IAEA-D4.40.01-CR-4
LIMITED DISTRIBUTION
LAST RESEARCH COORDINATION MEETING Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
Research Coordination Meeting on Exploring Genetic, Molecular, Mechanical and Behavioral Methods of Sex Separation in Mosquitoes
Scientific Secretary: Kostas Bourtzis
Ramada Plaza Hotel
Bangkok, Thailand
19–23 February 2018
Reproduced by the IAEA
Vienna, Austria 2019
______NOTE
The material in this document has been supplied by the authors and has not been edited by the IAEA. The views expressed remain the responsibility of the named authors and do not necessarily reflect those of the government(s) of the designating Member State(s). In particular, neither the IAEA not any other organization or body sponsoring this meeting can be held responsible for any material reproduced in this document.
Contents Summary 3
Background Situation Analysis: 3
To explore irradiation and classical genetic approaches for sex separation in mosquitoes - development of GSS based on irradiation and classical genetics 5
To explore molecular approaches for sex separation in mosquitoes - Development of GSS based on molecular genetics 7
To explore mechanical, behavioural, developmental and symbiont-based approaches for sex separation in mosquitoes 9
Selected References 11
Nuclear Component 19
Explanation / Justification: 19
Participation of Agency's laboratories (Yes/No) 19
Assumptions: 19
Related TC Projects 19
LOGICAL FRAMEWORK 21
Narrative Summary 21
Specific Objectives 22
Outcomes 23
Outputs 24
Activities 27
AGENDA 28
PARTICIPANT ABSTRACTS 31
LIST OF PARTICIPANTS 52
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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 prerequisite. In addition, male-only releases would improve SIT programme efficiency by increasing the frequency of sterile 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, nutritional, behavioural and developmental approaches, this CRP will primarily explore classical genetic and modern biotechnology techniques to accomplish female elimination in major mosquito vectors of disease. Whenever possible, these methods will be created with a view to application to a wide spectrum of mosquito species. Major beneficiaries will be operational AW-IVM programmes in Member States that plan to apply the SIT (classical, transgenic, and/or symbiont-based approaches) against mosquitoes. By the end of the CRP, methods for developing sexing strains will have advanced and some strains will be available for evaluation.
The development and evaluation of such methods through this CRP will have the following tangible benefits for SIT mosquito control programmes:
1.) Safety of SIT mosquito programmes will be enhanced. Mosquito SIT implementation cannot include the release of even sexually-sterile biting females. Therefore, mosquito SIT requires the exclusive release of sterile males, which is impossible on a large scale without sex separation methods.
2.) Male-only releases are several-fold more efficient than releases of both sexes. Consequently, when genetic sexing technology is available SIT programmes are significantly more efficient and therefore more cost-effective.
3.) As only the males are needed for the SIT, the production, handling and release costs can be reduced significantly if sexing strains are used and females are eliminated early in development. Background Situation Analysis: Among the major vectors of human diseases, mosquitoes are the most devastating ones. Urbanisation, globalisation, and climate change have further accelerated the spread and outbreaks of new mosquito-borne diseases. In view of the problems associated with insecticide-based mosquito control, such as resistance, health and environmental effects, major efforts are required to develop new and complementary control techniques, including the Sterile Insect Technique (SIT), for major mosquito species.
The 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
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(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 concerns the integration of sexing systems (including but not restricted to 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 will not be fully exploitable.
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. Several Wolbachia-related (semi)field trials (IIT alone or combined SIT/IIT) of various scope and size were conducted successfully in different countries and against different mosquito vector species. Field trials included interventions against Ae. aegypti in Australia, Thailand, Singapore and the USA, Ae. albopictus in China, Germany and the USA, and Ae. polynesiensis in French Polynesia. The success of IIT could be affected by the accidental release of fertile 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. To overcome this issue the combination of SIT with IIT has been proposed which eliminates the risk associated with accidental release of fertile females. Furthermore, the use of transmission blocking Wolbachia provides protection against pathogens like DENV, CHIKV and ZIKV. Altogether, the combined SIT/IIT approach represents a safe and secure genetic approach for the control of mosquito vector populations.
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 has been useful in this CRP for the development of GSS in mosquitoes.
There are several mosquito species that are major vectors of different pathogens in different countries. Of these, based on the severity of the disease, requests from Member States and the availability of scientific tools and information, the consultants recommended that the initial efforts to develop mosquito GSS should focus on the following species: Anopheles arabiensis (AAr), Anopheles stephensi (ASt), and Anopheles gambiae (AGa) (vectors of malaria), and Aedes albopictus (AAl), Aedes polynesiensis (APo) and Ae. aegypti (AAe) (vectors of dengue, chikungunya, zika and other viruses).
Mosquito GSS development was achieved using different approaches, all relying on some form of stable genetic change introduced and maintained in the developed strain. Genetic change was introduced using irradiation and classical genetics (as in the case of the Mediterranean fruit fly GSS) or modern biotechnology, specifically genetic transformation or gene editing approaches. Both approaches have advantages and disadvantages relating to transferability of systems between 4
species, stability in mass-rearing, regulatory approval, etc. Sex separation methods based on mechanical, nutritional, behavioural, developmental and symbiont-based approaches were also successfully developed.
The integration of all these methods will likely provide complete sex separation under large scale SIT-related operations.
Developing sex separation methods for mosquitoes is a prerequisite for the use of SIT in an AW- IVM approach. Four specific objectives have been addressed:
1. Classical genetic approaches including irradiation for sex separation in mosquitoes
2. Molecular approaches for sex separation in mosquitoes
3. Mechanical, nutritional, behavioral, developmental and symbiont-based approaches for sex separation in mosquitoes
4. Encouraging and attracting participants to the CRP in the field of classical genetics. 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).
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 genetic and molecular tools that are currently available in the toolbox and illustrates that despite recent advances in this field, there is an urgent need for further improvement.
The sequence of the genomes of some 20 species of mosquitoes is currently available and is an invaluable resource for the development of GSS using transgenic and genome modification technologies. For instance, such data will facilitate the determination of the exact position of chromosomal rearrangements in translocated strains, which can be considered as an interesting alternative to cytogenetics.
The first mosquito GSS were developed in ‘70s, using classical genetic approaches. However, these strains did not prove to be very useful for various reasons including instability of the genotypes produced in the lab and essential determinants of the effectiveness of the GSS. While none of the original strains are currently available, one of the original mosquito GSS was recreated based on dieldrin resistance in An. arabiensis. The use of insecticide resistance as a selectable marker is
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unlikely to be acceptable for large scale applications for a number of reasons including: (a) protection of the environment and human health; (b) accuracy and (c) potential contamination of the mass- rearing colony.
In view of the lack of highly efficient GSS there is a major demand for the development of new GSS for target mosquito species, which would be based on the successful strategy followed for Mediterranean fruit fly and other fruit fly species. An ideal GSS must have two features: (a) the ability to maintain desirable genotypes using selectable or morphological markers (b) a mechanism to ensure sex-specific selection. In addition, the development of GSS allowing early elimination of females in the mass production process (ideally at the embryonic stage), should be preferred to improve cost efficiency. Thus, the development of a mosquito GSS using conventional genetic technologies would demand the following:
(a) isolation and mapping of genetic markers (preferably morphological markers) from natural populations and/or laboratory populations following mutagenesis; most of these markers will be useful for genetic studies, some of which may meet the criteria for a selectable marker;
(b) irradiation-induced chromosomal rearrangements (i.e. inversions, translocations) and their characterization;
(c) isolation of conditional early lethal mutations (for example tsl mutation) and
(d) combination of the above components into a stable GSS. Making the decision about the most suitable selectable marker is crucial and will ultimately result in the construction of a robust GSS.
At this point, it should be emphasized that the isolation of genetic markers from natural populations or random mutagenesis is a rather challenging and labour-demanding serendipitous process. The achievement of this goal will require the recruitment of a dedicated and fully committed expert, as well as sufficient technical support, to join the mosquito group at the IPCL, Seibersdorf. Despite the fact that the principles of the classical genetic approach are easily applicable to most species, the actual tools that will be developed for a given species cannot be transferred to another mosquito species. On the other hand, the benefit of the classical genetic approach is that no legal restrictions or other regulations apply, and strains can be directly used for SIT application.
Expected outputs and achievements
1. Genetic markers from different sources including natural populations and / or mutagen-based screens. Such markers have been isolated or induced and have facilitated the development of mosquito GSS (AAr, AAe and AAl).
2. Chromosomal rearrangements in the target mosquito species. Chromosomal rearrangements have facilitated the development of stable mosquito GSS (AAr, AAe and AAl).
3. (Cyto)genetic data and analyses in support of sex separation. (Cyto)genetic analysis has facilitated genetic studies and characterization of the available GSS (AAr, AAe and AAl).
4. Morphological and selectable markers. These markers have allowed the construction of Aedes and Anopheles GSS (AAr, AAe and AAl).
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5. A classical GSS. Aedes and Anopheles GSS have been developed (AAr, AAe and AAl).
6. Lab and/or semi-field evaluation of a classical GSS. Lab and/or semi-field evaluation of the Aedes and Anopheles GSS has been performed and has provided data on their actual stability and overall fitness (AAr, AAe and AAl). Molecular approaches for sex separation in mosquitoes - development of GSS based on molecular genetics Enhancing SIT through the development of molecular-based genetic sexing strains has been proven successful for fruit flies, including the tephritid species C. capitata and A. suspensa, for which 100% conditional/repressible female lethality has been achieved through a well-understood apoptotic pathway combined with sex-specific splicing. A similar strategy was applied to animal pests including the New World screwworm Cochliomyia hominivorax and the Australian sheep blowfly Lucilia cuprina. The application of transgenic systems has the potential advantage of a faster development time and the ability to be transferred to other species. In addition, an approach to generate a male-only population by sex conversion of females to males has been tested in C. capitata, A. supsensa, B. oleae, and L. cuprina with very encouraging results. Some of the molecular-based GSS have been successfully evaluated under semi-mass rearing and field cage conditions with support from the FAO/IAEA Agriculture and Biotechnology Laboratories in Seibersdorf. These achievements will help to explore different options for molecular-based GSS in mosquito species.
