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

FIRST RESEARCH CO-ORDINATION MEETING Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Co-ordinated Research Programme on Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-Based Technologies Scientific Secretary: Kostas Bourtzis IAEA Headquarters, room M6, Vienna International Center, Austria 6-10 July 2015 1

Summary... 3 Background Situation Analysis:... 3 Selected References... 18 Nuclear Component:... 26 Explanation / Justification... 26 Participation of Agency s laboratories... 26 Other Resources required... 26 Assumptions... 26 Related TC projects... 27 LOGICAL FRAMEWORK... 28 Narrative Summary... 28 Specific Objectives... 29 Outcomes... 30 Outputs... 30 FUTURE ACTIVITIES... 33 AGENDA... 46 PARTICIPANT ABSTRACTS... 51 LIST OF PARTICIPANTS... 75 2

Summary: The application of the Sterile Insect Technique (SIT) in area-wide integrated pest management (AW-IPM) programmes continues to increase in response to requests from Member States (MS). However, programme efficiency can still be considerably enhanced when certain components of the technology are improved, such as the strains used to massproduce sterile males, which are the key component of SIT programmes. They can be produced by classical and modern biotechnology approaches and strains producing such males are now available for key insect pests. The pests targeted for SIT applications include species of agricultural, veterinary and medical importance such as the Mexican fruit fly, the oriental fruit fly, the Mediterranean fruit fly, the codling moth, the pink bollworm, the new world screwworm, as well as disease transmitting mosquitoes. This CRP will focus on comparing the performance of strains developed or improved by classical genetic, transgenic and symbiont-based approaches to a level where a decision can be made as to their suitability to produce high-quality sterile males for use in large scale SIT programmes. Major beneficiaries will be operational AW-IPM programmes in MS that apply the SIT against these major insect pests. By the end of the CRP several strains, including strains for new target species, producing high quality sterile males will be available with the following tangible benefits for pest control programmes in MS using SIT: 1.) As only the males are needed for the SIT, the production, handling and release costs can be reduced significantly if sexing strains are used. 2.) The efficacy, sustainability and the cost of SIT programmes depends on the performance of released sterile males. The availability of genetically stable strains producing high quality sterile males will increase the efficiency and will decrease the cost of SIT programmes. 3.) A considerable proportion of the cost of SIT programmes is used for monitoring sterile insects in the field and therefore a stable, fail proof genetic marking system for the released males and mated females will reduce costs considerably. 4.) Male-only releases are several-fold more efficient than releases of both sexes and are mandatory for disease transmitting insect species such as mosquitoes. Consequently, when the genetic sexing technology is available, SIT programmes are significantly more efficient, safe and cost effective. 5.) As horizontal transfer phenomena are of major ecological concern, strains producing males by transgenic or symbiont-based approaches for SIT applications will be assessed. Background Situation Analysis: Pest insect species result in an enormous burden to human health, agriculture and veterinary activities in every continent of the world. Their control is of the utmost importance for the survival and well-being of humankind. The control of insect pests using the Sterile Insect Technique (SIT) has been a major objective of past and present CRPs. These activities have resulted in significant progress in the development of SIT technologies, but gaps in knowledge remain and refinement of current approaches is still needed. Moreover, the introduction of measures that make the SIT more effective and cost efficient would be highly desirable. The SIT has also been used to mitigate the problem of introduction and establishment of invasive species in the Americas, Europe, Africa, Australia and Asia where several dipteran 3

and lepidopteran species are considered a major problem. This is reflected by the many requests for support by Member States in the area of insect pest control for these two groups of insects. Operational use of SIT continues to reveal areas where new technologies are needed to improve efficiency, and thus lead to more cost effective programmes. These technologies need to be expanded to other insects of economic and medical importance. There are many options to increase the efficiency of the SIT, e.g. improved mass rearing, release technology, quality control, etc., even when operational programmes are already being implemented (http://nucleus.iaea.org/sites/naipc/dirsit/sitepages/all%20facilities.aspx). However, one critical area identified by programme managers, where important advances can be made concerns the improvement of strains that are being reared and released. One example of how strain improvement can significantly enhance SIT applicability and efficiency has been the development and the use of Genetic Sexing Strains (GSS) of the Mediterranean fruit fly, Ceratitis capitata, and the Mexican fruit fly, Anastrepha ludens in area-wide integrated pest management (AW-IPM) programmes. These technologies were mainly developed through the Agency s CRP programme with support from the FAO/IAEA Agriculture and Biotechnology Laboratory in Seibersdorf. There are currently SIT programmes being implemented for several important dipteran and lepidopteran species where the development of improved strains would lead to major increases in applicability and efficiency of the SIT approach. Innovative methods to control agricultural, veterinary and human pest related problems were developed during the CRP entitled: Development and evaluation of improved strains of insect pests for SIT. These methods for pest control include the development of several new GSS using biotechnologies (i.e. genetic manipulation). This new CRP builds on the knowledge gained from that CRP and the availability of newly developed strains to a next phase of comparative assessment and validation of the performance of sterile males produced by classical genetic, transgenic or symbiont-based approaches and their suitability for integration into control programs. In addition to the comparative evaluation, refinement and validation of available and newly developed strains should be assessed for the potential of horizontal transfer phenomena. Overall, SIT programs would benefit from the: 1. Comparative evaluation of the performance and genetic stability of sterile males produced by classical genetic, transgenic or symbiont-based technologies 2. Refinement of existing technologies for the development and field application of strains for the control of agricultural pests and disease vectors 3. Assessment of potential horizontal transfer phenomena resulting from the use of strains developed by transgenic or symbiont-based approaches for SIT applications. The major outcome of these activities will be the availability of strains producing high quality males allowing efficient implementation of SIT and other related control strategies in area-wide programmes against some of the major insect pest populations of economic and medical importance (Table 1). Table 1: List of some of the major insect pests and disease vectors Region Agricultural pests - Agricultural pests - Fruit flies Moths Veterinary and human Africa Bactrocera dorsalis B. zonata Ceratitis capitata Cydia pomonella Grapholita molesta Ectomyelois ceroniae Anopheles gambiae, A. arabiensis, Aedes aegypti 4

