agricola Ambiente Giorgio Nicoli, Bologna. • In collaboration with Dr. David O’Brochta (University of Maryland), Dr. Zhijian Tu (Virginia Tech) has obtained transgenic Anopheles stephensi lines that produce florescent embryos. The florescent signal is strong enough to penetrate the dark egg shell thus can be readily detected. By modifying the marker gene, Dr. Tu is generating An. stephensi lines that produce sex-specific florescent signals detectable in the embryos. Dr. Tu will work with other groups to apply the technique to other target species (e.g. working with Drs. Bourtzis and Gilles on An. arabiensis ). We

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will work with Dr. Tu to develop software and hardware to selectively kill florescent embryos to achieve sex separation.

Contract 17926: Gulzamin Khan, 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: • Assess the use of Raman spectroscopy for sieving and scanning mosquito pupae as a mechanical sex separation method. • 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). • Test combination of diets with larval crowding for increased protandry, and difference in the pupal size. • Explore protandry in Culex quinquefasciatus for better sex-separation methods

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 31

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, WITS Research Institute for Malaria and National Institute for Communicable Diseases, South Africa (Collaborators: Pablo Tortosa, Jeremie Gilles)

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.

Our group is currently in the process of transferring the male dieldrin resistance into the local An. arabiensis population. We have mated females collected from Kruger National Park (AMAL) with GSS ANO IPCL1 males provided by the Insect Pest Control Laboratory (IPCL) FAO/IAEA Agriculture and Biotechnology laboratories, Seibersdorf, Austria, which carry dieldrin resistance on the Y chromosome. The aim is to continue with the back-crossing to obtain a colony that carries dieldrin resistance on the Y-chromosome but retains a genotype representing the South African population. The Introgressed strain is designated with the acronym GSS/AMAL.

Aims for the next 18 months: • Determine the stability of male-specific dieldrin resistance phenotype in An. arabiensis GSS/AMAL, obtained by introgression of IPCL1. • Since dieldrin is a banned insecticide, we intend to alleviate regulatory concerns by evaluating the effectiveness/efficacy of fipronil on the recently developed GSS An. arabiensis GSS/AMAL strain. We will also explore other environmentally-acceptable insecticides targeting GABA receptors. • Optimize dose and time of exposure, in order to better characterize the effects of radiation on induced sterility on the GSS/AMAL strain. • Investigate (concurrently) temperature sensitivity of the established GSS/AMAL An. arabiensis line and An. arabiensis populations collected from the field by exposing eggs at three different temperatures (35, 40 and 45ºC) in search of a temperature sensitive allele ( tsl ) for possible development of a more environmentally-friendly genetic sexing system.

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Contract 17953: Cyrille Ndo, Parfait Awono-Ambene (OCEAC, Cameroon), Igor Sharakhov (University of Virginia Tech USA) (Collaborators: IPCL, Jeremie Gilles)

Developing a genetic method for accurate sex separation and female elimination in An. arabiensis

The general aim of our project is to develop a new tool for sex separation and female elimination in the malaria vector An. arabiensis , based on the isolation and subsequent Y- autosome translocation of a temperature sensitive lethal ( tsl ) allele. Within the first part of the project, we will mainly focus on the identification of a tsl candidate both through identification of the tsl phenotype in natural populations and through EMS mutagenesis followed by a phenotypic screen.

Aims for the next 18 months: • Collect An. arabiensis in Sahelian regions of North Cameroon, and screen F1 mosquitoes for a tsl phenotype. • Determine the optimal dose of EMS mutagen for the An. gambiae homochrom 1 strain. • Perform mutagenesis of An. arabiensis male mosquitoes and screen isofamilies for the tsl phenotype and morphological markers. • Construction of tsl homozygous strains by standard crossing.

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. Munhengas and M. Calvitti)

Construction of an Rdl R Genetic Sexing Strain in Aedes albopictus .

CRVOI 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/ incompatible males. In the frame of the CRP “ Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquit oes”, we will create an Aedes albopictus Genetic Sexing Strain (GSS) using the rdl R allele, which confers dieldrin resistance. Since using a banned insecticide is not acceptable for mass production, we will also explore the possibility of using an alternative insecticide for selection.

Aims for the next 18 months: • Construct homozygous rdl R and rdl S lines • Measure the levels of fipronyl resistance conferred by rdl R allele.

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AGENDA Monday, 30 September 2013

OPENING SESSION

09.00-09.05 Jorge Hendrichs. Opening of the meeting

09.05-09.30 J. Gilles / K. Bourtzis: Administrative details and introduction of the participants

09.30-10.00 Mosquito Group. The use of the sterile insect technique to control mosquito populations, progress and challenges

10.00-10.30 COFFEE BREAK

SESSION 1 (K.Bourtzis)

10.30-11.00 F. Criscione, B. Hall, Y. Qi, V. Timoshevskiy, M. Sharakhova, I. Sharakhov, Z. J. Tu. Mosquito Y chromosome genes and sex-determination

11.00-11.30 P.A Papathanos. Sex-biased gene expression and sex determination in anopheline mosquitoes

11.30-12.00 F. Scolari, M. Manni, L. M. Gomulski, G. Gasperi, A. R. Malacrida. Development of sex-specific markers in the tiger mosquito Aedes albopictus .

LUNCH

SESSION 2 (Herve Bossin)

13.30-14.00 A. L. Costa-da-Silva, D.O. Carvalho, M.L. Capurro. Aedes aegypti sex conversion by transgenesis.

14.00-14.30 A. L. Smidler, W. R. Shaw, F. Catteruccia. Mosquito sex separation via TALEN-mediated female specific lethality

14.30-15.00 M. Schetelig and I. Hacker. Site-specific landing site systems to improve transgenic insects for SIT

15.00-15.30 N. Dennison, E. Krzywinska, V. Kokoza, M. Morris, E. Casa-Esperon, A. S. Raikhel, J. Krzywinski. Sex and development in mosquitoes

15.30-16.00 COFFEE BREAK

16.00-16.30 K. P. Hoang. Controllable meiotic drive system 34

16.30-17.00 A. Crisanti.