To generate molecular-based GSS in mosquitoes there are a number of options that are currently being considered which include female-specific lethality, sex ratio distortion, sex-conversion or sex- specific markers to be used with sorting. In all these systems, transgenic constructs enable genetic sexing using effector transgenes of different classes, and, if needed, combining them with sex- specific expression using validated sex-specific promoters. Alternatively, sex-specific expression of effector transgenes can be ensured through linkage to male-specific chromosomes or male-specific loci (e.g. M locus). Finally, to be suitable for implementation GSS will have to integrate conditional expression of the genetic sexing phenotype to permit mass rearing.
Transgene effectors, all using transgene-based approaches to genetically sex, have now been demonstrated in mosquito vectors. A female-killing transgene was recently generated in Anopheles stephensi by inserting an additional copy of the Y-chromosome linked male-determining factor Guy1 onto an autosome, which lead to dominant embryonic lethality of females but did not affect males. A similar female-lethal effect was observed in An. gambiae after embryonic microinjection with the mRNA of the male-determining factor Yob. Sex-conversion in the form of masculinization of genetic females has been shown in Aedes aegypti by transiently mis-expressing the primary sex-determiner Nix in females. Sex-ratio distorters using both naturally occurring endonucleases and more recently using CRISPR/Cas9 (see below) and expressed during spermatogenesis have been developed in Anopheles gambiae showing high degrees of male bias (~95%) in the offspring of transgenic males. Finally, cassettes consisting of fluorescent markers, under the control of male-specific promoters or inserted directly on the Y chromosome, have been developed for automated sorting and selection of transgenic male using a FACS-based technology. Recent integration of single molecule sequencing and long-range mapping strategies has resulted in significant improvements to the genome assemblies of both Ae. aegypti and Ae. albopictus. Significant progress has been made in the study of Anopheles Y chromosome and the Aedes M-locus. This has led to the identification and functional
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characterization of several primary male determining genes in mosquitoes, which have in turn acted as the effector transgenes in some of the above strategies.
Conditional or repressible expression systems will be required to consider the use of such GSS in a mass-rearing setting. In this context, the Q-system has now been successfully transferred to Anopheles gambiae and its transfer to additional mosquito species is now anticipated. In addition, the UAS-Gal4 and the tet-system are already available in a number of mosquito species. The suitability of these conditional systems will largely be dictated by the different sexing strategy and/or effector system used. To date, only the tet-system has been evaluated in the mass-rearing environment with some success but, reports regarding leaky expression and insertion site-specific variation suggest that alternative systems should be explored. For example, a temperature sensitive sex-specific lethal strain was recently produced by mutating the TRA2 protein in D. suzukii.
Creating genetically modified mosquitoes for biological control will require predictable and highly regulated gene expression and to achieve this kind of site-specific integration systems will be important. These systems enable the integration of transgenes into known sites within the genome, thereby eliminating the uncertainties associated with transposable element integrations and the variable gene expression that can arise due to ‘position effects’. A number of site-specific recombination systems are available and the phiC31/attP and Cre/LoxP systems have been introduced into Aedes aegypti, Ae. albopictus, Anopheles stephensi, Anopheles gambiae and Anopheles arabiensis. These systems have been shown to not only enable the integration of transgenes but in some cases for the exchange of transgenes by a method called Recombination Mediated Cassette Exchange (RMCE). The number of well characterized recombination sites in genomic locations known to result in minimal or no ‘position effects’ is currently quite small and the continued creation of mosquito lines with useful site-specific recombination ‘docking sites’ is essential.
RNA-guided endonucleases such as Cas9 from the CRISPR/Cas9 system have been widely adopted by insect biologists and there are now many published examples of its use in insects as both a somatic and germline mutagen. It is clear from these published data that the creation of null alleles can be quite efficient if the necessary guide RNAs are well designed and the Cas9 endonuclease can be effectively delivered to germ cells or presumptive germ cells. The use of these systems to insert transgenes by homology directed repair mechanisms has also been demonstrated in mosquitoes. The development of ‘gene editing’ systems is particularly significant because they provide insect biologists with a new powerful set of tools and capabilities that will facilitate the creation of genetic sexing strains.
The use of RNAi to control gene expression in insects has progressed with the discovery and development of new modes of dsRNA delivery to insect tissues and cells, either through feeding dsRNA, or the stable expression of dsRNA using transgenics Several feeding strategies have been explored, including feeding of naked dsRNA, dsRNA associated with inert carriers such as chitosan, or the delivery via dsRNA-expressing bacteria or yeast. All of these strategies have proven to be effective ways of regulating the expression of some mosquito genes. When the dsRNA-targeted genes were involved in sex determination, sex ratio distortion was observed indicating that the use of dsRNA may have some potential as a means for genetically sexing mosquito larvae.
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Expected outputs and achievements
7. Construction and characterization of transgenic strains with site-specific landing sites and known performance characteristics. Generation and assessment of transgenic strains harbouring site-specific landing sites (AGa, AAr, and AAe) have been achieved.
8. Methods, protocols and procedures for using RNA-guided endonuclease-based (‘gene editing’) technologies in mosquitoes. Genome editing technologies (mainly CRISPR-based gene editing) have been developed (AGa, AAe, ASt).
9. Data on the structure, organization and patterns of gene expression of the sex chromosomes of mosquitoes. Successful strategies have been developed and applied to a number of vector species that have provided data into the biology of sex chromosomes and sex determining genes (AGa, AAr, ASt, AAe, AAl). These strategies are applicable to other mosquito species.
10. Gene expression regulatory elements that confer sex-specificity. Regulatory sequences with male and female specific expression have been identified and characterized in transgenic strains in several mosquito species (AGa, AAr, ASt, AAe).
11. Effector molecules that can confer predictable phenotypes when expressed in mosquitoes. Effectors falling in the classes of female-specific lethals (ASt, AGa), sex ratio distorters (AGa), sex-converters (AAe) and fluorescent markers (AGa, AAr, ASt, AAe, AAl) have been identified, characterized and used in a number of species.
12. Strains of genetically modified mosquitoes created from elements generated from outputs 8, 10 and 11 that have the potential to be adapted to permit conditional genetic sexing. Combinations of regulatory elements and effector molecules have been used to generate female-specific effects (AGa, ASt).
13. Strains developed from output 12 assessed for performance characteristics in lab and/or semi-field conditions. Strains developed from output 12 have been assessed in respect to their performance characteristics under small-scale laboratory conditions. Mechanical, behavioural, developmental and symbiont-based approaches for sex separation in mosquitoes
Mechanical, behavioural, nutritional and developmental tools for sexing were developed or existing ones refined to further improve the removal of females at different developmental stages. The accessibility of such systems will provide a stop-gap measure that allows rapid start up with a minimum of investment. Moreover, their integration with genetic sexing systems will likely provide complete sex separation under large scale SIT-related operations.
The following sexing approaches were investigated by CRP participants. More than one method could be combined in order to attain full separation.
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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. Starting from previous trials which assessed differences in pupal body size, image analysis methods were developed that considerably improve male/female pupae separation under mass-rearing conditions for several mosquito species including Ae. aegypti, Ae. albopictus, and Ae. polynesiensis. Sexual dimorphism was used for automatic identification by computer vision algorithms. Once both sexes are identified, females have been mechanically removed using high speed positioning and accuracy precision laser systems driven by a dual axis galvanometer optical scanner.
Behavioural Tools. For all blood-feeding mosquitoes, sex separation could occur at the adult stage by spiking blood with insecticides (i.e. malathion, dieldrin) or other insects toxins (i.e. spinosad, ivermectin, plant based). Studies showed that all female Ae. aegypti and Ae. albopictus could be eliminated by spiking blood meals with 8 ppm ivermectin or spinosad within 2 days. Integration of this approach with other separation methods would increase female removal and the efficacy and safety of SIT-related applications.
Developmental Tools. Protandry is a well-known phenomenon in many mosquito species, where male develop more rapidly than female. 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, where it was possible to select a hyper-protandric strain showing an enhanced separation between sexes. Protandry was standardized at least for Ae. polynesiensis by adjusting factors such as rearing temperature, and larval density.
Symbiont-based Tools. Several mosquito species like Ae. albopictus, Ae. polynesiensis or Culex quinquefasciatus harbour endosymbiotic microorganisms like Wolbachia which influence several fitness parameters. We have found that the “wPip” Wolbachia strain, naturally occuring in Culex pipiens, significantly shortens the pupation onset time and increases pupal productivity when transferred to Ae. albopictus. These effects have been exploited for improving sexing at pupal stage.
On the contrary, the “wMel” Wolbachia strain does not seem to have any effect on immature development in Ae. albopictus.
The further investigation of the role of bacterial endosymbionts such as Wolbachia in influencing the duration of pre-imaginal development, protandry, sex ratio and pupal dimorphism, seems promising in the development of more effective sex separation methods using appropriate mosquito strains and mass rearing conditions. The dynamics of endosymbiont density in their host has also been investigated in view of its possible importance in SIT and/or IIT applications.
Expected outputs and achievements
14. Data that allows the use of Wolbachia symbiosis for sex separation. Wolbachia symbiosis has been shown to enhance protandry (wPip infection in AAl) and could thus improve existing sex separation methods.
15. A set of mechanical (e.g. size dimorphism), nutritional (e.g, larval diet), behavioural (e.g. phototaxis, toxicants) and developmental (e.g. protandry) tools for sex separation. Mechanical, nutritional, behavioural and developmental tools have been developed and tested for sex separation under laboratory conditions (AAe, AAl, APo) and in pilot trials (APo).
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16. Novel sex separation approaches exploiting mechanical, nutritional, behavioural and developmental properties. One novel sex separation tool (laser-based sorter) has been developed and evaluated under laboratory conditions (AAe, AAl, APo).