Americas Asia Australia and Oceania Europe C. rosa Glossina sp. Anastrepha ludens Diatraea saccharalis A. obliqua D. crambidoides A. grandis C. pomonella A. fraterculus G. molesta A. suspensa Pectinophora gossypiella A. striata Plutella xylostella A. serpentina Helicoverpa armigera B. carambolae B. oleae C. capitata Drosophila suzukii B. dorsalis B. carambolae B. correcta B. cucurbitae B. tryoni/b. aquilonis B. jarvisi C. capitata B. oleae C. capitata, D. suzukii C. pomonella H. armigera Spodoptera litura G. molesta P. xylostella E. ceroniae C. pomonella G. molesta C. pomonella G. molesta E. ceroniae Aedes aegypti, A. albopictus A. darlingi A. albimanus Cochliomyia hominivorax Culex quinquefasciatus A. stephensi A.sinensis A. aegypti A. albopictus C. pipiens C. tritaeniorynchous A. aegpyti A. albopictus Lucilia cuprina A. albopictus Phlebotomus perniciosus Classical Genetic Approaches The Mediterranean fruit fly, Ceratitis capitata, is a classic example of the sophisticated application of standard (non-transgenic) genetic manipulation for the development of GSSs and successful integration of these strains into operational programmes. For this species, a temperature-sensitive lethal based series of genetic sexing strains were developed by means of irradiation and classical genetic approaches. Several of these strains (Vienna-7 and Vienna-8) have been thoroughly evaluated and are currently being used in mass rearing facilities for large scale AW-IPM programmes that include an SIT component. In the Mexican fruit fly, Anastrepha ludens, a genetic sexing strain has been developed that is based on an autosomal black pupae (bp) colour mutation and a translocation Y-A based genetic sexing system, in which females homozygous for the recessive mutation have black pupae, while genetically heterozygous males have brown pupae due to the wild-type allele being translocated onto the Y chromosome. These characteristics allow the sex separation in the pupal stage using mechanical means, followed by male-only irradiation and release. The performance of this strain was tested under laboratory conditions, small-scale field-cage experiments and, recently, large-scale production has been initiated in Mexico. The performance of irradiated males in the field remains to be evaluated. Moreover, several new strains carrying recessive temperature-sensitive lethal mutations have been developed using classical genetic approaches, and these strains also need to be evaluated for their rearing properties and field performance. Y-chromosome-autosome translocations and recessive white colour mutations of the puparium have also been used to construct several GSSs in the oriental fruit fly (Bactrocera dorsalis), two related species, B. correcta and B. carambolae, as well as in B. cucurbitae. Two of the strains, Salaya1 in B. dorsalis and Salaya5 in B. carambolae, have a high reproductive capacity comparable to wild-type strains. In addition, males produced by these 5