(Wednesday) D. O’Brochta. Forward Genetic Analysis of Sex-specific Gene Expression in Anopheles Mosquitoes.

Tuesday, 1 October 2013

SESSION 3 (Marc Schetelig)

08.30-09.00 C. Ndo. Developing genetic method for accurate sex separation and female elimination in the malaria vector Anopheles arabiensis.

09.00-09.30 P. Tortosa , C. Atyame, L. C. Gouagna. Towards the development of a genetic sexing strain: construction of an Aedes albopictus rdl R homozygous line.

09.30-10.00 L.L. Koekemoer, G. Munhenga , L. Essop, L. 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

10.00-10.30 COFFEE BREAK

10.30-11.00 C. Tur Lahiguera , G. Salvador. Development of procedures to sex separate Aedes albopictus in a mass production facility by means of mechanical and behavioural methods.

11.00-11.30 R. Bellini, A. Puggioli , F. Balestrino, R. Veronesi, M. Carrieri. Exploiting sex dimorphism and protandry for Aedes albopictus sex separation.

11.30-12.00 W. Abeyewickreme , M. Hapugoda, N. Gunawardena, J. Harischandra. Exploring Tools for Sex Separation of Dengue Vector Mosquitoes in Sri Lanka.

LUNCH

SESSION 4 (Pablo Tortosa)

13.30-14.00 G. Z. Khan, I. Khan . Exploring mechanical and nutritional methods of sex separation in Aedes albopictus

14.00-14.30 R.G. Armas . Conditional lethality by insecticide resistance for sex separation of Aedes aegypti .

14.30-15.00 H. C. Bossin , M. A. Cheong Sang, T. Paoaafaite. Wolbachia -based vector control strategy in French Polynesia: addressing the issue of sex-separation in Aedes polynesiensis.

15.00-15.30 M. Calvitti , R. Moretti, F. Marini, A. Desiderio. Effect of different Wolbachia types (absence, native and artificial) in Aedes albopictus on pre-imaginal and sexual maturity 35

15.30-16.00 COFFEE BREAK

16.00-17.00 Discussions and composition of the working groups

Wednesday, 2 October 2013

08.30-10.00 Revision of the CRP documents (introduction, contracts) by 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, 3 October 2013

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, 4 October 2013

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

14.00-16.00 Presentation of the RCM report 16.00-16.15 Closing 36

PARTICIPANT ABSTRACTS

Exploring Tools for Sex Separation of Dengue Vector Mosquitoes in Sri Lanka

W. Abeyewickreme, Menaka Hapugoda, Nilmini Gunawardena and Jeevanie Harischandra

Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Ragama, and Anti Malaria Campaign, Colombo 5. Sri Lanka.

With the increase in dengue infections/epidemics in Sri Lanka and its effect on human health and mortality there is an urgent need for strict control measures to be implemented to contain the disease burden. Further, in the absence of specific drugs or vaccines, control focuses on suppressing the principal mosquito vectors, Aedes aegypti and Aedes albopictus . Yet, current methods have not proven adequate to control the disease. New methods are therefore urgently needed, for example genetics-based sterile-male release methods. However, this requires that lab-reared, modified mosquitoes be able to survive and disperse adequately in the field (Lacroix et al , 2012).

The Sterile Insect Technique (SIT) is an increasingly important component of area-wide integrated vector control (AW- IVC) programmes for key insect vectors such as mosquitoes. At present the process for the development of SIT for dengue vector mosquitoes in Sri Lanka has been initiated by the Anti-Malaria Campaign (AMC) and Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya with the help of the IAEA (IAEA TC 5024).

One of the major challenges for any large scale area-wide control programme of mosquitoes using SIT is to separate the males from the females. This has to be done because females bite and could potentially transmit diseases and they can distract the males from dispersing and mating with the target wild type females (Myers et al ., 1998). This female distraction has been demonstrated in large scale field trials using Medfly (Rendón et al ., 2004) and male-only release produced a three- to five-fold improvement compared to male-only release. Therefore the above highlights the fact that either female sterilization or stringent, if not absolute, sex separation approaches are even more critical for the successful application of SIT for the control of vector mosquitoes. In mosquitoes of the Genus Aedes separation of males and females can be performed using genetic, molecular, mechanical and behavioural methods.

In this study attempts will be made to establish and improve existing methods for sex separation and identify novel selectable genetic markers with the potential to be used for transgenic methods of sex separation.

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Development of procedures to sex separate Aedes albopictus in a mass production facility by means of mechanical and behavioural methods

Carlos Tur Lahiguera, Gustavo Salvador Centro de control biológico de Plagas (TRAGSA). In Spain, Aedes albopictus is an aggressive biting species that causes a severe nuisance to humans and can potentially be a vector for transmission of viruses such as Chikunguya. This exotic mosquito was initially reported in the Barcelona province in 2004 and its spread has been very fast, reaching the province of Alicante in 2005 and Castellón in 2011. The pest is still absent in most parts of its predicted distribution area in Spain, although it will likely be established in the near future.

Currently, integrated control programmes of the pest are being operated at a municipal level in some of the affected areas. These programmes include raising social awareness combined with larviciding and adulticiding treatments. However, although there are still large areas of its predicted distribution currently free of the pest, no emergency eradication strategy has been developed to be initiated upon early detection. Since 2007, in the Valencian Community, Tragsa is in charge of an operational AW-IPM programme against Ceratitis capitata using SIT as the major component, releasing 400 million sterile males per week in a 180,000 ha citrus growing area. During this period we have automated many processes for mass rearing, releasing and monitoring in the field.

