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THIRD RESEARCH COORDINATION MEETING

Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture

Research Coordination Meeting on

Comparing Rearing Efficiency and Competitiveness of Sterile Male

Strains Produced by Genetic, Transgenic or Symbiont-Based

Technologies

Scientific Secretary: Kostas Bourtzis

Mahidol University

Bangkok, Thailand

18–22 June 2018

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

Classical Genetic Approaches 5

Selected References 24

Nuclear Component 37

Explanation / Justification 38

Participation of Agency's laboratories 38

Assumptions 38

Related TC Projects 38

LOGICAL FRAMEWORK 40

Narrative Summary 40

Specific Objectives 41

Outcomes 42

Outputs 43

AGENDA 45

PARTICIPANT ABSTRACTS 50

LIST OF PARTICIPANTS 72

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 mass-produce 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 Queensland fruit fly, the Mediterranean fruit fly, the olive fly, the cherry vinegar

fly, the codling moth, the pink bollworm, the New World Screwworm fly, 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 that eliminate females early in development 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 and will bring a higher certainty to the monitoring

systems, allowing an unequivocal discrimination between sterile and fertile males in the field.

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.

Improvements of the SIT have been major objectives 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 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 genetic breakdown and/or 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 -

Fruit flies

Agricultural pests -

Moths Veterinary and human

Africa Bactrocera dorsalis

B. zonata

Ceratitis capitata

C. rosa

Cydia pomonella

Grapholita molesta

Ectomyelois ceroniae

An. gambiae,

An. arabiensis,

Ae. aegypti

Glossina sp.

Musca domestica

Americas Anastrepha ludens

A. obliqua

A. grandis

A. fraterculus

A. suspensa

A. striata

A. serpentina

B. carambolae

B. oleae

C. capitata

Drosophila suzukii

Diatraea saccharalis

D. crambidoides

C. pomonella

G. molesta

Pectinophora gossypiella

Plutella xylostella

Helicoverpa armigera

Ae. aegypti,

Ae. albopictus

An. darlingi

An. albimanus

Cochliomyia hominivorax

Culex quinquefasciatus

Stomoxys calcitrans

Haematobia irritans

Dermatobia hominis

Asia B. dorsalis

B. carambolae

B. correcta

B. cucurbitae

C. pomonella

H. armigera

Spodoptera litura

G. molesta

P. xylostella

E. ceroniae

An. stephensi

An. sinensis

Ae. aegypti

Ae. albopictus

Cx. pipiens

Cx. tritaeniorynchous

Musca domestica

Australia

and

Oceania

B. tryoni

B. aquilonis

B. jarvisi

C. capitata

D. suzukii

C. pomonella

G. molesta

P. xylostella

Epiphyas postvittana

H. armigera

Ae. aegypti

Ae. albopictus

Lucilia cuprina

H. irritans

Europe B. oleae

C. capitata

D. suzukii

C. pomonella

G. molesta

E. ceroniae

Ae. albopictus

Ae. aegypti

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 Y-autosomal translocation-based genetic

sexing system, in which females homozygous for the recessive mutation have black pupae (bp-), while

genetically heterozygous males have brown pupae due to the wild-type allele (bp+) 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. Since 2012 a large-scale production

has been initiated in Mexico reaching a production of 30 million male pupae per week by 2018. Field

evaluation showed a detriment in the quality of male flies in the field, that prompted the refreshment of

the mass-reared strain with wild flies resulting in a significant improvement of quality control

parameters and mating competitiveness. Using the same sexing mechanism, a new Mexfly strain

(Family 10) has been developed in Guatemala, evaluated, and implemented at the Petapa (Guatemala)

mass rearing facility. New strains carrying recessive temperature-sensitive lethal mutations have been

developed using classical genetic approaches, and these strains also require evaluation for 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), and 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 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 WZ/ZZ sex chromosome system, in which females

are the heterogametic sex.

Genomic studies in support of pest insect evaluation and control

The whole genome sequence, assembly and annotation of the Mediterranean fruit fly, C. capitata, has

been completed with the participation of several CRP members. This study and new sequencing projects

on pest species will provide an enormous resource for methods development for the identification of

genome-wide polymorphisms that can be used for population genetic analysis and source determination

of medflies identified in ports of entry. The extensively annotated gene set for medfly, in particular,

will facilitate identifying the molecular basis of mutations in strains used for SIT (e.g. tsl sexing strain),

and the identification of novel targets that can be utilized to facilitate higher efficiency and efficacy of

IPM programs. These will include genes and regulatory systems important to conditional sexing and

sterility, ligands and receptors critical to courtship/mating, host plant/animal detection and lures for

trapping and mating disruption, the basis for insecticide resistance, and the genetic basis and

involvement in invasiveness and adaptation. A new genome assembly system currently being produced

for medfly at the Baylor College of Medicine will significantly improve automated annotations and the

physical map for more advanced analysis.

From sex determination studies to transgenic sexing strains in flies, butterflies and

mosquitoes.

One main objective is to isolate sex determining genes which can be used to improve existing genetic

sexing strategies. In particular, male determining factors can be exploited for generating conditional

male-only strains. Several primary signals controlling male sex determination have thus far been

identified in dipteran insects: nix, a tra-2 related splicing factor on homomorphic sex chromosomes in

Aedes aegypti and Aedes albopictus, Guy-1, a Y-linked gene in Anopheles stephensi, a Y-linked Yob

gene in Anopheles gambiae and, more recently, Mdmd, a paralog of the spliceosomal factor CWC22 in

the common housefly Musca domestica.