strains showed satisfactory performance in small-scale field experiments. Characteristics of these two strains in large mass rearing and performance of released males in large-scale field experiments remain to be evaluated. Population genetic studies have been performed, comparing Salaya5 with B. carambolae populations across species range in South East Asia and Suriname. There has been an interest in developing GSSs using classical genetic approaches in pest moths (Lepidoptera). However, the sexing system developed in two model species, the Mediterranean flour moth (Ephestia kuehniella) and silkworm (Bombyx mori), which is based on balanced sex-linked recessive lethal mutations, was found to be only marginally applicable in mass rearing. GSSs amenable to mass rearing conditions, such as those constructed in the Mediterranean fruit fly, could not be developed in any lepidopteran species owing to their sex chromosome system being the WZ/ZZ system, in which females are the heterogametic sex. From sex determination studies to transgenic sexing strains in flies, butterflies and mosquitoes. Genetic studies of sex determining genes in Ceratitis capitata led to exploit female-specific splicing of Cctra gene to obtain transgenic sexing strains having either conditional femalespecific lethality or masculinization of XX individuals. Further improvements can be envisioned in the near future taking advantages of both novel genomic sequencing technologies and reverse genetics tools. Key sex determining genes composing primary signals have been isolated only in three insect species, including the dipteran Drosophila and Aedes, and the hymenopteran Apis mellifera. The Y-linked M factor in C. capitata, which represses maternal activation of Cctra needs to be identified. Like in C. capitata, genetic evidence suggests that an M factor, that resides either on the Y chromosome as in Anopheles mosquitoes or in the M-locus of a homomorphic sexdetermining chromosome as in Aedes mosquitoes, triggers male development in mosquitoes. Y chromosome and M-locus tend to be repeat-rich and thus difficult to assemble. As a result, Y- and M-linked genes are often absent from genome assemblies. A few methods have been developed to identify Y- or M-linked genes. One such method is called chromosomal quotient (CQ) and it was successfully used to identify Y chromosome genes in Anopheles mosquitoes as well as M-linked genes in Aedes aegypti. An M-locus gene Nix, encoding a splicing factor, was also discovered in A. aegypti using the CQ method. A new search for male determining genes in relevant pest insects which can be targeted by SIT, started recently by taking advantage of de novo sequencing technologies, cytogenetic micro dissection of sex chromosomes, sexed embryonal transcriptomes, availability of genome sequences from male and female sex of various species, and novel bioinformatic tools including chromosome quotient, edger statistical analysis for differentially expressed genes in the two sexes. In the insect order Lepidoptera, chromosome mechanism of sex determination is of the WZ type. However, the actual role of the W and Z chromosomes remains unknown except for the silkworm, Bombyx mori. However, after years of fruitless search for a W-linked proteincoding gene that could be the primary trigger of female development, only recently Kiuchi et al. (2014) made a surprising discovery that the feminizing factor in B. mori is a W-encoded small PIWI-interacting RNA named Fem pirna. The authors also showed that the Fem pirna downregulates the expression of a Z-linked gene, Masculinizer (Masc), which promotes male development at the absence of the W chromosome. In other words, the Fem pirna controls female-specific splicing of the B. mori doublesex (Bmdsx) gene by 6

downregulating expression of the Masc gene. However, it is not yet known (i) whether the Fem pirna-masc sex-determining pathway is conserved in other lepidopteran species with the W chromosome and (ii) whether Masc plays a role in species with a Z0/ZZ sex chromosome system that are thought to have the Z-counting mechanism of sex determination. Transgenic approaches to population control The ability to genetically manipulate many of the species subject to SIT now presents the possibility to create transgenic strains that will significantly enhance the efficiency and costeffectiveness of SIT. During the previous CRP on Development and evaluation of improved strains of insect pests for SIT, multiple new systems and strains were developed that allow marking to detect males released into the field and females that have mated to released males, conditional lethal systems that result in reproductive sterility, female-lethality and female-tomale sex reversal for male-only strains. Reproductive sterility systems. Sterility systems for fruit fly pests were created based on tetracycline (Tet)-suppressible conditional lethality. The first Tet-dependent transgenic method to improve SIT was the RIDL system (release of insects carrying a dominant lethal) that renders males genetically sterile in the absence of Tet (Ceratitis capitata). Although promising, a critical shortcoming for RIDL is that lethality depends on the Tet-transactivator (tta) accumulating to toxic levels during development resulting in late larval and pupal lethality. While useful for adult stage pests, this allows survival of larvae that are most damaging to crops when used for sterile-release. CRP members have improved upon this technology by developing a Tet-suppressible embryonic lethality system for both sexes (reproductive sterility). This system, tested in both C. capitata and Anastrepha suspensa, is based on a driver component that uses a promoter active during early embryogenesis to induce a lethal effector gene resulting in early embryonic lethality. The development of this system for other insect pests has also been improved by new methods to pre-evaluate newly isolated driver and effector components using a cell culture assay for cellular lethality, in addition to quantitative PCR. This will save considerable time and effort in the validation of these components and, importantly, their ability to function together previous to laborious germ-line transformation experiments. These cell culture assays have allowed not only the evaluation of promoters from embryonic genes, such as serendipity alpha, together with the endogenous pro-apoptotic genes hid, reaper, and grim, but also the determination of the most-efficient driver-effector cassette combinations for use in A. suspensa transformants, resulting in two hybrid strains exhibiting 100% lethality. In C. capitata two strains with 100% lethality were developed and evaluated at the IPCL in Seibersdorf with one strain showing good performance and character stability under semi-mass rearing conditions. The isolation and in vitro validation of species-specific promoters and lethal effector genes greatly improved the efficiency of creating highperformance conditional lethality strains, which can be extended to other insect pest species. Tetracycline-repressible female-lethal sexing strains. Another critical component for enhancing SIT has been the development of transgenic sexing strains (TSS) in fruit flies and livestock pests to eliminate the costs of mass-rearing females and to eliminate mating competition when sterile females must be released with males. Moreover, male-only releases are a prerequisite for mosquito-sit programmes, as released females will increase the risk of disease transmission. Unfortunately, a highly effective genetically-based (non-transgenic) technology to eliminate females as early embryos is only available for SIT programmes 7