17. Data on the efficacy and reproducibility of mechanical, nutritional, behavioural, and developmental tools under low to mid production scales using standard quality control parameters. Data on the efficiency, reproducibility, and applicability of the laser-based sorter were collected using pupae from low to mid scale productions (AAe, AAl, APo). Selected References Alphey L., Nimmo D, O'Connell S, Alphey N (2008) Insect population suppression using engineered insects. Transgenesis and the Management of Vector-Borne Disease, 627:93-103 [http:// www.springer.com]. New York: Springer Science+Business Media, LLC Landes Bioscience
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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, zika, and malaria outbreaks.
That IAEA Member States continue requesting the development of the SIT package for mosquitoes, and that enough scientists and institutions are willing to participate in the CRP and can be motivated to apply classical genetic approaches to develop sex separation systems in mosquitoes.
The demand for area-wide integrated insect pest management approaches, including SIT and augmentative biological control as non-polluting suppression/eradication components, continues to increase, mandating expansion and improvement in cost-effectiveness of these environment- friendly, sustainable approaches.
Related TC Projects MEX5031: Using the Sterile Insect Technique to Control Dengue Vectors.
PHI5033: Building Capacity in Using the Sterile Insect Technique against Dengue and Chikungunya Vectors.
SAF5014: Assessing the Sterile Insect Technique for Malaria Mosquitos in a South African Setting, Phase II.
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SRL5047: Establishing a National Centre for Research, Training and Services in Medical and Molecular Entomology for Vector-borne Disease Control (on going).
SUD5038: Implementing the Sterile Insect Technique for Integrated Control of Anopheles arabiensis, Phase II.
RAS5066: Promoting the Sharing of Expertise and Infrastructure for Dengue Vector Surveillance towards Integration of the Sterile Insect Technique with Conventional Control Methods among South and South East Asian Countries.
RER5022: Establishing Genetic Control Programmes for Aedes Invasive Mosquitoes.
RLA5074: Strengthening Regional Capacity in Latin America and the Caribbean for Integrated Vector Management Approaches with a Sterile Insect Technique Component, to Control Aedes Mosquitoes as Vectors of Human Pathogens, particularly Zika Virus.
INT5155: Sharing Knowledge on the Sterile Insect and Related Techniques for the Integrated Area- Wide Management of Insect Pests and Human Disease Vectors.
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LOGICAL FRAMEWORK:
Narrative Summary Objective Means of Verification Important Assumptions Verifiable Indicators Overall Objective
To explore genetic, N/A N/A Requests by Member States in the area molecular, behavioural of mosquito control using the SIT are and mechanical methods increasing. To make this nuclear of sex separation in technology available to Member States mosquitoes for several mosquito species, the development of male-only releases is either a requirement to reduce programme costs or an essential precondition. Given the large number of different target species and the lack of any efficient tools for mosquito control, strategies based on genetic, molecular, mechanical and behavioural techniques are amenable. Biological material is available. An expert on classical genetics and a technician in the mosquito group in Seibersdorf are recruited.
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Specific Objectives
1. To explore Classical GSS for at Reports and or published Irradiation and classical genetics can be irradiation and classical least one mosquito papers. applied for the construction of genetic approaches for species constructed. mosquito GSS. sex-separation in mosquitoes
Molecular-based Molecular approaches are applicable to 2. To explore Reports and or published GSS for at least one target mosquito species. molecular approaches for papers. mosquito species sex separation in mosquitoes constructed.
Development and improvement are Mechanical, Reports and or published 3. To explore possible. behavioural, mechanical, behavioural, papers. developmental and developmental and symbiont-based symbiont-based approaches for sex approaches for sex separation for at least separation in mosquitoes one mosquito species developed or improved.
Relevant scientists can be identified Agreements or contracts Geneticists pursuing and encouraged to join the CRP. 4. To continue a classical approach issued and signed. encouraging and attracted to the CRP. attracting participants to the CRP in the field of classical genetics
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Outcomes
1. Classical genetic Irradiation protocols Data collected and Facilities and resources available. approaches for sex and classical genetic feasibility analysis separation in mosquitoes approaches assessed determined
Data collected and 2. Molecular Methods for Facilities and resources available. approaches for sex molecular-based sex feasibility analysis separation in mosquitoes separation developed developed
3. Mechanical, Protocols developed Facilities and resources available. behavioural, Data collected and for improved sex developmental and feasibility analysis symbiont-based separation methods approaches for sex separation in mosquitoes improved and validated
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Outputs
1. Genetic markers Genetic markers Reports and or published Biological material and methods from different sources isolated or papers. available. including natural developed. populations and / or mutagen-based screens.
Chromosomal 2. Chromosomal Reports and or published Scientists working on classical genetics rearrangements rearrangements in the papers. and methods are available. selected. target mosquito species.
Reports and or published (Cyto)genetic methods, chromosome 3. (Cyto)genetic (Cyto)genetic markers and papers. maps and experts are in place. data and analyses in chromosomal support of sex separation. rearrangements described.
4. Morphological Morphological and Reports and or published Morphological and selectable markers and selectable markers. selectable markers papers. are identifiable. confirmed.
Classical GSS Reports and or published Suitable genetic markers, irradiation 5. A classical GSS. constructed and papers sources, chromosomal rearrangements characterized are available.
Classical GSS in 6. Lab and semi- Laboratory and semi-field testing is laboratory and semi- Evaluation data field evaluation of a feasible. classical GSS. field testing identified and published evaluated
7. Construction and characterization of Strains with landing Landing sites and performance Reports and or published transgenic strains with sites constructed and characteristics are available. papers site-specific landing sites performance and known performance characteristics characteristics. assessed
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8. Methods, Accurate genome Reports and or published Genome editing technologies, genomic protocols and procedures editing in target loci papers data are available and endogenous for using RNA-guided and species achieved repair mechanisms are functional. endonuclease-based
(‘genome editing’) technologies in mosquitoes.
9. Data on the structure, organization Sex determination Tools and omics data for the analysis of Reports and or published and patterns of gene pathways and sex complex sex determination pathways papers expression of the sex specific expression and mechanisms are available. chromosomes of in mosquitoes better mosquitoes. understood. Content of sex chromosomes studied.
10. Gene expression Regulatory elements Reports and or published regulatory elements that tested Regulatory elements can be identified papers confer sex-specificity. and tested.
11. Effector Effector molecules molecules that can confer Reports and or published tested Effector molecules are available and predictable phenotypes papers when expressed in can be tested.
mosquitoes.
Molecular-based 12. Strains of Reports and or published sexing strains Components for the application of genetically modified papers developed molecular-based technologies are mosquitoes created from available elements generated from outputs 8, 10 and 11 that have the potential for conditional genetic sexing.
13. Strains developed Reports, and or published Laboratory and semi-field testing of Molecular-based from output 12 assessed papers GSS is feasible. for performance GSS performance characteristics in lab characteristics 25
and/or semi-field evaluated in lab or conditions. semi-field scale.
Reports and or published Wolbachia infection influences 14. Data that allows Wolbachia influence papers developmental parameters in a sex- the use of Wolbachia on sex separation specific manner symbiosis for sex exploited separation.
Tools for characterization are available. 15. A set of Reports and or published Factors influencing mechanical (e.g. size papers dimorphism), nutritional the efficiency of sex separation tools (e.g, larval diet), characterized behavioural (e.g. phototaxis, toxicants) and developmental (e.g. protandry) tools for sex separation.
16. Novel sex Reports and or published A prototype can be made Novel sex separation separation approaches papers tools tested based on output 15.
17. Data on the Reports and or published Tools and methods from output 16 are efficacy and Efficacy and papers reproducibility of reproducibility of in place. mechanical, nutritional, tools assessed behavioural, and developmental tools for lab and/or semi-field testing.
18. Publication of Papers drafted and Journal issue with results in a peer reviewed Data for publication available. journal. submitted. published papers
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Activities
1.Form a network of Proposals evaluated Signed contracts and Suitable proposals submitted, funding research collaborators and 6 Research agreements. available and approval of Contracts and Contracts, 11 Agreements by CCRA-NA committee. Research
Agreements and 2 Technical Contracts awarded.
Contracts and Agreements signed by 2. Organise 1st RCM to st Participants’ activities 1 RCM held 2013. counterpart organisations. refine the logical and logical framework framework and plan the revised. overall activities of the
CRP.
3. Organise 2nd RCM to nd analyse progress in 2 RCM held 2015. Participants and RCM Progress satisfactory. delivering research Progress Reports. outputs and plan the next phase of the project.
4. Organise 3rd RCM to rd Participants and RCM 3 RCM held 2016. Progress satisfactory and mid-CRP analyse progress in Progress Reports. evaluation approved by CCRA-NA delivering the research committee. outputs and plan the final phase of the project.
5. Organise final RCM to Participants and RCM Final reports are submitted to the assess the success of the 4th RCM held 2018. Final Reports Agency. CRP in reaching its objectives and review the final publication.
6. Publish the results of Scientific publication. Consensus can be found on appropriate the CRP in a special issue international journal and acceptance by of an international journal obtained. journal.
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FINAL FAO/IAEA RESEARCH COORDINATION MEETING ON “Exploring genetic molecular, mechanical and behavioural methods of sex separation in mosquitoes” 19-23 February 2018 Ramada Plaza Hotel, Bangkok, Thailand AGENDA
MONDAY, 19 FEBRUARY 2018
08:45 – 09:00 Kostas Bourtzis: Welcome statement, administrative issues and goals of the meeting.