We propose to use this experience to develop a systematic approach for obtaining males with a predefined sex purity level in laboratory conditions and to scale the methodology to mass rearing conditions in order to produce male-only mosquitoes with limited production costs. Mechanical and behavioural sex separation methods involving protandry and larval/pupal separation will be studied. Varying larval rearing conditions such as suboptimal nutrition in the final stages of larval development, quality of diet, decreasing water temperature in pre- pupation days and feeding the schedule will be tried in order to assess their influence on protandry, in order that the gap between male and female pupation might be enlarged. Sexual dimorphism of pupae means that the image analysis artificial vision could be an effective tool for sexing of pupae and it would be scalable to mass rearing processes.

Exploiting sex dimorphism and protandry for Aedes albopictus sex separation

Romeo Bellini, Arianna Puggioli, Fabrizio Balestrino, Rodolfo Veronesi, Marco Carrieri Centro Agricoltura Ambiente “G. Nicoli”.

Since 1999, the Centro Agricoltura Ambiente “G. Nicoli” in Crevalcore, Italy (CAA) has been conducting research to develop the sterile insect technique (SIT) to suppress Aedes albopictus (Diptera: Culicidae). One of the main constraints the SIT technology currently suffers is the lack of an efficient sexing method to be applied in mass rearing facility in order to release male-only sterile insects. 38

Currently our system of sexing Ae. albopictus for SIT pilot studies is based on the sieving of pupae in 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 pupal productivity with this method is in the range 25-30% (calculated from the initial number of reared males), with an acceptable residual presence of females around 1%. These values are of course not satisfactory in the case of mass rearing and a better performing system should be developed. During the CRP we will look for possible solutions which are applicable considering the current EU legislation (requiring non-transgenic approaches). Therefore our proposed studies will aim to: i) check if the natural sexual dimorphism in Ae. albopictus is an inheritable character and therefore selectable for the production of a strain with more marked differences in the size of the pupae as a function of sex, in order to improve the sex separation and increase the male productivity; ii) analyze the natural protandry of Ae. albopictus and verify the possibility of enhancing it by artificial selection of a strain obtained by crossing the males that pupate earlier with the females that pupate later on; iii) assess the influence of the larval diet components and doses, in relation to the larval density, both on sexual dimorphism and protandry. The following parameters will be regularly evaluated: male pupae size, male pupal productivity at 24 h from the beginning of pupation, and percentage of residual females present after the collection of male pupae and sieving.

Exploring Mechanical and nutritional methods of sex separation in Aedes albopictus

G. Z. Khan, I. Khan Nuclear Institute for Food and Agriculture (NIFA), G.T Road, Tarnab, Peshawar, Pakistan.

Pakistan is experiencing dengue cases each year and the disease circulates throughout the year with a peak incidence in the post-monsoon period (August to November). Dengue is now a reportable disease in Pakistan. Usually, insecticides are used for vector control. Due to environmental constraints, health hazards and development of resistance in mosquitoes, other environmentally friendly vector control strategies are needed. The sterile insect technique (SIT) is a species-specific and environmentally friendly control technique but it requires technical knowhow about the separation of male females during early stages of development. We have achieved progress towards a feasibility trial of SIT and have initiated extensive surveillance of vector species from over 170 sites in six districts of the Khyber Pukhtum Khwa (KPK) province of Pakistan. Data recorded from larval collection at these sites indicated that Anopheles were present in stream bed pools in fully exposed and partially shaded areas but absent from waters with a foul smell. Aedes albopictus was found in temporary standing pools, construction site water tanks, discarded cans, empty water drums, tyres and desert coolers. Culex quinquefasciatus was the most abundant species found in almost all types of habitat except in the very small disposable containers. The mean monthly population of both Aedes albopictus and Culex quinquefasciatus was highest in the months September and October. Our ovitrap data showed significantly higher numbers of female mosquitoes in the outdoor traps than in the indoor traps. 39

In our preliminary studies on the photosensitivity behaviour of males and females for mechanical separation, tendencies of female pupae to a flush of inflorescent and incandescent 100 watt light was assessed. Female pupae were more photosensitive to the lights causing them to dive to the bottom thereby making them easily separated mechanically. The males on the water surface were separated by sieving through different sized meshes. At least 14 amino acids, sugars, polyunsaturated fatty acids (PUFAs) especially C18, C20, and C22, sterols, nucleotides and some vitamins have been reported as being essential for larval development, survival and adult flight. Studies on differential feeding preference for proteins, starches and vitamins for male and female larvae are lacking. Our future strategy for continuation of the SIT programme will focus mainly on the differential feeding strategy for male and female requirements at the larval stage and the subsequent mechanical separation of dimorphic pupae during their attempts to rise to the surface for oxygen. Mechanical separation of pupae will be done by exploiting the repeated attempt of dimorphic pupae to pass through the grid; larger female pupae will be trapped under the grid.

Wolbachia -based vector control strategy in French Polynesia: addressing the issue of sex-separation in Aedes polynesiensis

Hervé C. Bossin, Michel A. Cheong Sang, Tuterarii Paoaafaite Institut Louis Malardé, Papeete, Tahiti, French Polynesia

Aedes mosquitoes (principally Ae. aegypti, Ae. polynesiensis , and Ae. albopictus ) severely affect the health and well-being of Pacific island communities by transmitting infectious diseases such as lymphatic filariasis (LF), dengue and chikungunya. For Ae. polynesiensis , the principal vector of LF, the integration of vector control with multiple drug administration campaigns would ensure elimination of the parasitic disease from the region. However, none of the classical control methods are efficient against this exophilic, diurnal mosquito, which breeds in a diversity of both domestic and natural habitats. The development and (field) assessment of novel control methods is therefore critical.