targeting the Mediterranean fruit fly, and it will be costly and take many years to replicate this system for other insects. Thus, among the many pest species currently subject to SIT programmes, or in the planning stages, Anastrepha species in Central and South America and Bactrocera species in Asia, Europe, and Africa, could specifically benefit from the much more rapid implementation of TSS technology, especially those based on tetracyclinerepressible (Tet-off) female-specific embryonic lethality. Although these species are much less well studied than the species for which TSS has been developed, the application of the basic genetic components and methodologies should be straightforward. Recently, early embryonic Tet-off TSS have been developed for the tephritid fruit flies C. capitata and A. suspensa and the livestock-relevant species, L. cuprina, all resulting in 100% female lethality. All three species use similar endogenous components of their respective genomes to induce lethality through a well-understood pro-apoptotic cell death pathway. The transgenic sexing approaches are highly effective and cost-efficient by eliminating female insects early in embryogenesis. Female-specific Tet-off pupal sexing systems due to tta overexpression lethality (based on RIDL) have been also developed in C. capitata, the New World screwworm Cochliomyia hominivorax, the Australian sheep blowfly Lucilia cuprina, the diamondback moth (Plutella xylostella), pink bollworm, and in the silkworm. The C. hominivorax pupal TSS produce 100% males when reared on diet that lacks tetracycline and most are comparable to the current production strain in various fitness parameters that are important for production. Further, male aggression and male competitiveness of some of the strains are comparable to the production strain. These would be the first sexing strains available in the over 50 year history of the highly successful screwworm SIT program. However, such late sexing systems do not eliminate the costs for female larval rearing. Other TSSs have been successfully evaluated under semi/mass rearing and field cage conditions with support from the FAO/IAEA IPCL in Seibersdorf. These achievements will help to explore different options for TSS in other important agricultural and livestock pest insects (see Table 1). In mosquitoes, currently no GSSs are available that have the potential for use in SIT programs. However, promising research results and technologies have been reported recently that, with further research and development, could lead to the development of mosquito TSSs. A "flightless female" transgenic strain of A. aegypti exists, which carries a transgene that destroys the female flight muscles when raised without tetracycline added to the diet. However, this strain had poor fitness characteristics in large open field cage trials in Mexico. In A. gambiae a sex distortion approach was developed, which destroys X-bearing sperm and resulted in 95-97% male progeny, while in A. aegypti, double-stranded RNA against the female-specific variant of the doublesex gene was fed to larvae resulting in up to 97% adult males (by death of females). Moreover, the development of an early embryonic femalespecific lethality system like in Medfly and Caribfly should be possible, once endogenous candidate genes for the establishment of such a system are identified in mosquitoes. Temperature-sensitive conditional lethality. CRP members also developed a new dominant temperature-sensitive (DTS) conditional lethality system based on a heat-sensitive mutant allele of the D. melanogaster proteasome 20S subunit gene, Prosβ2 (first described as DTS7). The Prosβ2 cognate from A. suspensa was isolated and mutagenized in vitro to create the AsProsβ2 1 (AsDTS7) mutant allele, which was transformed into Caribfly. Transformants had normal viability at 25 C, but exhibited lethality rates of 96-100% in four lines at 29 C. While highly encouraging as a conditional lethal system, lethality was limited to the pupal stage making its use for larval pests inefficient, though use in adult pests, such as mosquito disease vectors, should be considered. The DTS7 system may also be used for redundant 8

lethality to ensure that any, albeit rare, survivors from the Tet-off embryonic lethality system are eliminated before propagating in the field to ensure ecological safety. Transgenic sexing based on sex reversion by sex-determination gene repression. An approach to generate a male-only population by sex reversion of females to males has been successfully tested in C. capitata, which could result in doubling the mass production of male-only progeny, and avoiding the need for female-specific lethality. A transgenic sexreversion line of C. capitata that shows 98% conversion of XX individuals into fertile males, with 2% intersexes was generated. In vivo RNAi against Cctra driven by a transgene can be very effective if the parental female deposits dsrna molecules into oocytes, as has been revealed by an RNAi maternal effect. When a parental female carrying one transgene copy (+/-) is crossed with a non-transgenic XY male (-/-), the Cctra-specific dsrna maternally deposited can act efficiently to switch off the Cctra gene in both transgenic (+/-) and nontransgenic embryos (-/-). Hence, 50% of male only progeny is composed of XX and XY individuals, which are non-transgenic. This is a first preliminary proof of principle for the possibility ofdeveloping insect transgenic technologies leading to non-transgenic male only progeny. Such strategiesmay be used for SIT in countries having restrictions on the use of GM insects in the wild. However, a rather complex design will be required to obtain a similar transgenic sexing system operational for a mass rearing. Another alternative would be to develop a transgenic sexing strain bearing a maternally masculinizing Cctra-IR transgene, homozygous in both sexes, under conditional Tet-off control. Genetic tools for genome manipulation Genomic targeting of transgene insertions. Transposable elements are widely used as vectors for integrating transgenes into the genome of insects. However, the random nature of transposon vector integrations often results in mutations and makes transgene expression subject to variable genomic position effects. This makes reliable quantitative comparisons of different transgenes difficult and development of highly fit transgenic strains laborious. Tools for site-specific transgene genomic targeting are essential for functional genomic comparisons and to develop the most advanced transgenic insect strains for applied use. Improved genomic targeting systems for non-drosophilid insects were tested as integration and recombinase-mediated cassette exchange (RMCE) systems based on phic31-attp/b and Cre/loxP, respectively. For C. capitata, A. suspensa, and A. ludens, the phic31-attp/b system was established and used for the stabilization of transgenes in the genome of C. capitata as well as the generation of target-site lines with high fitness in A. ludens. In addition, the system was proven to be functional in the mosquitos A. aegypti, A. gambiae, and A. stephensi. The Cre/loxP targeting system has been established in A. suspensa and allowed a comparison of the Drosophila constitutive polyubiquitin promoter and the artificial 3xP3 tissue-specific promoter in the same genomic context within each species, showing that the widely used 3xP3 promoter is apparently non-functional in the tephritid fly. Both systems will help to improve the safety, efficiency and variety of transgenic systems by allowing the functional comparison, combination and exchange of essential elements required for transgenic strain development. The transfer of RMCE site-specific integration systems to other pest insects should therefore be a high priority. RNAi for invertebrate pest control. In insects, as in other organisms, RNAi is a powerful tool for experimental studies aiming to determine gene function. This commonly involves the 9