09:00 – 09:30 Danilo Carvalho, Jorge Torres, Antonios Augustinos, Panagiota Koskinioti, Gulizar Pillwax and Kostas Bourtzis: Development and characterization of Aedes strains for SIT- based applications
09:30 – 10:00 Hervé C. Bossin, Hereiti Petit, Michel A. Cheong Sang and Jérôme Marie: Successful, Wolbachia-driven suppression of the mosquito vector Ae. polynesiensis on the atoll of Tetiaroa, French Polynesia
10:00 – 10:30 Maurizio Calvitti, Riccardo Moretti and Elena Lampazzi: Managing unperfected sex separation in suppression methods for Aedes albopictus: Wolbachia based benefits and innovative strategies
10:30 – 11:00 COFFEE BREAK
11:00 – 11:30 Cyrille Lebon, Aude Benlali1, Célestine Atyame, Patrick Mavingui and Pablo Tortosa: Construction of a Genetic Sexing Strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito
11:30 – 12:00 Cyrille Ndo, Yacouba Poumachu, Danale Metitsi, Herman-Parfait Awono- Ambene, Timoléon Tchuinkam, Jeremie Lionnel Roger Gilles and Kostas Bourtzis: Isolation and characterization of heat-sensitive strain of Anopheles arabiensis using ethyl methanesulfonate
12:00 – 12:30 Givemore Munhenga, Thabo Mashatola, Lerato Malakoane, Leonard C Dandalo, Oliver R Wood, Leanne N Lobb, Basil D Brooke, Maria Kaiser and Lizette L Koekemoer: Development of sex separation tools to eliminate female Anopheles arabiensis during mass production
12:30 – 14:00 LUNCH
14:00 – 14:30 Romeo Bellini, Arianna Puggioli and Marco Carrieri: Exploiting protandry for Aedes albopictus sex separation
14:30 – 15:00 Gustavo Salvador, Carlos Tur Lahiguera, David Almenar Gil, Mario Zacarés González and Ignacio Plá Mora: Aedes female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer
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15:00 – 15:30 Nayana Gunathilaka, Tharaka Ranathunge, Lahiru Udayanga, Asha Wijegunawardena, Jeremie Roger Lionel Gilles and Wimaladharma Abeyewickreme: Use of mechanical and behavioural methods to eliminate female Aedes aegypti and Aedes albopictus: Potential use of a combined approach in the context of sex separation for Sterile Insect Technique and Incompatible Insect Technique
15:30 – 16:00 COFFEE BREAK
16:00 – 16:30 Gul Zamin Khan, Inamullah Khan, Alamzeb, Tahir Badshah and Muhammad Nisar: Exploring nutritional, behavioural and mechanical methods of sex separation in Aedes albopictus and Aedes aegypti species of mosquitoes
16:30 – 17:30 Open discussion on the presentations
18:00 IAEA Reception at Ramada Plaza Hotel
TUESDAY, 20 FEBRUARY 2018
09:00 – 09:30 F. Bernardini, Roya Elaine Haghighat-Khah, Roberto Galizi, Andrew Marc Hammond, Tony Nolan and Andrea Crisanti: Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes
09:30 – 10:00 Elzbieta Krzywinska and Jaroslaw Krzywinski: Promoters to drive expression of female embryonic-lethal constructs in the Anopheles gambiae complex mosquitoes
10:00 – 10:30 Sean Scott, Enzo Mameli, W. Robert Shaw, Andrea Smidler and Flaminia Catteruccia: The Plugin regulatory region mediates sexual transfer of proteins in the malaria vector Anopheles gambiae
10:30 – 11:00 COFFEE BREAK
11:00 – 11:30 Irina Haecker and Marc Schetelig: Site-specific landing site systems to improve transgenic mosquitoes for their use in SIT
11:30 – 12:00 Zhijian Jake Tu: Sex determination in mosquitoes: mechanism, evolution and application
12:00 – 12:30 Margareth Lara Capurro: Construction and characterization of genetic sexing strain (Aedes aegypti and Ae. albopictus)
12:30 – 13:00 Scolari F, Gomulski LM, Mariconti M, Manni M., Gabrieli P, Savini G, Malacrida AR, Gasperi G and Giuliano Gasperi: Sexing of the tiger, Aedes albopictus
13:00 – 14:00 LUNCH
14:00 – 14:30 Open discussion on the presentations
14:30 – 15:30 Special Issue on Parasites & Vectors
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Working Group Discussions
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WEDNESDAY, 21 FEBRUARY 2018
08:00 – 13:00 Field trip (TBC)
13:00 – 14:00 LUNCH
14:00 – 15:30 Working Group Discussions
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Working Group Discussions
THURSDAY, 22 FEBRUARY 2018
08:30 – 10:30 Working Group Discussions
10:30 – 11:00 COFFEE BREAK
11:00 – 12:30 Working Group Discussions
12:30 – 14:00 LUNCH
14:00 – 15:30 Drafting Report
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Drafting Report
FRIDAY, 23 FEBRUARY 2018
08:30 – 10:30 Reports of Working Groups and Revision of Logical Framework
10:30 – 11:00 COFFEE BREAK
11:00 – 12:30 Drafting of Final Report, and self –evaluation report
12:30 – 14:00 LUNCH
14:00 – 15:30 Drafting of Final Report, and self –evaluation report
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Presentation and approval of the Final Report - General discussion
17:30 End of the meeting
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ABSTRACTS
FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Successful, Wolbachia-driven suppression of the mosquito vector Ae. polynesiensis on the atoll of Tetiaroa, French Polynesia AUTHOR (S): Hervé C. BOSSIN, Hereiti PETIT, Michel A. CHEONG SANG, Jérôme MARIE ORGANIZATION: Medical Entomology Laboratory, Institut Louis Malardé, Papeete, Tahiti, French Polynesia SHORT SUMMARY OF PAPER Abstract: The rapid emergence and expansion of Zika (Musso & Gubler, 2016) and other arboviruses (e.g., dengue, chikungunya) in the last decade have become major public health issues. The situation is particularly critical in Latin America, in several U.S. and European overseas territories and in small island developing states (SIDS) in the Caribbean and the Pacific. Aedes aegypti and, to a lesser extent Ae. albopictus have been linked with most known arbovirus outbreaks but the ecology of transmission in the Pacific may prove uniquely diverse, as other species within the Stegomyia subgenus also play a role in transmission to humans. For example, the native species Ae. hensilli and Ae. polynesiensis have been implicated in Zika outbreaks in Yap (Duffy et al., 2009) and French Polynesia (Cao-Lormeau et al., 2014) respectively. Ae. polynesiensis is widely distributed across the Pacific (incl. French Polynesia, Wallis and Futuna, Fiji, Samoa, American Samoa, Tuvalu, Tokelau, Cook Islands, Kiribati, and Pitcairn) where it is the primary vector of lymphatic filariasis and of zoonotic diseases, such as the dog heartworm Dirofilaria immitis. Overall, no less than 12 potential dengue vectors have been identified in the Pacific Islands. With no vaccine currently available, nor targeted viral therapeutics, vector control plays a critical role in protecting individuals against infection by Zika and other existing (dengue, chikungunya) or future mosquito-borne diseases. Yet, control or elimination of Ae. aegypti, and especially its sister species, Ae. albopictus and Ae. polynesiensis remains difficult due to their day-biting activity as well as the heterogeneity and cryptic nature of the containers they use. New approaches are urgently needed. For Ae. polynesiensis, a significant suppression trial (AeLIMIN+) conducted by Institut Louis Malardé (ILM) was being implemented on an isolated islet (75 ha) of the atoll of Tetiaroa, French Polynesia. This integrated control approach relied for the most part on the release of Wolbachia incompatible males mosquitoes that seek, court and mate with wild females, thereby reducing their reproductive capacity. Entomological and environmental data were collected at the field site to characterize and monitor the dynamics of the Ae. polynesiensis mosquito before, during and after the IIT intervention. Initial control measures consisted mostly in the removal of domestic breeding containers and the deployment of gravid Aedes traps (GAT) in the built environment. Wolbachia male releases were then initiated for population suppression and possibly elimination. Ca. 55,000 incompatible males
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were released once weekly between Sept. 2015 and Sept. 2016. In total, over 3 million incompatible males were successfully produced, transferred and released at the Tetiaroa treatment site. The sustained, inundative releases resulted in the significant suppression of the targeted Aedes polynesiensis population as determined by adult trap data and ovitrap indices compared to adjacent no-release control islets. Wolbachia-induced sterility was detectable within a few weeks of treatment, and reduction of the adult female population became manifest after only 4 months of intervention. After 13 months of treatment the treated population had crashed almost completely. Lasting benefits were observed post-treatment with little to no biting nuisance felt in the area for over a year. Signs of population recovery were only recently observed, which encourage more regular Wolbachia male releases to prolong suppression. Taken together, these results further support the evaluation of novel genetic control strategies in larger islands of French Polynesia, which provide an ideal context for studying ecological questions (Cressey, 2016, Davies et al. 2016).
In the absence of a genetic sexing strain for Ae. polynesiensis, male/female sorting relied upon both developmental (protandry) and mechanical sex separation techniques. QC activities (rearing/sorting parameters, male mating competitiveness, lab and field Wolbachia molecular monitoring) were undertaken throughout the project. To assess the level of sex separation and minimize the risk of accidental female release, over 1 500 pupae were randomly sampled each week following mechanical separation and their sex was verified individually under the microscope. Under ILM standard laboratory rearing conditions, the sexing accuracy averaged 99,99% male purity (0.01% ± 0.0003 females, mean ± SD) with rare female contamination events detected during intervention. While these limited female releases did not result in population replacement, they highlight the safety benefit that combined SIT-IIT (irradiation of male batches at doses that specifically sterilize contaminating females) can bring (Bourtzis et al., 2014). These results also support the critical need for developing more efficient sexing systems for Ae. polynesiensis and other (Aedes) mosquito vectors.