Recent male release trials have been performed in French Polynesia to assess the potential of a Wolbachia -based autocidal strategy (incompatible insect technique or IIT) for the control of Ae. polynesiensis . Field trials have relied upon mechanical sex separation techniques combined with visual sorting of individuals to safeguard against female release. While appropriate for feasibility trials, manual inspection is unlikely to be cost effective at a larger scale.

The significant sterility induced by Wolbachia in the targeted mosquito population encourages the implementation of a population suppression trial requiring the production and sorting of larger numbers of incompatible males. The scale of such operations will require a progressive transition from classical laboratory rearing (standard larval trays, conventional diet) toward (semi-)industrial mosquito mass production and sex separation using tools and procedures specifically designed for male-only mosquito releases. Further optimization of the rearing conditions and improvement of the sorting methodology is thus warranted.

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To that end, we propose to: i) optimize existing mosquito sorting methods, and ii) explore classical genetic approaches toward the development of genetic sexing strains (GSS) for Ae. polynesiensis as a contribution to this CRP.

Sex-biased gene expression and sex determination in anopheline mosquitoes

Philippos A Papathanos

Polo d’Innovazione di Genomica, Genetica e Biologia

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 tackle the disease burden. A nearly identical repertoire of genes is present in each sex, so the dramatic differences observed between the sexes, ranging from morphology to physiology to behaviour, are largely governed by differential gene expression. Mechanistically this differential expression can be achieved either by varying the levels of expression of genes with sex-specific cis and/or trans regulation or by the alternative splicing of sex-specific isoforms. The male-limited Y chromosome likely also plays some role in causing sex-specific and sex-biased gene expression of genes located elsewhere, but the specific role of the Y chromosome in Anopheles mosquitoes is not yet known.

Our work is focused on characterizing the repertoire of sex-biased genes in anopheline mosquitoes. We hope to dissect the evolutionary constraints and outcomes of sex-biased genome evolution, identify specific genes involved in sex determination and ultimately, through functional validation of these, generate innovative tools for vector control. Our approach combines the latest in state-of-the-art of mosquito transgenic technologies for sex discrimination during ontogeny with high-throughput transcriptome analysis. The approach benefits from the fact that at early time-points of development, the analysis that is directed to uncover variation between males and females, is not likely to be confounded by the presence of sex-specific tissues such as adult germlines, which have previously hampered the identification of candidate genes for the sex determination pathway. Initially, our efforts will be directed at An. gambiae s.s. (G3 strain). Within the next year, we are also planning to analyse the sex-biased gene repertoire in 4 other species, including An. arabiensis , An. miminus , An. dirus and An. albimanus .

I will describe our current progress and planned output for the project.

Mosquito sex separation via TALEN-mediated female specific lethality

A. L. Smidler, W. R. Shaw, F. Catteruccia Harvard School of Public Health, Boston MA, USA. The focus of the Catteruccia laboratory is to understand the molecular events controlling the reproduction of a major African malarial vector Anopheles gambiae . During mating males transfer sperm, which is produced by the testes and then stored in the female body in a

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specialized sperm storage organ. Males also transfer a mating plug, a gelatinous rod produced by the male accessory glands and inserted into the female reproductive tract, and required for effective sperm storage. After mating, females change their behaviour, becoming refractory to further mating and laying eggs after taking a blood meal (virgin females do not lay eggs). Our work is unraveling the female pathways that translate the mating signals into behavioural and physiological changes that are key to reproductive success. Using a combination of high- throughput studies followed by in depth functional analyses, we have identified male-female molecular interactions that regulate egg development and fertility. Moreover we have characterized the sperm storage organ, and determined that genes involved in energy and protein metabolism, active transport and protection of sperm from oxidative stress play key roles in maintaining sperm viability and function for the female lifetime in maximizing long- term fertility. As well as mating biology expertise, we have the ability to generate transgenic mosquitoes in the laboratory. Recently, a targeted mutation in the genome of Anopheles gambiae was achieved by expression of two Transcription Activator-Like Effector Nucleases (TALENs) from transgene insertion (‘docking’) sites in the genome (Smidler et al ., 2013). When both were simultaneously expressed in the same organism, TALENs generated mutations at low frequency. We propose to create an X-chromosome-linked docking line for transgenesis and use it to create a new sex-separating strain, whereby female offspring, inheriting 2 X- chromosomes and both TALENs, will die through a somatic mutation, leaving only viable males. Initial work will test candidate X-chromosome docking lines for reproductive fitness. Further work will identify suitable target genes to be mutated by TALENs and suitable promoters to drive TALEN expression in these transgenes. Such a strain could potentially generate 100% males for release in irradiated-male programmes.

Effect of different Wolbachia types (absence, native and artificial) in Aedes albopictus on pre-imaginal and sexual maturity

Maurizio Calvitti, Riccardo Moretti, Francesca Marini and Angiola Desiderio

ENEA- Italian National Agency For New Technologies, Energy and Sustainable Economical Development.

The scientific objective of ENEA, as part of the CRP on “Exploring Genetic Molecular, Mechanical and Behavioural of Sex Separation in Mosquitoes”, is to investigate the role of the endosymbiotic bacterium Wolbachia pipientis in the pre-imaginal development of the mosquito vector Aedes albopictus , in response to varying mass rearing conditions in terms of temperature and larval density.

To achieve this we are going to study three laboratory lines of Aedes albopictus which have the same genetic background but different Wolbachia infection type: the first one deprived of the bacterium by an antibiotic treatment, the second one naturally harbouring two bacterial 42

strains wAlbA and wAlbB and the third generated by microinjecting the wPip strain of Wolbachia , taken from Culex pipiens eggs, into aposymbiotic embryos.

The practical goal of this research is to examine the effects of the endosymbiosis on sex development (i.e, strong protandry, pupal sexual dimorphism) and to evaluate the possibility of selecting the best combination of rearing conditions and mosquito line to maximize the efficiency of the sexual separation at the pupal stage for SIT-IIT programmes.