microinjection of dsrna into the target organism, often directly into the target tissue. The dsrna is cut by endogenous Dicer proteins into a population of small interfering RNAs (sirnas), which in turn bind and degrade complementary mrna sequences. Careful dsrna design can ensure highly specific silencing in terms of both individual gene targets and species. In plants and some invertebrates (eg. C. elegans), the efficacy of RNAi is improved through a combination of signal amplification and systemic spread, such that the entry of one dsrna or sirna molecule into a single cell can lead to effective silencing of the target gene throughout the target organism. In some insects, RNAi appears to be cell-autonomous, with no amplification or cell to cell communication of the gene silencing signal.insect pest control methods are being developed through dsrna oral delivery. The efficacy varies depending on the insect species and genes. Some examples of delivery include paper soaked in dsrna for termites, plants coated with or expressing dsrna and bacteria expressing dsrna, fed with the insect diet. In the case of vectors that transmit diseases, triatomines can be fed live symbiotic bacteria that express constitutively dsrna, mosquito larvae can be soaked in dsrna solutions, fed chitosan-coated dsrna and fed live or dead bacteria previously induced to express dsrna delivered in food particles. The lack of a mechanism for amplification and systemic spread of a dsrna signal (in some insects) has implications for the development of RNAi as a control tool for insect pests. To achieve effective control, dsrna/sirna must be delivered to the appropriate tissue in the target pest at a sufficient dose to produce the necessary level of gene silencing to achieve the desired objective. There is considerable variation across insect species in their sensitivity to RNAi, and the evidence to date suggests that this is largely due to the relative acquisition, durability and transport efficiency of dsrna or sirna within insects. The effectiveness of RNAi could be improved by technologies that provide (1) more effective transport across the integument (cuticle or gut), (2) greater protection against degradation by UV and enzymes, and/or (3) active transport to the target tissues; in addition, there are continuous efforts to improve the effectiveness of transgenic-based RNAi applications. RNAi can be potentially used to achieve genetic sexing as part of SIT programs by targeting female-specific transcripts during the developmental stages of the generation to be released. This application of RNAi offers a greater level of control of delivery than other RNAi applications, but unlike other applications demands near 100% efficacy. Depending on the target organism, oral and/or topical delivery is possible. CRISPR/Cas9 gene-editing (Clustered Regularly Interspersed Short Palindromic Repeats. A wide variety of bacteria and archaea have a surprisingly complex adaptive immune system based on clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 nuclease (Cas9) genes. The bacterial type II CRISPR-Cas9 system was very recently adapted as a genome-engineering tool in many different organisms, including various insect species, and in vitro preparations, dramatically expanding the possibility to modify, at single nucleotide level, specific genes in the genomes. CRISPR/Cas9 genome editing in insects was first reported in B. mori and has since been successfully used to modify the genomes of D. melanogaster, A. aegypti, and Tribolium castaneum. The Cas9 knock-in homologous DNA repair strategy developed in Drosophila took advantage also of a loss of function mutation in the ligase 4, a gene required for DNA non-homologous repair. Homozygous lig4 flies showed a 5-7 fold increase of mutagenic Cas9 effect. The high precision and accuracy of gene editing technologies enables the creation and assembly of genotypes identical to those created and assembled using classical mutagenesis and genetic approaches but without necessarily requiring large genetic screens. This is a potential benefit of using genome editing technologies in the creation of genetic 10