Cited references 1. Bourtzis K, Dobson SL, Xi Z, Rasgon JL, Calvitti M, Moreira LA, Bossin HC, Moretti R, Baton LA, Hughes GL, Mavingui P, Gilles JR (2014) Harnessing mosquito-Wolbachia symbiosis for vector and disease control. Acta Trop 132S: 150S–163S. doi: 10.1016/j.actatropica.2013.11.004 2. Cao-Lormeau, V. M., C. Roche, A. Teissier, E. Robin, A. L. Berry, H. P. Mallet, A. A. Sall, and D. Musso. 2014. Zika virus, French polynesia, South Pacific, 2013. Emerg Infect Dis 20: 1085- 1086. 3. Cressey D. Tropical paradise inspires virtual ecology lab. Nature 2015; 517(7534): 255-6. 4. Davies, N., D. Field, D. Gavaghan, S. J. Holbrook, S. Planes, M. Troyer, M. Bonsall, J. Claudet, G. Roderick, R. J. Schmitt, L. A. Zettler, V. Berteaux, H. C. Bossin, C. Cabasse, A. Collin, J. Deck, T. Dell, J. Dunne, R. Gates, M. Harfoot, J. L. Hench, M. Hopuare, P. Kirch, G. Kotoulas, A. Kosenkov, A. Kusenko, J. J. Leichter, H. Lenihan, A. Magoulas, N. Martinez, C. Meyer, B. Stoll, B. Swalla, D. M. Tartakovsky, H. T. Murphy, S. Turyshev, F. Valdvinos, R. Williams, and S. Wood. 2016. Simulating social-ecological systems: the Island Digital Ecosystem Avatars (IDEA) consortium. GigaScience 5: 1-4. 5. Duffy, M. R., T. H. Chen, W. T. Hancock, A. M. Powers, J. L. Kool, R. S. Lanciotti, M. Pretrick, M. Marfel, S. Holzbauer, C. Dubray, L. Guillaumot, A. Griggs, M. Bel, A. J. Lambert, J. Laven, O. Kosoy, A. Panella, B. J. Biggerstaff, M. Fischer, and E. B. Hayes. 2009. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 360: 2536-2543. 6. Musso, D., and D. J. Gubler. 2016. Zika Virus. Clin Microbiol Rev 29: 487-524.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Managing unperfected sex separation in suppression methods for Aedes albopictus: Wolbachia based benefits and innovative strategies AUTHOR (S): Maurizio Calvitti, Riccardo Moretti, Elena Lampazzi ORGANIZATION: ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Biotechnology and Agroindustry Division SHORT SUMMARY OF PAPER Abstract: Within this CRP, my ENEA research group has been involved in two main research topics. Firstly, we have investigated the effect of foreign, artificially established, Wolbachia infections on Ae. albopictus larval development and pupation dynamics. Four different mosquito lines (with the same geographical background) were investigated with this purpose: APO100 (Wolbachia-cured Ae. albopictus); Rimini67 (wild type, adapted to mass rearing protocols); ARwP (line with artificially replaced Wolbachia infection).
Basically, we found that the wPip Wolbachia strain, in particular, significantly accelerates the pupal development of Ae. albopictus. In order to exploit this effect, we have also observed that collecting the pupae at 22 hours from the pupation onset, instead of 24 hours, we are able to maintain high level of pupal productivity (about 35% of the initial number of larvae) with minimum percentages female contamination after mechanical separation of pupae (~0,5%). APO100 showed to suffer significant fitness costs, as confirmed by an increased pupation time and a reduced immature survival.
Concurrently, we set up laboratory-scale IIT experiments (we analysed 5 discrete generations) to evaluate the effect of incompatible male releases without female contamination (IIT-P) or with a 1- 2% of female contamination (IIT-F) at a 3:1 incompatible: fertile males ratio (one per generation). Results were evaluated by monitoring the dynamics of the mean number of biting females, per cage, over 3 generations subjected to the releases and three subsequent generations since releases were stopped. On average, despite a high variability of data, a stronger short term suppression action was observed in replicates in which we applied IIT-P. However, as the male releases were stopped, the residual wild-type population immediately started to grow fast. Where IIT-F with 1% of females had been applied, suppression was equally strong, though, on average, about 5% less effective compared to IIT-P). However, in this last case, a significant long lasting effect of reduction of the population growth rate was observed, due to the coexistence of two bidirectionally incompatible Wolbachia infections. (wPip frequency up to 30%). This slowing effect can make the mosquito population more susceptible to the action of other control strategies, included a conventional SIT.
The outcome of this study supports that, in the context of a “suppression program” aiming at maintaining the vector population below the density required to sustain transmission of human
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pathogens, it is worth investigating further the feasibility of applying of the “cytoplasmic incompatibility management (CIM) strategy (Dobson et al., 2002) [1].
Of course, infection type frequencies need to be monitored [2] during the subsequent generations. In this way, we could gradually adjust the ratio of incompatible males to be released to save costs and artificially sustain the coexistence of two incompatible Wolbachia infections. However, we look at this strategy with great interest to the extent that the mosquito strain we are using for male releases would be proved to be a significantly attenuated human virus vector.
[1] Dobson et al., 2002- Proc. R. Soc. Lond. B (2002) 269, 437–445 437 - 2002 The Royal Society DOI 10.1098/rspb.2001.1876
[2] Khoshmanesh et al., 2017 DOI: 10.1021/acs.analchem.6b04827 Anal. Chem. 2017, 89, 5285−5293
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Construction of a Genetic Sexing Strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito AUTHOR (S): Cyrille Lebon1*, Aude Benlali1,2*, Célestine Atyame3, Patrick Mavingui2,3,4 and Pablo Tortosa2,3 ORGANIZATION: 1Groupement d’Intérêt Public Cyclotron Océan Indien (CYROI), 2 rue maxime Rivière, 97490 Ste Clotilde, France. 2Symbiosis Technologies for Insect Control (SymbioTIC). Plateforme de Recherche Cyroi, 2 rue Maxime Rivière, 97490 Ste Clotilde, France. 3Université de La Réunion, Unité Mixte de Recherche Processus Infectieux en Milieu Insulaire Tropical (UMR PIMIT). CNRS 9192, INSERM 1187, IRD 249. Plateforme de recherche CYROI, Sainte Clotilde, Ile de La Réunion, France. 4Université de Lyon, Lyon, France; Université Lyon 1, CNRS 5557, INRA 1418, Villeurbanne, France.
SHORT SUMMARY OF PAPER Abstract: Background Aedes albopictus is an invasive mosquito species of global medical concern as its distribution has recently expanded to Africa, the Americas and Europe. In the absence of prophylaxis protecting human populations from emerging arboviruses transmitted by this mosquito species, the most straightforward control measures rely on the suppression or manipulation of vector natural populations. A number of environmental-friendly methods using innundative releases of sterilizing males are currently under development. However, these strategies are still lacking an efficient sexing method required for mass production of males. Results We present the first Genetic Sexing Strain (GSS) in Ae. albopictus, hereafter referred as TiCoq, obtained by sex linkage of rdl gene conferring dieldrin resistance. Hatching rate, larval survival and sex ratio were followed during twelve generations. The use of dieldrin at third larval stage allowed selecting 98% of males on average. Conclusion A good production rate of TiCoq males makes this GSS suitable for any control method based on mass production of Ae. albopictus sterilizing males. Despite limitations resulting from affected egg hatch as well as the nature of the used insecticide, the construction of this GSS paves the way for industrial sex separation of Ae. albopictus.
Keywords: Aedes albopictus, Genetic Sexing Strain, sex separation, Sterile Insect Technique, Incompatible Insect Technique. 35
FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Isolation and characterization of heat-sensitive strain of Anopheles arabiensis using Ethyl methanesulfonate
AUTHOR (S): Cyrille NDO, Yacouba POUMACHU, Danale Metitsi, Herman-Parfait AWONO-
AMBENE, Timoléon TCHUINKAM, Jeremie Lionnel Roger GILLES and Kostas BOURTZIS ORGANIZATION: OCEAC
SHORT SUMMARY OF PAPER Abstract: Background: Malaria is still a global health problem and vector control is the cornerstone of disease control strategies through the use of indoor residual insecticide spraying (IRS) and insecticide-treated nets. The situation is becoming acute with widespread resistance to the limited arsenal of available insecticide classes. Therefore, new and innovative tools to reduce Plasmodium transmission are in dire need and this situation rised considerable interest in using SIT against human pest insects, particularly Anopheles malaria vectors. When considering a mosquito release programme, one of the first issues to be addressed is how to eliminate/separate the hematophagous vector females. In this paper we report the develoment of an A. arabiensis heat-sensitive strain which will further be used for the development of a Genetic sexing Strain (GSS). Methods: Anopheles arabiensis male mosquitoes originated from North Cameroon were treated with 0.05% ethyl methanesulfonate. The mutagen was added to 10% sucrose solution and mosquitoes were allowed to feed ad-libitum for 24h and 48h on a cotton wool soaked with sucrose-mutagen solution placed at the top of the cup. Treated males were then allowed to mate with wild virgin females and their progenies were screened for heat sensitivity from F3 to F8 until isolation of a heat-sensitive strain which was further characterized by assessing it productivity (fecundity and fertility), larval development, adult longevity as well as nature and inheritance pattern of the heat sensitive lethal. Results and conclusions: Observations showed that the number of eggs laid and their hatch rate were similar between females mated with mutagenized males (range: 25 to 153; Mean: 72.22; Median: 69) and those from the control (range: 30 to 162; Mean: 73.23; Median: 59.5), suggesting that mutagenesis didn't affect A. arabiensis male fecundity and fertility. At F3, fourteen (15.91%) isofamilies out of 88 tested which showed mortalities ranged between 50 and 80% were selected as possibly containing a hsl. From F4 to F8, a heat-sensitive strain was isolated by screening L1 larvae at 41°C for 3 hours. This strain showed almost comparable life traits compared to the control in term of eggs fertility, larval development time and adults emergence. Crossing experiments to further assess the nature and inheritance pattern of the hsl isolated are ongoing and preliminary data suggest that it is recessive and would be located on an autosome. Keywords: Anopheles arabiensis, heat-sensitive lethal, Ethyl methylsulfonate
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FINAL RESEARCH COORDINATION MEETING ON “EXPLORING GENETIC, MOLECULAR, MECHANICAL AND BEHAVIOURAL METHODS OF SEX SEPARATION IN MOSQUITOES”
RAMADA PLAZA HOTEL, BANGKOK, THAILAND
19 - 23 FEBRUARY 2018
TITLE: Development of sex separation tools to eliminate female Anopheles arabiensis during mass production AUTHOR (S): Givemore Munhenga1,2, Thabo Mashatola1, Lerato Malakoane2, Leonard C Dandalo1, Oliver R Wood1, Leanne N Lobb1, Basil D Brooke1,2, Maria Kaiser1and Lizette L Koekemoer1. ORGANIZATION: 1Wits Research Institute for Malaria, MRC Collaborating Centre for Multi- Disciplinary Research on Malaria, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. 2 Centre for Emerging Zoonotic & Parasitic Diseases, National Institute for Communicable Diseases, Johannesburg, Sandringham 2131, South Africa.