Since the study is designed to test the specific role of Wolbachia, each experiment, carried out under different temperature and larval density conditions, will be supported by quantitative monitoring of the bacterial density by real time PCR. Furthermore, considering that the integrated use of irradiation and Wolbachia induced Cytoplasmic Incompatibility is an option under evaluation to improve the efficiency of SIT, the effects of pupal irradiation on Wolbachia-infected and uninfected lines will also be evaluated in terms of adult emergence.

Mosquito Y chromosome genes and sex-determination

Frank Criscione 1, Brantley Hall 1, Yumin Qi 1, Vladimir Timoshevskiy 2, Maria Sharakhova 2, Igor Sharakhov 2, and Zhijian Jake Tu 1

1Department of Biochemistry, Virginia Tech, Blacksburg, VA, 2Department of Entomology, Virginia Tech, Blacksburg, VA.

Y chromosomes are responsible for the initiation of male development, male fertility, and other male-related functions in diverse species. However, Y genes are rarely characterized outside a few model species. In this presentation, I will discuss the recent work from my laboratory that establishes a novel method to discover Y genes by sequencing males and females separately and by comparing genomic sequences with RNA-seq data. We have identified seven Y genes that are expressed in the early embryos and adult testis of Anopheles mosquitoes. Analysis indicates rapid Y chromosome evolution between An. stephensi and An. gambiae . Functional analysis showed that one of these genes, Guy1, is likely the Male- determining factor (M factor) in An. stephensi . Transgenic lines are established in which all or most of the transgenic individuals are males. These Y genes provide unique markers for population and phylogenetic analysis, and opportunities for novel mosquito control measures through the manipulation of sexual dimorphism and fertility.

Controllable meiotic drive system

Kim. P. Hoang

Vina-UK Business Ltd, 13 Golden Cross, OX1 3EU, Oxford.

Arboviruses transmitted by mosquitoes represent a widespread threat to human health, against which an effective control solution is urgently required due to widespread insecticide resistance.

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We developed a method to target the Transformer-2 gene and its orthologs in Culicine mosquitoes which is conditionally regulated under various insect spermatogenesis-specific promoters. The outcome of this genetic system is a controllable meiotic drive effect in the progeny generation; hence the sex ratio of offspring can be controlled.

We are looking for a chance to develop this technique and with the possibility of applying it for Anopheles mosquitoes.

Sex and development in mosquitoes

Nathan Dennison, Elzbieta Krzywinska, Vladimir Kokoza, Marion Morris, Elena de la Casa- Esperon, Alexander S. Raikhel, Jaroslaw Krzywinski

Liverpool School of Tropical Medicine, UK; Pirbright Institute, UK; University of California, Riverside, USA; Albacete Science and Technology Park and University of Castilla-La Mancha, Spain.

Development into males and females in insects is controlled by a short cascade of sex determination genes. The cascade is triggered by a primary signal, which in mosquitoes comes from a dominant maleness ( M) factor, and whose presence or absence determines the sex. In anophelines M is linked to the Y chromosome, while in culicines, in which sex chromosomes are not differentiated, it is located on the autosomal chromosome 1. The molecular nature of M remains unknown. Here, we report progress in identification and characterization of the factor in Anopheles gambiae and Aedes aegypti . In An. gambiae , we detected a Y chromosome-linked transcript, whose onset of expression coincides with maternal-to-zygotic transition and precedes the establishment of sex-specific splicing pattern of doublesex , the last gene of the sex determination cascade. Experiments aimed at understanding the Y gene’s function led to death of all females in the embryonic stage, likely due to an upset of dosage compensation. Thus, the Y gene has the characteristics of the primary maleness factor. For Ae. aegypti , we are characterizing the chromosomal region encompassing the M locus. Experiments involving crosses of the transgenic strain with the transformation marker tightly linked to the M locus indicate the existence within the vicinity of M of at least three factors with different sex-specific lethal effects manifested during development. Our findings may have a great direct impact on the development and implementation of SIT strategies to control Anopheles and Aedes mosquitoes.

Aedes aegypti sex conversion by transgenesis

André Luis Costa-da-Silva, Danilo Oliveira Carvalho & Margareth Lara Capurro

Universidade de São Paulo.

The sterile insect technique (SIT) is an increasingly important component of pest management programmes. SIT has the ability to eradicate new outbreaks of pests so as to prevent their establishment. Globalization and climate change also are leading to an increase in new outbreaks of mosquito-borne diseases and major efforts are underway to develop new control techniques, including the SIT, for the mosquito species responsible for these outbreaks. Operational use of the SIT continues to reveal areas where new technologies are needed to

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improve efficiency and thus lead to more cost-effective programmes. There are many options for increasing the efficiency of SIT, improved mass rearing, release technology, quality control, etc., even when operational programmes are already underway. However, one critical area identified by programme managers and in which important advances can be made is the improvement of the strains that are being reared and released. The development of improved strains would lead to major increases in applicability and efficiency of a SIT programme. Mosquito strain improvement can be achieved using different approaches but all rely on some form of stable genetic change being introduced and maintained in the improved strain. Genetic change can be introduced either using 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. This includes transgenic insects that have been sterilized by irradiation for use in SIT programmes. The idea is to have: 1) strains that allows the production of males only for sterilization and release (genetic sexing strains), and 2) strains that incorporate a genetic marker to reliably and cheaply differentiate released insects from wild insects (marker strains). Salvemini and collaborators (2012) showed molecular characterization of the sexual gene homologues in Drosophila sp (DSX) in Aedes aegypti (Aeadsx ). This gene produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Also, Aeadsx produces a novel female-specific splicing variant. These proteins were present in early stages of development and their regulation starts at least in late larval stages.