sexing strains. Because the organisms produced using gene-editing technologies can be genetically similar to those produced using classical approaches, their transition from the laboratory to the field and adoption by end-users could follow current technology transfer strategies for non-gm organisms. It must be noted, however, that how organisms created with gene-editing technologies will be viewed by regulatory agencies is unclear, including whether insects produced using the specific mutagenesis tools of gene-editing will be considered equivalent to those produced using non-specific mutagens (chemicals and radiation). Symbiont-based approaches One aspect currently being explored is the potential role that insect microbiota may play in insect reproduction, physiology, fitness and their ability to transmit pathogens. For instance, it is now well established that Wolbachia, an intracellular bacterium that infects a large variety of insects, has the ability to induce reproductive abnormalities like cytoplasmic incompatibility (a kind of male sterility) as a strong inhibitory effect on the ability of mosquitoes to transmit human pathogenic viruses (e.g. dengue, chikungunya) and other important pathogens (e.g. Plasmodium sp.). Incompatible insect technique (IIT) is referred to as population suppression and entails the release of male mosquitoes infected with Wolbachia, resulting in sterile matings and a reduction in the insect population. Wolbachia transinfection (or transfer of Wolbachia between different insect host species through embryonic microinjection) to generate a stable novel Wolbachia infection in the target pest species is the first step in developing a Wolbachia-based IIT for the control of both agriculturally and medically important insect pests. In C. capitata and B. oleae, stable Wolbachia transinfections have been achieved, using the Wolbachia wcer2 and wcer4 strains of R. cerasi (Table 2). In C. capitata, the Vienna-8 strain has been transinfected and the potential of Wolbachia as an additional component in SIT is studied under laboratory conditions. Since Wolbachia was first introduced into the primary dengue vector A. aegypti in 2005, extensive efforts have been dedicated to developing Wolbachia as a novel genetic tool for controlling dengue, malaria, and the other vector-borne diseases, with a number of stable transinfected lines being available at present (Table 2). An integration of IIT with SIT is currently developing to enhance the effectiveness of population suppression for A. albopictus. The minimum irradiation dose for the sterilization of A. albopictus females escaped from sex separation has been established, without affecting the male mating performance. In addition, the presence of different strains of Wolbachia in laboratory strains and natural populations of the A. fraterculus species complex is currently being characterized and evaluated. Ongoing analysis points to the presence of different Wolbachia strains in this species complex. The characterization of the phenotype induced by Wolbachia in its host is also under study. Table 2: Stable transinfected medfly, olive fly and mosquito lines with the potential to be used for agricultural and medical IIT/SIT applications. Transinfected line Recipient embryos Wolbachia strain Donor embryos 88.6 C. capitata (Benakeio strain) wcer2 Rhagoletis cerasi S.10.3 C. capitata (Benakeio strain) wcer4 R. cerasi 56S2 C. capitata (Vienna 8 strain) wcer2 R. cerasi 11

B. oleae[wcer2] B. oleae (Democritus strain) wcer2 C. capitata (Vienna 8-E88) WB1 A. aegypti walbb A. albopictus (Hou strain) PGYP1 and 2 A. aegypti wmelpop D. melanogaster MGYP2 A. aegypti wmel D. melanogaster HTB A. albopictus, aposymbiotic walbb A. albopictus (Hou strain) HTR A. albopictus, aposymbiotic wri D. simulans ARwP A. albopictus, aposymbiotic wpip C. pipiens HouR A. albopictus walba, walbb, wri D. simulans HTM A. albopictus, aposymbiotic) wmelpop D. melanogaster Uju.wMel A. albopictus, aposymbiotic wmel D. melanogaster HC A. albopictus walba, walbb, wpip C. pipiens HM A. albopictus walba, walbb, wmel A. albopictus MGYP2 LB1 A. stephensi walbb A. albopictus More recently, it has been demonstrated that specific components of the mosquito microbiota can be engineered to secrete anti-plasmodium effector molecules and in this way dramatically reduce the mosquito s vectorial competence as a paratransgenic approach. The life cycle of most insect-vectored pathogens starts in the insect gut. In most cases, parasite numbers in this compartment are at their lowest point (bottleneck), making this the most vulnerable stage of the pathogen s cycle in the insect. Importantly, insects harbour a microbiota composed of well-defined bacterial genera that share the same insect compartment (the midgut lumen) with the most vulnerable stages of the pathogens they transmit. This proximity between microbiota and pathogen suggests a new possible strategy for control of transmission, namely the engineering of resident bacteria to secrete antipathogen molecules also known as paratransgenesis. Alternatively, the insect midgut could be populated with bacteria that naturally inhibit pathogen development. In proof-of-principle experiments, mosquito bacteria (Pantoea agglomerans) have been engineered to secrete a variety of anti-plasmodium molecules and this resulted in a dramatic inhibition of vectorial competence. In another proof-of-principle set of experiments, an Enterobacter sp. bacterium has been identified that strongly inhibits the development of Plasmodium in anopheline mosquitoes. While these initial findings are encouraging, a major challenge for field implementation of this strategy is to develop means to spread the inhibitory bacteria into mosquito populations in the field. This remains a high priority item for future research. One possible mechanism is 12