SHORT SUMMARY OF PAPER Abstract: Over the past six years South Africa has been optimising various technical aspects of the sterile insect technique (SIT) for control of the malaria vector Anopheles arabiensis. One of the critical steps needed for applying a mosquito SIT is having an efficient sex separating system to eliminate female mosquitoes from the production line before irradiation and field releases. Female mosquitoes cannot be released, because of their capacity as disease vectors. Additionally, male only releases increase SIT programme efficiency because released sterile males will only focus on mating with wild females. Our group is exploring the use of both classical genetics and exclusive blood feeding behaviour of females in anophelines to accomplish female elimination in a laboratory An. arabiensis strain targeted for use during a planned SIT pilot release in northern KwaZulu-Natal Province, South Africa.
This work describes the successful development of an An. arabiensis genetic sexing strain (GSS) utilising dieldrin resistance as a selectable marker while containing a South African genetic background. Results on both the reproductive and physiological fitness as well as strain stability will be presented. Data on the effectiveness/efficacy of other environmentally-acceptable insecticides targeting GABA receptors in eliminating females from the newly developed strain will be discussed. In view of the challenges being faced in using the newly developed sexing strain, results on investigations of alternative sex-sorting systems based on (i) blood feeding behaviour of adult females, and (ii) sex linkage of a selectable temperature sensitivity gene in which temperature conditions favour the production of males only will also be presented.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Exploiting protandry for Aedes albopictus sex separation AUTHOR (S): Bellini Romeo, Puggioli Arianna, Carrieri Marco ORGANIZATION: Centro Agricoltura Ambiente “G. Nicoli”, Crevalcore, Italy
SHORT SUMMARY OF PAPER Abstract: Our efforts have been focused on the possibility to increase the Aedes albopictus male separation in mass rearing by enhancing the natural protandry. We operated by continuous selection of a strain obtained from crossing the early pupating males with the latter pupating females. In our standard procedure, Ae. albopictus egg hatching is conducted with a nutrient broth culture overnight, for around 14-16 hours, which cause asynchronous hatching and may therefore shadow protandry.
We worked on the egg hatching procedures aiming to increase synchronicity in larval development by testing 1-2-3-4 hours permanence of the eggs in the hatching container, instead of the usual 14- 16 hours. First evidence showed the convenience to reduce the hatching time without significant reduction in the hatching rate.
The outcome of the continuous selection of an enhanced-protandry Ae. albopictus strain, performed on more coetaneous L1, has been evaluated by checking the following parameters: pupation dynamics, male pupae productivity, percentage of residual females after the male pupae sieving on the F10.
In cooperation with ENEA, we also continued the rearing under SOP of different lines of Ae. albopictus ARwP by evaluating the pupation time and the male production by sieving.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Aedes female removal based on pupal dimorphism using artificial vision algorithms and computer controlled laser beamer AUTHOR (S): Gustavo Salvador2, Carlos Tur Lahiguera1, David Almenar Gil1, Mario Zacarés González3, Ignacio Plá Mora1 ORGANIZATION: Centro de control biológico de Plagas (TRAGSA)1, Universidad CEU Cardenal Herrera2, Universidad Católica de Valencia San Vicente Mártir3 SHORT SUMMARY OF PAPER Abstract: The purpose of this work is to develop mechanical tools for sexing Aedes sp pupae based in sexual dimorphism. This subject is included in the frame of the Co-ordinated Research Programme on “Explore mechanical, molecular, behavioral or genetic methods of sex separation in mosquitoes”. The proposed method is based on identification of both sexes by artificial vision algorithms due to the existing pupae sexual dimorphism in size. Previous data analysis have shown substantial difference in size between male and female pupae when analysis is performed within the same cohort. This enables an artificial vision system to separate up to 90 percent of males with a negligible risk of female presence. First, pupae are identified and measured using specific developed software. Then, the frequency curve of pupae size is obtained, typically showing two differentiated peaks. Each peak represents the most frequent size for each sex. The frequency curve is fitted to a bimodal model using an Expectation-Magnification algorithm. This model allows the estimation of the specific size threshold needed to assure a certain level of purity in the final male sample. Both the frequency curve and the threshold are updated in real time with any new pupae analysis, which allows the continuous analysis of large amounts of pupae in a very short time. Once the threshold size has been calculated, the female centroid coordinates are obtained and a laser beam is directed using a system driven by a dual axis galvanometer optical scanner. The laser beam will kill all the pupae bigger than the threshold (mostly female) and mainly male pupae will survive. During the last 18 months great advances have been made in the image analysis development. The system is now available to identify and follow the pupae in different pictures to improve the calculation of size. First tests have been done with Aedes albopictus in Valencia, and Aedes aegypti and Ae. polynesiensis in IPCL-NAFA to validate the statistical prediction model. Using pupae until 24h after the first pupation and based on the prediction model 95% of males can be recovered with 99.80% sex purity in all tested species. Male percentage recovery decreases to reach the same sex purity after 24 h from the first pupation. Finally, a system for administration and removal of pupae from the device has been also improved, and an industrial version of the prototype is being built and it will be tested in the pilot field trial in Valencia during next months.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Use of mechanical and behavioural methods to eliminate female Aedes aegypti and Aedes albopictus: Potential use of a combined approach in the context of sex separation for Sterile Insect Technique and Incompatible Insect Technique. AUTHOR (S): Nayana Gunathilaka1*, Tharaka Ranathunge2, Lahiru Udayanga2, Asha Wijegunawardena2, Jeremie Roger Lionel Gilles4, Wimaladharma Abeyewickreme4 ORGANIZATION: 1Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 3Department of Parasitology, Faculty of Medicine, Sir John Kotelawala Defense University, Sri Lanka, 4Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna, Austria
SHORT SUMMARY OF PAPER Abstract: Introduction
Sex separation of different stages of mosquitoes are currently being attempted to ensure successful release of male mosquitoes in novel vector control approaches. Among them, mechanical and behavioural methods have been tried mostly.
Methods
Batches of (n= 300) Aedes aegypti and Aedes albopicus pupae were used for mechanical separation methods namely; standard sieving method (1.12 mm, 1.25 mm, 1.40 mm and 1.60 mm mesh sizes) and Fay and Morlan glass plate separator. The experiments were repeated five times. Male and female separation by each method was calculated. In behavioural separation, a blood meal (cattle origin) was spiked with different concentrations (2, 4, 6, 8 and 10 ppm) of Ivermectin (Ivotec, 1% w/v) and Spinosad (Spinosyn, 12% w/v). Spiked blood meal of each concentration was exposed to a batch (n= 300) of adult Ae. aegypti and Ae. albopictus (1:1 sex ratio). An additional blood meal with the initially fed concentrations of Ivermectin and Spinosad was provided after 24 hours, followed by a 48-hour observation of mortality. This experiment was repeated with blood containing a mixture of Ivermectin and Spinosad in 1:1 ratio from each concentration.
Results:
The percentage of males and females separated at different pore sizes differed significantly (p <0.05). Majority of the male pupae were separated at 1.12 mm pore size in both Ae. aegypti (73%, n= 438) and Ae. albopictus (69%, n= 483) while, females were separated mainly by the pore size of 1.25 mm. In Fay and Morlan glass plate separation, 99.0% (n= 643) of the Ae. aegypti and 99.2% 40
(n= 714) Ae. albopictus male pupae were separated in the lower band. Feeding rate of both Ae. aegypti and Ae. albopictus decreased gradually with the increasing concentrations of Ivermectin and Spinosad. The maximum feeding rate of the females of both species were observed after providing two blood meals at initial and followed by 24 hours’ duration. The 100% Female mortality (n= 750) for Ivermectin was observed for both species at 8 ppm as the minimum concentration within 48 hours. It is suggestive that the use of spiking blood meal approach as the second screening for sexes after applying mechanical glass plate method may provide 100% separation of sexes, since the females selected by the mechanical processes, being the smallest of the sample, may be effectively eliminated through a spiking blood meal.
Keywords: Aedes, Sex separation, Ivermectin, Spinosad, Fay and Morlan.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Exploring Nutritional, Behavioural and Mechanical methods of sex separation in Aedes albopictus and Aedes aegypti species of Mosquitoes. AUTHOR (S): Gul Zamin Khan, Inamullah Khan, Alamzeb, Tahir Badshah and Muhammad Nisar ORGANIZATION: Nuclear Institute for Food and Agriculture (NIFA), Peshawar, Pakistan
SHORT SUMMARY OF PAPER Abstract: Rationale: Aedes albopictus and Aedes aegypti are the key vectors responsible for the deadly dengue disease in Pakistan and need environment friendly vector control strategy as no vaccine for the disease is available globally. Reliance mainly on the use of insecticides for vector control in Pakistan has caused health hazards, entomological problems and environmental constraints thus development of an environment friendly vector control strategies, such as Sterile Insect Technique (SIT) become indispensable for integration with available conventional control methods.
Exploring nutritional means for sexual dimorphism and subsequent mechanical sex separation in Aedes species: In SIT practice, only males are required for releasing into the environment, consequently, sex separation at pupal stage is a vital element in launching successful and risk free SIT program. Effect of different locally available suitable food ingredients were tested at the larval stage of Ae. albopictus and Ae. aegypti for observing the desired sexual dimorphism that may favour subsequent mechanical sex separation with precision and accuracy. Additional investigations like; behavioural response to different stimuli (light, temperature etc.) and exploitation of natural protandory were also conducted for working out successful sex separation. Larvae of Ae. albopictus and Ae. aegypti were reared on different diets and concentration to determine their effect on male/female sexual dimorphism. After the observance of sexual dimorphism, the pupae were subjected to subsequent mechanical sex separation through different seives of mesh sizes as 1250µm, 1400µm and 1600µm in replicated trials. Results revealed that among the various tested diets, Stevian diet produced significantly higher sexual dimorphisim with female having cephalothrax size of (1300µm with average range of >1250µm) than in male pupae having significalty less size with an average range of (800µm-900µm) for the Ae. albopictus. This size difference was quite enough to increase accuracy of the ultimate mechanical sex seperation. Seimilarly, in case of Ae. aegypti, the female and male size was in range of 1285 µm and 1000 µm respectively. Among the tested sieves, the mesh size of 1250µm separated both the sexes efficiently with 100% precision for Ae. albopictus. While in case of Ae. aegypti, the sex seperation was 99.5% at 10 minutes standard time provision.