Besides sex-specific gene characterization, early embryonic promoter genes such as AAEL008851, AAEL001543 and AAEL008722 (Biedler et al. 2012), need to be isolated. Also, the products of the maternal-effect genes nanos (nos) and oskar (osk) are important for the development of germ cells in insects. Furthermore, these genes have been proposed as candidates for donating functional DNA regulatory sequences for use in gene drive systems to control transmission of mosquito-borne pathogens.

This project will evaluate the possibility of sex-reversion in Aedes aegypti . We are going to discuss how to produce this transgenic line using RNAi for the doublesex gene in embryonic stages. Also we are going to test the best embryonic promoters to drive this interference.

Site-specific landing site systems to improve transgenic insects for SIT

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. To improve the SIT, new strategies based on genetic modifications of pest insects have been developed or are currently under investigation.

The goal is to further improve the development and ecological safety of genetically engineered (GE) insects created for enhanced biological control programmes. A major concern for GE insect release programmes is transgene stability, and maintenance of their

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consistent expression. 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. To address potential transgene instability, site-specific integration systems with the possibility of subsequent post-integration stabilization have been created and tested in several insect species. Examples are the in vivo tests of attP systems in medfly and mosquitoes or lox systems in Drosophila melanogaster and Caribfly. The best combinations will be tested in mosquito species .

In addition, random genomic insertion is challenging for GMO strain development due to genomic position effects that suppress transgene expression or insertional mutations that negatively affect host fitness and viability. Diminished transgene expression for example could result in the unintended survival of conditional lethal individuals, or the inability to identify them. To target transgene vectors to defined genomic insertion sites with minimal negative effects on gene expression and host fitness, a recombinase-mediated cassette exchange (RMCE) strategy will be developed. RMCE will also allow for stabilization of the target site, will be tested in mosquito species, and will aid the development of stabilized target-site strains for conditional lethal biocontrol.

Development of sex-specific markers in the tiger mosquito Aedes albopictus

Francesca Scolari, Mose’ Manni, Ludvik M. Gomulski, Giuliano Gasperi, Anna R. Malacrida Dept. Of Biology And Biotechnology ‘Lazzaro Spallanzani’ University Of Pavia, Italy.

Efficient applications of the SIT for mosquito AW-IPM programmes currently need fundamental components to be developed, validated and optimized. The Asian tiger mosquito Aedes albopictus , with its rapid expansion and vectorial capacity for several arboviruses, is becoming a worldwide emerging threat and the expansion of the biological information as well as the toolbox for improved control approaches is becoming an impelling necessity (Bonizzoni et al 2013, Trends Parasitol 29:460-8). To this aim, we intend to transfer knowledge and technologies developed in recent years for different tephritid pests to Ae. albopictus . These efforts will contribute to generate new tools which, in the long term, may be useful for engineering efficient GSS strains to be used in mass production for female elimination. In this regard, we initiated a project aimed at the identification and characterization of DNA sequences specific for the male sex chromosome in Ae. albopictus , a species currently under investigation in our laboratory also for what concerns its invasive process, taking advantage of our recently identified microsatellite and ITS markers. This work is following the Representational Difference Analysis (RDA) approach already applied successfully in our laboratory in Bactrocera oleae (Gabrieli et al 2011, PLoS ONE 6: e17747), and which is currently giving promising results also in B. dorsalis . The RDA data will provide male- specific sequences which will allow the development of male/female-specific primers for unambiguous molecular sexing and, in addition, will be investigated in terms of their potential functionality in sex determination, male fertility and male specific behaviour. The ongoing cytogenetic characterization of the mitotic and polytene chromosome complement of Ae. albopictus , a necessary premise for the development of any genetic sexing system, will be of key importance not only for the physical mapping of the identified sex

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chromosome-specific sequences, but also for the efficient location of genes and contigs (Fluorescent in situ hybridization, FISH), which will be soon available thanks to the upcoming publication of the Ae. albopictus genome. In addition, high-throughput genomic DNA resources, combined with transcriptome sequencing data, have been recently shown to be essential for the identification of Y chromosome genes in the other mosquito species Anopheles stephensi and An. gambiae (Criscione et al 2013, Insect Mol Biol 22:433-41; Hall et al 2013, BMC Genomics 14:273) and may greatly support the discovery of sequences specific to the male sex chromosome in Ae. albopictus too.

Developing genetic method for accurate sex separation and female elimination in the malaria vector Anopheles arabiensis

Cyrille Ndo Organisation de Coordination pour la Lutte contre les Endémies en Afrique Centrale (OCEAC), Yaoundé-Cameroon

I am a holder of a PhD in medical entomology with a thesis defended in May 2011 at the University of Yaoundé I (Cameroon, Central Africa). My scientific expertise covers various aspects of field and molecular entomology notably mosquito collection, identification and rearing, mosquito biology and ecology, population genetics, cytogenetic, bioinformatics, and insecticide resistance.

My research project entitled “Developing genetic method for accurate sex separation and female elimination in the malaria vector Anopheles arabiensis ” is part of the CRP axis: “Explore molecular approaches for sex separation in mosquitoes - Development of GSS based on molecular genetics”. It aims to develop a new genetic method for sex separation and female elimination in the malaria vector An. arabiensis by linking a temperature sensitive allele on Y-autosome rendering males resistant to relatively high temperatures while females remain sensitive.

The newly developed method should facilitate sexing operations by exposing eggs to heat shock. It will allow treatment of large number of eggs without several manipulations, reducing losses. Moreover, sexing operations will no longer involve chemicals, therefore no additional effort will be required for waste management, and no environmental pollution will be possible as was the case with the dieldrin (insecticide)-based sexing method.

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Towards the development of a genetic sexing strain: construction of an Aedes albopictus rdl R homozygous line.