to use bacteria that are vertically transmitted, such as Asaia. However, in addition to vertical transmission, the bacteria should have an advantage over existing insect bacteria to allow their spread into insect populations. Moreover, issues such as transgene stability, pathogen resistance to the effector molecules, potential harm of the bacteria to humans and the environment and toxicity of the effector molecules also need to be evaluated A relatively new area of research has been the role played by the microbiota in insect fitness. This is an important aspect, since increased insect fitness could be highly beneficial for SIT activities. As shown in recent studies mainly for mosquito vectors and C. capitata (using both culture dependent and culture independent high throughput approaches), there can be a complexed symbiotic community in natural populations that seems to be absent in long established laboratory populations. This is also evident by ongoing studies in IPCL, in a variety of colonized populations representing different species. Studies in different Tephritidae species, such as C. capitata and B. dorsalis, have shown that the addition of bacteria (like Klebsiella sp., Enterobacter sp., Citrobacter sp.), either as probiotics or as live bacteria in the diet can have a positive impact in a variety of parameters. Comparison of different probiotics in adult diet to evaluate its effects on the fitness, rearing efficiency and competitiveness of mass-reared sterile males of B. dorsalis to find best probiotics bacteria has been done. Regulating mechanism of intestinal microflora homeostasis of B. dorsalis is currently being investigated. The hypothesis that the addition of probiotics to the larval diet could increase pupal weight in mass rearing and consequently the size of sterile adult of B. dorsalis and C. capitata males, as well as their dispersal, longevity, mating competitiveness and sperm transfer to females is being studied. The incorporation of probiotic supplements in the mass rearing protocols should be clearly described and taken into account when comparing strains efficiency. Standardization of such approaches would help in the replication of experiments among different groups. The utilization of live bacteria, either as effective components of control (such as Wolbachia) or as beneficiary supplements in diet, raises the concern of horizontal transfer events. In this direction: a) the documented transfer of parts (or even the whole) Wolbachia genome in the host genome (as evident at least in Drosophila and tsetse species), b) horizontal transfer of symbionts among different species and, c)) the naturally occurring hybridization of different species, are issues that should be also taken into consideration. Evaluation Technologies Technological advances are also allowing for more efficient evaluation of strains produced for SIT, and for improved monitoring before and after release. The application of these technologies as part of SIT programs should provide valuable information to improve rearing and release practices against other species. Domestication under mass rearing conditions. A major concern is the domestication of both strains used in SIT applications and populations introduced in the lab from the wild for comparative reasons (including mating competitiveness and compatibility experiments). Studies in B. oleae, B. dorsalis, B. tryoni and A. fraterculus show that there is probably a species-specific way of adaptation, accompanied by either drastic changes in the very few first generations (as in B. oleae and B. tryoni) or less severe changes (like in B. dorsalis and A. fraterculus). Adaptation could have also a severe impact on the structure of the symbiotic communities, affecting therefore fitness and performance. As evident, such changes are affecting the efficiency of the different strains and the interpretation of evaluation 13

experiments. The appropriate tools to study in depth the structure and complexity of the symbiotic communities are now available (although standardization is ongoing) and include Next Generation Sequencing (NGS) approaches, focusing on the 16S rrna gene. The monitoring of the status of the strains used in SIT applications and accompanying experiments, both in genetic and symbiotic level should be considered. Universal (if possible) approaches should be used in experiments testing the efficiency of strains used in SIT, regarding both their diets and the tests performed to evaluate their competitiveness. Complete genome assemblies are available for many of the target mosquito species, as well different Tephritidae species, such as C. capitata, B. dorsalis and Drosophila suzukii. Moreover, draft genomes have recently been completed for the melon fly (B. cucurbitae), olive fly (B. oleae), Qfly (B. tryoni) and the Australian sheep blowfly (L. cuprina). A reference genome sequence does not contain the full genetic diversity of a species, which can be better captured by sequencing individuals from various populations or strains. Inexpensive Illumina sequencing methods can now be employed to quantify this genetic variation at a genome-wide scale, which can be used to examine variation that exists among wild populations, or changes that occur during the domestication or release processes as part of an SIT program. Genome-wide markers are also extremely useful to generate high density linkage maps in target species. In addition to their value in improving genome assemblies by joining and ordering scaffolds into chromosomes, these markers can also be associated with phenotypes of relevance to SIT and hence can be used both to understand and to monitor the fitness of strains used in SIT programs. Varying levels of transcriptome data accompany these genomes, and with the greater accessibility of RNAseq methodologies to laboratories worldwide, the quantity and quality of transcriptome data is likely to increase for all target species. As such, transcriptome assessments may become standard practice as part of strain evaluation procedures in the near future. Genetic-based marking. Genetic-based marking is also a critical component of the SIT providing the ability to monitor released males to distinguish them from the field population when collected in traps, and to monitor the frequency of sterile male matings to females in the field. In addition, markers are important tools for the production of high quality insects in mass rearing facilities. While phenotypic markers have been isolated as visible mutations useful for SIT, their identification has often been serendipitous, they are species-specific, and optimal markers can take years to be developed, if at all, for some species. In contrast, transgenic fluorescent protein markers have been shown to be widely applicable, with the same genetic constructs functional in many species, including green or red fluorescent proteins introduced into A. suspensa, A. ludens, Bactrocera dorsalis, B. oleae, B. tryoni, C. capitata, D. suzukii, C. hominivorax, L. cuprina and several mosquito species. Through the use of different tissue-specific promoters and transgene integration sites, hundreds of transgenic lines with different tissues expressing the fluorescent protein could be established. In particular, tissue-specific sperm or Y-linked markers were developed for C. capitata, A. suspensa, A. ludens, B. tryoni, B. mori, and the mosquitos A. aegypti and A. stephensi. These markers may be used for sexing in some species (using fluorescence-based sorters), and allow identification of females that have mated with released males based on the spermathecal storage of fluorescent sperm. Moreover, they have been proven to be successful in the medfly for tracing differential sperm use in presence of multiple mating, which is particularly relevant to SIT applications. 14