Stevian larval diet dose/larvae/day was calculated as 400mg. The dose was standardized after repeated trials. Biological parameters; larval and pupal periods was also compared to standard larval diet of IAEA. The standard IAEA diet was best in producing the shorter larval and pupal periods.
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However, these parameters when recorded in Stevian diet (6, 2 days) was at par to IAEA diet (5.27, 2 days)
Behavioural means as helping tools in sex separation: The female pupae were found highly photosensitive and found a trend to move in opposite direction to males. This tendency that favored the mechanical separation through different flush of lights were tested in a mixed culture of the male and females in the rearing containers. The pupae on the top and bottom were kept separately and the percent number of sexes (male/female) were recorded. Among the different light intensities tested, 100 watt of Inflorescent and 1000 watt of Incandescent light were found effective in separating the sexes with significantly high percent accuracy ratio. Among the low temperatures tested, 5-7 degree Celsius in combinations of saw dust was found effective for induction of sheathing in female pupae and thus increase in sexual dimorphism needed for mechanical separation.
Natural protandory i.e., the natural trend in the eclosion of male earlier than female was exploited in the production of strain that may have significant sexual dimophisim. For this purpose, male pupae were selected from the early pupal cohorts and were crossed with the female of the late cohorts and vice versa. Different tests was subjected for few generations and is in progress. Data on the production of male/female ratio and sexual dimorphisim were recorded. In these trials we found the possibilty of producing the 100% male pupae upto 6 hours continously with fifth filial (F5) generation. Data regarding the succeeding generations are being recorded. This natural protandory will be utilized for the sex seperation and development of desirable sex strain subsequently.
Conclusion: The successful sex separation of the key dengue vectors (Ae. albopictus and Ae. aegypti) at pupal stage via nutritional, behavioral and mechanical methods had offered opportunities in launching of SIT of Aedes species of mosquitoes in Pakistan and elsewhere. Further studies are being accomplished by integrating the multi-aspects mentioned above for the sexual dimorphism and subsequent sex separation on mass scale of Ae. albopictus and Ae. aegypti SIT program.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes. AUTHOR (S): Federica Bernardini, Roya Elaine Haghighat-Khah, Roberto Galizi, Andrew Marc Hammond, Tony Nolan, Andrea Crisanti. ORGANIZATION: Imperial College London
SHORT SUMMARY OF PAPER Abstract:
Malaria is a serious global health burden, affecting more than 200 million people each year in over 90 countries, predominantly in Africa, Asia and the Americas. Since the year 2000, a concerted effort to combat malaria has reduced its incidence by more than 40%, primarily due to the use of insecticide-treated bednets, artemisinin-based combination therapies and indoor residual spraying. Nevertheless, the cost of control is expected to nearly triple over the next decade and the current downward trend in disease transmission is threatened by the rise of resistance to drugs and insecticides. Novel strategies that are sustainable and cost-effective are needed to help usher in an era of malaria elimination. The most effective strategies thus far have focussed on control of the mosquito vector. The sterile insect technique (SIT) is a potentially powerful strategy that aims to suppress mosquito populations through the unproductive mating of wild female mosquitoes with sterile males that are released en masse. Unfortunately, the technique and its derivatives are currently not appropriate for malaria control because of the inability to effectively sterilise males without compromising their ability to mate, and because males cannot be easily separated from females that could contribute to disease transmission. Advances in genome sequencing technologies and the development of transgenic techniques provide the tools necessary to produce mosquito sexing strains, which promise to improve current malaria-control programs and pave the way for new ones. In this review, the progress made in the development of transgenic sexing strains for the control of Anopheles gambiae, a major vector of human malaria, is discussed.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Promoters to drive expression of female embryonic-lethal constructs in the Anopheles gambiae Complex mosquitoes. AUTHOR (S): Elzbieta Krzywinska, Jaroslaw Krzywinski ORGANIZATION: The Pirbright Institute, UK
SHORT SUMMARY OF PAPER Abstract: Transgenic sexing strains of pest insects, in which sex determination pathway genes are mis- expressed, may constitute a very powerful tool to produce purely male populations for genetic approaches to insect control. In an earlier work we have identified the primary sex determination gene Yob, which confers maleness in the Anopheles gambiae mosquitoes. Ectopic delivery and transient presence of Yob mRNA in embryos leads to female embryo lethality in An. gambiae, and its sister species, An arabiensis, due to improper activation of the dosage compensation mechanism. Also, previously, we created proof-of-concept transgenic An. gambiae strains with stable ectopic Yob expression under the control of vasa2 promoter to test the feasibility of Yob as a female-killing effector molecule. None of the strains had fully penetrant female-lethal phenotype; however, surviving females were strongly masculinized at the morphological and molecular level and did not feed on blood. Incomplete penetrance is likely attributable to a relatively weak and restricted activity of the vasa2 promoter. Therefore, we focused on the identification and isolation of other promoters that would drive the expression of Yob during the embryo stage. We have isolated promoters of five genes with either embryo-specific or ubiquitous constitutive expression. After validation of their activity through transient assays in embryos, the promoters were used for engineering of transgenic constructs, which in turn were used for embryo microinjection. The transgenic strains carrying these new constructs are currently being established. In addition to the above work, I will present progress on the search for components of the dosage compensation complex in An. gambiae.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: The Plugin regulatory region mediates sexual transfer of proteins in the malaria vector Anopheles gambiae AUTHOR (S): Sean Scott, Enzo Mameli, W. Robert Shaw, Andrea Smidler, Flaminia Catteruccia ORGANIZATION: Harvard T.H. Chan School of Public Health, Boston, USA
SHORT SUMMARY OF PAPER Abstract: Mass releases of mosquitoes for the suppression of vector populations via sterile insect technique (SIT) or incompatible insect technique (IIT) require the specific release of males, as releasing females would increase disease transmission. Efforts to separate male from female mosquitoes prior to release require the characterization of promoters for sex-specific gene expression that can be used to generate fluorescently-labelled transgenic mosquitoes, allowing reliable separation of the sexes. In a bid to identify sex-specific expression, we characterized a promoter for the Anopheles gambiae reproductive gene Plugin (AGAP009368). This gene is expressed exclusively in the male accessory glands (MAGs) and is not expressed in females. We used this promoter to drive a transgene fusing the red fluorescent protein TdTomato to the C-terminus of the Plugin coding region, and characterized its expression pattern. We observed expression of Plugin-TdTomato in the anterior compartment of the MAGs from pupal stages throughout the lifetime of male mosquitoes. Labelled Plugin-TdTomato was functional and incorporated into the mating plug, a gelatinous rod rich in proteins and lipids that is transferred to females during mating. Mating to these transgenic males induced normal oviposition behavior in mated females, and did not adversely affect female fertility or fecundity. The Plugin promoter could therefore be useful in sex separation strategies, the assessment of transgenic male competitiveness in mating swarms, and the development of novel control strategies involving the transfer of chemosterilizants to females.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Site-specific landing site systems to improve transgenic mosquitoes for their use in SIT programs
AUTHOR (S): Irina Haecker, Marc Schetelig ORGANIZATION: Justus-Liebig University Giessen, Germany
SHORT SUMMARY OF PAPER Abstract: Molecular genetic tools can be used to support the SIT, e.g. by creating sexing strains or conditionally lethal strains to be used in SIT-based pest control programs. Such GMOs can be used to improve existing programs, for example with respect to efficacy, or to develop new programs for additional species.
Genetic modification is often achieved by random genomic insertion of a transgene construct via transposons. This is challenging for GMO strain development due to genomic position effects that could suppress transgene expression or by insertional mutations that negatively affect host fitness and viability. Recombinase-mediated cassette exchange (RMCE) allows for site-specific integration of transgene constructs into a well-characterized integration site of so-called landing site lines. RMCE has been successfully established in Drosophila and in Caribfly, and was recently also achieved in Aedes aegypti by us and others using the phiC31 recombinase (phiC31-RMCE). Moreover, the so-called iRMCE was established in Ae. aegypti. While both methods allow the site- specific integration, iRMCE, in contrast to phiC31-RMCE is not a true cassette exchange. And both systems are not reversible, i.e. the landing site can only be modified once. In contrast, Cre- and Flp- recombinase systems allow for the repeated modification of the landing site due to their reversibility. We therefore wanted to establish the reversible Cre-RMCE system in Ae. aegypti. Moreover, our goal was to improve the efficiency of phiC31-RMCE. As previously reported, we first created several landing site lines for Cre-RMCE and phiC31-RMCE.
Cre-RMCE in Aedes aegypti was successfully established in a two-step protocol, in which the first step comprises the integration of the whole donor plasmid at the genomic landing site via recombination of one of the two recombination sites flanking the landing site cassette. The reaction is completed by the excision of the plasmid backbone in the 2nd step, thus resulting in a completed cassette exchange. We also performed extensive fitness tests with the Cre-RMCE landing site lines to evaluate the fitness costs due to transgene insertion at different genomic positions. We included one of the integration lines (1st step product) to evaluate the effect of integration of additional exogenous sequence. Fitness costs were not uniform for all tested life traits within a line. One line, AH0212-F1, showed greater overall fitness across all parameters and is therefore recommended as 47
landing site line for downstream Cre-RMCE modifications. Moreover, the fitness tests showed that 1) genomic locations annotated as intergenic can still have negative impacts on line fitness when interrupted by transgene insertion; and that 2) the chosen experimental conditions can have a strong influence on the fitness performance of the analysed lines.