Pablo Tortosa, Célestine Atyame and Louis Clément Gouagna Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Océan Indien (www.crvoi.org)

Arboviruses are of great concern to the islands of the Western Indian Ocean and notably on Réunion Island where Dengue dynamics are currently strictly surveyed following the 2005- 2006 Chikungunya epidemics that dramatically impacted economies and human health. Investigation of insecticide resistance have shown that Culex pipiens quinquefasciatus and, to a lesser extent Aedes albopictus, exhibit strong levels of insecticide resistance thus strengthening the need for the development of innovative vector control methods. CRVOI is currently developing 2 innovative strategies for the control of natural Ae. albopictus populations: the sterile insect technique (SIT), and incompatible insect technique (IIT). Although the corresponding methods used for impairing males ‘fertility’ are clearly different, both techniques rely on the inundative release of males with the aim of sterilizing females in the wild. Therefore they require the availability of an efficient sexing method in order to be implemented in large scale. Taking advantage of the investigations of insecticide resistance evolution in natural mosquito populations carried out in La Réunion, we propose the construction of an Ae. albopictus isofemale line homozygous for the rdl R allele, conferring resistance to dieldrin, required for the subsequent construction of a genetic sexing strain. The drawback of the use of insecticide resistance conferring genes will be discussed.

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

L.L. Koekemoer, G. Munhenga, L. Essop, L. National Institute for Communicable Diseases Anopheles arabiensis is one of the major vectors of malaria in South Africa (besides An. funestus ) and is responsible for seasonal malaria transmission during the summer season. It is difficult to control this species using residual insecticides owing to its behavioural variation. Anopheles arabiensis will feed on humans (anthropophilic) and animals (zoophilic) and will feed both indoors (endophilic) and outdoors (exophilic). Insecticide resistance in vector mosquitoes is a major concern for the vector control programme in South Africa. During 1999/2000 South Africa experienced an epidemic and more than 65,000 cases of malaria were reported due to insecticide resistance in a malaria vector. With appropriate resistance management this epidemic was quickly suppressed, however the threat of insecticide resistance is not over. During 2002, a DDT-resistant population of An. arabiensis was detected at Mamfene, KwaZulu/Natal. A few years later we also identified pyrethroid resistance in this population from KwaZulu/Natal. Additional vector control strategies are urgently needed to strengthen the current control interventions.

Additional strategies such as entomopathogenic fungi and the sterile insect technique (SIT) are currently investigated for this purpose. SIT has been successfully used to combat other 48

insect pests. We are currently investigating the feasibility of SIT for An. arabiensis in South Africa. Through collaboration with the Insect Pest Control Laboratory (J. Gilles) in Austria, South Africa has colonized the genetic sexing strain (GSS-ANO IPCL1) in the local insectary in Johannesburg. This strain has since been introgressed with the South African local wild type strain AMAL. The strain (GSSxAMAL) is stable and doing well and is now available to conduct additional studies with. This proposal aims to investigate the effect of radiation on the fitness of the newly established GSSXAMAL strain as well as investigate the use of alternative, more environmentally friendly insecticides such as fipronil on this strain.

Conditional lethality by insecticide resistance for sex separation of Aedes aegypti .

René Gato Armas Institute of Tropical Medicine Pedro Kourí. Havana, Cuba.

The first haemorrhagic dengue epidemic in the Americas was reported in Cuba in 1981. The country remained dengue-free for years. However, in current context the control programme has failed in reducing the populations of Ae. aegypti in a sustainable way. New integrated strategies are imperative, particularly given the increasing travel between Cuba and endemic countries in the region. The sterile insect technique (SIT) is a remarkably promising vector control method in terms of efficacy, efficiency and environmental compatibility. One of the critical points for the success of SIT is the need for a method to separate sexes, since released sterile females would be able to transmit disease and to produce a biting nuisance. In order to reduce the production cost in mass rearing facilities, sex separation should take place as early as possible. A number of methods have been developed for sexing based on pupal sexual dimorphism, male-swarming, female blood-feeding, classical genetic methods and transgenic approaches. Under the current world economic conditions, it is also important to develop cost- effective tools that can be implemented inside undeveloped countries. For this year we plan to start the development of sex-separation methods based on conditional lethal systems. A strain with a high level of resistance to temephos, developed under selection pressure with this insecticide, will be used. Experiments for translocation of the temephos resistant gene to the male-determining chromosome will be carried out in order to induce conditional lethality. Additionally, a prototype device for sex separation based on blood feeding behaviour will be presented.

Forward Genetic Analysis of Sex-specific Gene Expression in Anopheles Mosquitoes.

David A. O’Brochta

Institute for Bioscience and Biotechnology Research & The Department of Entomology, University of Maryland, College Park; 9600 Gudelsky Drive, Rockville, MD 20850, USA.

Advanced transposon-based genetic technologies are now available for use in Anopheles mosquitoes that provide new opportunities for performing forward and reverse genetic

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analyses leading to new advances in our understanding of Anopheles gene structure and function. Transposon-based gene vectors can be configured to carry specific transgenes that can act as sensors to permit the detection of genetic elements such as enhancers, promoters and genes that are in proximity to the inserted vector. Here I report that the piggyBac transposon following its introduction into Anopheles stephensi can be remobilized at high frequencies by expressing piggyBac transposase in trans. piggyBac remobilization is the basis for new piggyBac -based enhancer- and gene-trap technologies. Remobilizing piggyBac elements configured to detect enhancers results in about 1.3% of the progeny harbouring the enhancer-trap element in a new position with a unique enhancer-specific pattern of reporter gene expression. A functional gene-trap system has also been created consisting of a promoter-less reporter gene with a 3’ splice-acceptor site that serves as a gene-detector. The reporter gene contained within this gene-trap element will be expressed only when the element integrates into the intron of an actively transcribed gene. Both systems will be used to identify and isolate sex-specific gene-regulatory elements in An. stephensi . Enhancer-trap and gene-trap detection experiments will be conducted in which larvae and adults will be screened for the presence of sex-specific trap-dependent reporter gene expression. Individuals with patterns of reporter gene expression of interest will be used to establish lines whose patterns of reporter gene expression will be further analysed in more detail. Those enhancer- and gene-trap lines deemed potentially useful based on reporter gene expression patterns will be used to physically identify and isolate the functional enhancers and promoters responsible for the gene expression patterns originally detected. Sex-specific gene regulatory elements from Anopheles mosquitoes could be useful in designing and constructing genetic and molecular methods for sex separation of mosquitoes.