Horizontal transfer - transposon- and/or symbiont mediated A critical concern for transposon vector-based and symbiont-based strain manipulations is the potential for horizontal interspecies transfer (HT) of the transposon vector or symbiont (or symbiont-mediated transfer of a transformation vector). This presents a critical ecological safety concern for associated insect and non-insect species within a field release site, and especially for beneficial species that might be negatively impacted. Symbionts may have a non-specific, if not a broad host range, and autonomous transposons are thought to utilize HT as a natural mechanism for their maintenance and proliferation. Non-autonomous transposon vectors are normally incapable of self-mobilization (in the absence of functional transposase), but the unintended or unrecognized presence of the same or cross-mobilizing transposase may allow their transmission directly into closely associated predators and symbionts, or through indirect transmission via symbionts or viral systems. However, establishment of stable horizontal transfer requires the introduction into the germ line. Moreover, most of the considered constructs are probably evolutionary neutral or even have negative selection characteristics (lethality, sterility), which would not favour the selection of rare horizontal transfer events. Nevertheless, it should be a high priority to evaluate potential HT between transgenic and/or symbiont infected host species and closely associated predatory (e.g. parasitoid) organisms or natural symbiont populations. Evaluation guidelines for the creations and analysis of transgenic strains for eventual contained field release applications Investigators should be aware of requisite information relevant to the genetic modification protocols that may be necessary for eventual applications for the contained field release of their organisms, especially as it relates to risk assessment. In particular is the required information outlined in the NAPPO agreement in the following sections: 2.1.2.3 Description of the genetic construct; 2.1.2.4 Characterization of the transgene inserted into the transgenic arthropod; and 2.1.2.5 Description of the phenotype of the transgenic arthropod. Investigators should also be aware of potential risk issues associated with the modification of particular insect species modified with particular genetic elements released into particular ecological environments, and modifications that could diminish risk, and in some cases be prerequisites for approved release. These considerations should include: 1) Robust and stable genetic marking systems that allow identification of released transgenics after field trapping, both by visible inspection and sensitive molecular tests. 2) Use of post-integration vector immobilization systems integrated into the vector, or a means to evaluate potential cross-mobilization within a host genome, to assess and mitigate potential re-mobilization of transposon-based vector systems by the unintended presence of the mobilizing enzyme (e.g. transposase). This helps ensure strain stability and potential horizontal transgene vector transmission into unintended associated organisms, including symbionts. 3) Use of robust species-specific intracellular lethality systems to ensure that survivors do not occur normally, that lethality is confined to the host organism leaving predatory organisms unaffected, and is also confined to the released species. 4) Use of genomic targeting systems where possible to avoid potential genomic site-specific effects including insertional mutations and modification of transgene expression. 5) Specific use of cassette exchange systems (i.e. RMCE) for both primary and secondary transgene integrations to avoid introduction of plasmid DNA including antibiotic resistance genes. 6) Potential use of dual redundant lethality systems to ensure that genetic breakdown of either system does not result in lethal revertant survivors in the field. 15

Evaluation guidelines - Quality control of insect strains Evaluation of strains for use in SIT programmes should be conducted by documenting the two most important parameters: (a) rearing performance (production and quality control) and (b) field performance (field cage or open field). Rearing performance of a strain: Before any strain is used in small or large scale applications, any new strain should be evaluated and, ideally, be compared with currently used strains, if available. During the rearing process, there are several relevant performance parameters that need to be evaluated using as a reference the classical genetics Mediterranean fruit fly TSL strain (see below tables with production parameters). Stability of the strains: This parameter measures the number of aberrant insects that appear during the rearing process. In order to clean a strain from aberrant/recombinant insects, a filter rearing system has been designed. This process allows documenting the number of recombinant flies in the initial colony, their removal and starting a new colony free of aberrant insects. The suggested value is: recombinants < 2% for classical genetic sexing strains like that of the Mediterranean fruit fly, while for transgenic strains it is expected to be < 1%. Production Parameters: The initial and most important parameter for the rearing process is the evaluation/comparison of strain fertility, fecundity including the pre-ovipository and ovipository phase. These filter systems should be extended to accommodate transgenic strains. Regarding symbiont-based strains, the infection titre and type should be regularly verified through molecular techniques. PRODUCTION PARAMETERS ( CURRENTMINIMUM Quality control medfly tsl strain) TRANSGENIC Manual V.6 2014 Fluorescence reference (page marker scoring number) efficiency Pre-oviposition period (days) 4 4 NA Oviposition profile (days) 10-14 10-14 NA FECUNDITY and fertility Number of eggs / female (release colony) 14.9 ± 2.18 > 15 NA NA Information not available in the QCM The recommended values of the additional parameters for comparing the rearing process, based on the Mediterranean fruit fly model, are: PRODUCTION PARAMETERS ( medfly tsl CURRENTMINIMUM Quality strain) TRANSGENIC control Manual V.6 2014 reference (page number) Egg to pupae recovery (male only) 25% > 40% NA Liters of Pupae/kg larval diet 0.18 ± 0.01 > 0.18 ± 0.01 NA LARVAL DEVELOPMENT PERIOD Colony larval development time at 25 C (days) 10 10 NA PUPAE DEVELOPMENT Percent pupation at 24hr 90 90 NA 16