In a proof of principle experiment, we obtained one-step phiC31-RMCE events in Aedes aegypti. In addition, we also obtained several pools of transformants with RMCE intermediate products, which occurred by recombination of only one of the two attP sites flanking the landing site cassette, thereby integrating the whole donor plasmid like in Cre-RMCE. Using optimized reaction conditions in this proof-of-principle and successive experiments, we regularly achieved a strongly increased efficiency in cassette exchange compared to published phiC31-RMCE data from Drosophila, Bombyx mori and Aedes aegypti. Landings site lines are currently being characterized with regard to their integration number and integration site.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Sex Determination in Mosquitoes: Mechanism, Evolution and Application AUTHOR (S): Zhijian Jake Tu ORGANIZATION: Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, USA
SHORT SUMMARY OF PAPER Abstract: Sex is critical to the survival and evolution of many organisms including mosquitoes. Only female mosquitoes feed on blood and transmit disease-causing parasites or viruses while the non-biting males are harmless in this context. Genetic evidence suggests that a dominant male-determining factor (M factor) from the male-specific sex-determining chromosome provides the primary signal that initiates male development and controls sex determination in mosquitoes. Mosquitoes that lack the M factor develop into the default female sex. We have recently discovered a male determining factor Nix in Aedes aegypti and a strong M factor candidate Guy1 in Anopheles stephensi mosquitoes. In this presentation, I will discuss our recent studies on the functions of these genes and compare and contrast the outcome of ectopic expression of Nix and Guy1 in Aedes and Anopheles mosquitoes in the context of the need for dosage compensation. I will present our recent survey of the distribution and turnover of M-locus and Y-chromosome genes among divergent mosquito species. I will also discuss the potential applications of using these “maleness” genes to control mosquito-borne infectious diseases such as malaria, dengue, and Zika.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Construction and characterization of genetic sexing strain (Aedes aegypti and Ae. albopictus) AUTHOR (S): Margareth Lara Capurro Guimarães ORGANIZATION: Associate Professor, São Paulo University
SHORT SUMMARY OF PAPER Abstract
Mosquito-borne diseases as dengue, zika, and chikungunya have achieved the world attention due to the high number of cases and deaths reported annually. Given the deficit of effective vaccines for these diseases, our best defense has been the mosquito control. Sterile or disease-refractory mosquitoes are promising weapons to reduce the population densities or replace the wild population respectively. Both approaches involve mass production and the constant release of large numbers of mosquitoes in a given target area. In mosquitoes, the male-only release is an essential prerequisite since females are blood feeders and transmit disease. When considering a male mosquito mass release program, one of the first issues to decide is how to eliminate or separate females in the mass rearing facility. To achieve this goal, we exploited the natural RNA interference pathway in the mosquito to construct an effector gene expressing a double-stranded RNA using an inverted repeat of the exon 5b sequence from Aedes aegypti doublesex gene (Aeadsx) to induce post-transcriptional gene silencing. Moreover, our research group along with a partnership with Dr. Chun-Hong Chen from National Health Research Institutes (NHRI, Taiwan) designed a GSS construction using a CRISPR/Cas9 tool for genome editing. Our proposal is to disrupt Aeadsx by generating a mutant by the insertion of a visible fluorescent marker.
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FINAL RESEARCH COORDINATION MEETING On “Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes”
Ramada Plaza Hotel, Bangkok, Thailand
19 - 23 February 2018
TITLE OF WORKING PAPER: Sexing of the tiger, Aedes albopictus AUTHOR (S): Scolari F, Gomulski LM, Mariconti M, Manni M., Gabrieli P, Savini G, Malacrida AR, Gasperi G ORGANIZATION: Department of Biology & Biotechnology, University of Pavia, 27100 Pavia, Italy
SHORT SUMMARY OF PAPER Abstract: Using morphological, cytogenetics and molecular approaches, we have developed markers that can be used to sex Aedes albopictus at different developmental stages, from embryos to adults. We used the morphological dimorphism of the developing larval gonads to sex larvae and to derive mitotic chromosomes. These mitotic karyotypes display a sex-related banding pattern on chromosome 1. Both homologues of one arm of chromosome 1 bear a band in females, and only one homologue bears a band in males. FISH of the 18S ribosomal DNA indicated that the 18s locus is on the other arm of chromosome 1 compared to the sex-related band. FISH using a 883 bp Nix sequence, indicated that the Nix locus is also on the same arm of chromosome 1 as 18s but only in males and it is present on the homologue that does not carry the sex-related band.
We have characterized the Ae albopictus male-specific Nix locus at the level of the gene and in terms of its transcriptional activity in developmental stages. The gene has two exons separated by a short intron.
DNA and RNA were extracted from the same individual embryos at different time-points after deposition. Successful amplification of a Nix fragment from the DNA enabled us to identify male embryos. cDNA derived from male embryos was used to determine the transcriptional profile of Nix during development.
Given that Nix is present only in males and given its precocious transcriptional activity it may represent a putative male-determining factor (M) like its orthologue in Ae. aegypti.
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List of Participants BRAZIL
Ms DE LARA CAPURRO-GUIMARAES Margareth
Universidade de Sao Paulo Instituto de Ciencias Biomedicas Av. Prof. Lineu Prestes, 2415 Butantan 05508-000 SAO PAULO BRAZIL Email: [email protected]
CAMEROON
Mr NDO Cyrille
Organisation de Coordination pour la lutte contre les endémies en Afrique Centrale (OCEAC) 2.0 Rue du Centre Pasteur P.O. Box 288 YAOUNDÉ CAMEROON E-Mail: [email protected]
FRANCE Mr TORTOSA Pablo
Institut de recherche pour le développement (IRD) Representation de La Reunion, CS41095 Parc Technologique Universitaire 2 rue Joseph Wetzell 97495 SAINTE CLOTILDE CEDEX FRANCE Email: [email protected]
Mr BOSSIN Herve Christophe (French Polynesia)
Institut Louis Malarde(ILM) Laboratoire de recherche en entomologie médicale B.P. 30 98713 PAPEETE TAHITI FRENCH POLYNESIA Email: [email protected]
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GERMANY
Ms HAECKER Irina
Justus-Liebig-Universität Giessen (JLU) Angewandte Entomologie Winchesterstr. 2 35394 GIESSEN GERMANY Email: [email protected]
ITALY
Mr GASPERI Giuliano
Dip di Bio Animale Via Ferrata 1 Univ di Pavia 27100 PAVIA ITALY Email: [email protected]
Mr CALVITTI Maurizio
BIOTEC/AGRO Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA) Lungotevere Thaon di Revel, 76 00196 ROMA ITALY E-Mail: [email protected]
Mr BELLINI Romeo
Centro Agricoltura Ambiente G. Nicoli Via Argini Nord 3351 CREVALCORE ITALY Email: [email protected]
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PAKISTAN
Mr GUL ZAMIN Khan
Nuclear Institute for Food and Agriculture (NIFA) G.T. Road P.O. Box 446 25000 PESHAWAR PAKISTAN Email: [email protected]
SPAIN
Mr TUR LAHIGUERA Carlos
Empresa de Transformacion Agraria, SA (TRAGSA) Instituto Valenciano de Investigaciones Agrarias -IVIA Carretera Moncada a Naqueda Km 4,5 46113 VALENCIA SPAIN Email: [email protected]
SOUTH AFRICA
Mr MUNHENGA Givemore
National Health Laboratory Service Private Bag X4, Sandringham JOHANNESBURG 2131 SOUTH AFRICA Email: [email protected]
SRI LANKA
Mr ABEYEWICKREME Wimaladharma
University of Kelaniya, Faculty of Medicine Thalagolla Road P.O. Box 06 RAGAMA, GAMPAHA SRI LANKA Email: [email protected]
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UNITED KINGDOM
Ms BERNARDINI Federica
Imperial College London Crisanti Lab, 5th Floor Sir Alexander Fleming Building SW7 2AZ LONDON UNITED KINGDOM Email: [email protected]
Mr KRZYWINSKI Jaroslaw
The Pirbright Institute Ash Road, Pirbright GU24 0NF WOKING UNITED KINGDOM Email: [email protected]
UNITED STATES
Mr TU Zhijian
Virginia Tech 4044 Derring Hall - Mail Code 0120 24061 BLACKSBURG, VA UNITED STATES Email: [email protected]
Mr SHAW William Robert
Harvard School of Public Health 677 Huntington Avenue 02115 BOSTON, MA UNITED STATES Email: [email protected]
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Observers
FRANCE
Mr MAVINGUI Aime Patrick
Director of Research Unit PIMIT Processus Infectieux en Milieu Insulaire Tropical Infectious processes in tropical environments Reunion University, INSERM U1187, CNRS 9192, IRD 249, Plateforme Technologique CYROI - 2, rue Maxime Rivière 97490 Sainte-Clotilde, Île de La Réunion, France Email: [email protected]
GERMANY
Mr SCHETELIG Marc
Justus-Liebig-Universität Giessen (JLU) Institute for Insect Biotechnology Winchesterstrasse 2 35394 GIESSEN GERMANY Email: [email protected]
SINGAPORE
LU Deng
Radiation Protection & Nuclear Science Department, National Environment Agency (NEA) 40 Scotts Road, Environmental Building #03-00 228231 SINGAPORE SINGAPORE Tel:65 67119119 Email: [email protected] THAILAND
Ms KITTAYAPONG Pattamaporn
Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University 999 Phutthamonthon 4 Road, Salaya 73170 NAKHON PATHOM THAILAND Tel:0066 2 4410 227 Email: [email protected]
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Mr VICHASRI GRAMS Suksiri
Center of Excellence for Vectors and Vector-Borne Diseases 2nd Floor, Science Building 2, Faculty of Science Mahidol University at Salaya 999 Phutthamonthon 4 Road NAKHON PATHOM THAILAND Tel: Email: [email protected]
SPAIN
Mr HERRARZ Salvador Gustavo
University CEU Cardenal Herrera Escuela Superior de Enseñanzas Técnicas, C/.San bartolome, 55 46115, Alfara del Patriarca, Spain Email: [email protected]
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