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LIST OF PARTICIPANTS

BRAZIL Email: [email protected] Ms Margareth CAPURRO- Tel: +689 53 3156 GUIMARRAES Universidade de Sao Paulo GERMANY Instituto de Ciencias Biomedicas Mr Marc SCHETELIG Av. Prof. Lineu Prestes, 2415 Fraunhofer-Institute for Molecular Biology Butantan, 05508-000 Sao Paulo and Email: [email protected] Applied Ecology (IME) Project Group “Bioresources” CAMEROON Winchesterstr. 2 Mr Cyrille NDO 35394 Giessen Organisation de Coordination pour la lute Email: [email protected]; contre les endémies en Afrique Centrale [email protected] (OCEAC) Tel: + 49 641 9939504 2.0 Rue du Centre Pasteur ITALY P.O.Box 288, Yaoundé Mr Philippos PAPATHANOS Email: [email protected] Polo d'Innovazione di Genomica, Genetica Tel: +237 22 23 22 32 e Biologia, Edificio D 3 Piano Via Gambuli CUBA 06132 Perugia Mr Rene GATO ARMAS Email: [email protected] Instituto de Medicina Tropical "Pedro Tel: + 39 2134531398 Kourí" Apartado 601, Autopista Novia del Ms Ariana PUGGIOLI Mediodía Km 6,5 (replacing Mr Romeo BELLINI) Marianao 13, La Habana Centro Agricoltura Ambiente Email: [email protected]; Giorgio Nicoli s.r.l. [email protected] Via Argini Nord, 3351 Tel: +53 7 2 55 36 19 40014 Crevalcore BO Email: [email protected] FRANCE Tel: + 39 051 6802240 Mr Pablo TORTOSA Institut de recherche pour le Ms Francesca SCOLARI développement (IRD) Dipartimento di Biologia e Biotecnologie Representation de La Reunion, CS41095 Universita degli Studi di Pavia Parc Technologique Universitaire Via A. Manzoni 8 2 rue Joseph Wetzell 25020 Seniga (BS) 97495 Saine Clotilde Cedex Email: [email protected] Email: [email protected] Tel: + 39 382 986023 Tel:+ 262 262 48 33 50 Mr Maurizio CALVITTI Mr Hervé BOSSIN BIOTEC/AGRO Institut Louis Malardé Italian National Agency for New BP 30 98713 Papeete Technologies, Energy and Sustainable Tahiti, French Polynesia Economic Development (ENEA) 51

Lungotevere Thaon di Revel, 76 UNITED KINGDOM 00196 Roma Mr Jaroslaw KRZYWINSKI Email: [email protected] The Pirbright Institute Tel: +39 0630484597 Pirbright, Woking, GU24ONF Email: [email protected] Mr Giuliano GASPERI Tel: + 44 1483 232441 Department of Biology and Biotechnology University of Pavia Mr Andrea CRISANTI Via Ferrata 9 Imperial College London 27100 PAVIA Biological Sciences Dept. ITALY SAF Building Email: [email protected] Imperial College Road http://genmic.unipv.eu/site/home.html London SW72AZ Tel: +39 (0382) 986 294 Email: [email protected] Tel: + 44 207 594 5426 PAKISTAN Mr Inamullah KHAN UNITED STATES OF AMERICA (Replacing Mr Khan GULZAMIN) Mr Zhijian TU Nuclear Institute for Food and Agriculture Virginia Tech (NIFA) G.T. Road, Tarnab, Peshawar 4044 Derring, Hall-Mail code 0120 Email: [email protected] 24061 Blacksburg, VA Tel: +92 912964059 Tel: + 540 231 8062 SOUTH AFRICA Email: [email protected] Mr Givemore MUNHENGA (r eplacing Ms Lizette KOEKEMOER) Mr William Robert SHAW Medical Entomology Research Group (replacing Ms Flaminia CATTERUCCIA ) Harvard School of Public Health Wits Health Consortium 665 Huntington Avenue 8 Blackwood, Parktown Boston, Massachusetts 02115 2131 Johannesburg Email: [email protected]; Email : [email protected] [email protected] Tel: +27 11 386 6484 Mr David O’BROCHTA SPAIN University of Maryland Institute for Mr Carlos TUR LAHIGUERA Bioscience and Biotechnology Research Centro de Control Biologico de Plagas (IBBR-SG) (Tragsa) 9600 Gudelsky Drive Poligono 9, Parcela 13, Paraje la Cantina Rockville 20850-3467 46315 Caudete de las Fuentes Email: [email protected] Email : [email protected] Tel : +240 314 6309 Tel : +34 962319515

SRI LANKA OBSERVERS Mr Wimaladarma ABEYEWICKREME Molecular Medicine Unit SPAIN Faculty of Medicine Mr Gustavo SALVADOR HERRANZ University of Kelaniya Universidad CEU Cardenal Herrera Email: [email protected] 46113 Moncada, Valencia Tel: +94 112 960483 SPAIN Email: [email protected] 52

GERMANY Ms Irina HÄCKER Fraunhofer Projektgruppe Bioresources Winchesterstr. 2 35394 Giessen Email: [email protected] Tel: +641-9939504

UNITED KINGDOM Mr Kim Phuc HOANG Director Vina-UK Business Ltd 13 Golden Cross OX1 3EU, Oxford Email: [email protected] Tel: + 44 1865 7909

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