Non-recombining regions such as heteromorphic or homomorphic chromosomes carrying male

determiners tend to be highly repetitive and difficult to assemble from genomic reads. Several methods

have been developed to identify Y- or M-linked genes. One such method is the chromosomal quotient

(CQ) which was successfully used to identify the male determiner in Anopheles mosquitoes and in Ae.

aegypti. Based on the Ae. aegypti sequence, Nix has been also recognized in Ae. albopictus. However,

it is still unclear if mosquito genomes contain a transformer ortholog to transduce these signals to the

conserved doublesex gene.

In the insect order Lepidoptera, sex determination is based on female heterogamety, ZZ/ZW. However,

the underlying molecular mechanism of the W and Z chromosomes is unknown except for the silkworm,

Bombyx mori. Recently Kiuchi et al. (2014) discovered that the feminizing factor in B. mori is a W-

linked gene which encodes ad small PIWI-interacting RNA named Fem piRNA. The authors also

showed that the Fem piRNA downregulates the expression of the Z-linked gene, Masculinizer (Masc),

which, when active, promotes male expression of the B. mori doublesex gene (Bmdsx) that in turn

directs male development when active. Presence of Fem promotes female-specific expression of Bmdsx

by downregulating expression of the Masc gene. Studies of sex determining genes in Ceratitis capitata

led to the discovery of an evolutionary novelty. Different from the Drosophila transformer gene (tra),

the Medfly ortholog (Cctra) is the first switch in the pathway and is regulated by a positive feedback

mechanism. Orthologues of Cctra are also present in other dipteran, coleopteran, and hymenopteran

species and regulated by an autocatalytic function. Hence it has been proposed that this positive

feedback mechanism, which maintains the female determining activity of tra, is an ancestral feature of

the pathway. This loop serves as a cellular memory to enforce the proper execution of the female

program. It has been postulated that the function of M factors is to prevent activation or disrupt this

loop at an early stage. Once the loop collapses, cells lose their female identity and become

reprogrammed to resume a male fate. Thus, male development presumes an irrevocable loss of tra

activity. The intrinsic instability of an autoregulatory loop makes it a facile target for destruction. A

single blow can decrease the activity needed to sustain the loop below a critical threshold from which

it cannot recover. For instance, transient embryonic RNAi experiments show that a shift to male splicing

can be very efficiently provoked by injections of tra dsRNA during early embryogenesis. There may

not be much restriction on the performance range of a male determinant. Interference at any regulatory

level, e.g., transcriptional, post-transcriptional, post-translational, complex formation, or even nuclear

import/export may be sufficient to let the loop collapse. For instance, M factors can emerge from

dominant antimorphic mutations in positive regulators of female expression of tra as it has been

postulated for Mdmd in the housefly.

The Y-linked M factor in C. capitata, which represses female-specific expression of Cctra, like Mdmd

in Musca, is still elusive and needs to be identified. This effort may also assist in the identification of

corresponding M factors in other Tephritid species, such as the olive fly, Bactrocera oleae, if the male

instructive signal is conserved among the various members of this family.

Speculations on the molecular nature of the medfly male determining factor led to propose different

possible mechanisms disrupting the sensitive feedback loop: the M factor could be 1) a translational

repressor of the Cctra maternal mRNAs, 2) a splicing factor inhibiting CcTRA/CcTRA-2 and leading

to male-specific Cctra splicing (see Musca domestica case), 3) an RNA belonging to novel special

classes (ncRNA, piRNA, miRNA; see the Bombyx mori case), 4) a Y-linked DNA sponge sequestering

CcTRA and/or CcTRA-2 proteins. Hence, the molecular nature of M can vary widely and an unbiased

validation of candidates is necessary.

The first list of candidate male determining factors is under investigation by a novel functional test

developed in embryos by injections of either dsRNAs or DNA genomic fragments and detection of

splicing changes in Cctra by RT-PCR after few hours from injections. The exploitation of female-

specific splicing of Cctra gene to generate transgenic sexing strains with conditional female-specific

lethality in C. capitata and A. suspensa. Similarly, the sex-specific intron from the C. hominivorax tra

gene was used to build TSS for L. cuprina and C. hominivorax. Further improvements can be expected

in the near future from exploiting latest DNA sequencing technologies and new and powerful reverse

genetics tools, and from a better understanding of the molecular mechanisms underlying sex

determination and sexual differentiation.

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 cost-effectiveness 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-to-male 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 C. capitata, Anastrepha

suspensa, and Lucilia cuprina, 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 high-performance 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 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 a rapid implementation

of TSS technology, especially those based on tetracycline-repressible (Tet-off) female-specific

embryonic lethality. Although these species are 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, A. suspensa, A. ludens, and a livestock-relevant species, L. cuprina, all resulting in 100%

female lethality. All four species use similar endogenous components of their respective genomes to

induce lethality through a well-understood pro-apoptotic cell death pathway, however, in some species

the embryonic promoter has pre-zygotic activity in the maternal ovary resulting sterility. This has been

ameliorated in these females by short-term Tet-diet feeding that restored female fertility, with only male

progeny surviving after removal of the Tet-diet. These transgenic sexing approaches are highly effective

and cost-efficient by eliminating female insects early in embryogenesis.

Figure 1. Embryonic sexing using female-specific splicing under the control of the repressible tTA-system (Eckermann

et al. 2014). The depicted transgenic sexing system uses a sex-specifically spliced intron and a hyperactive pro-apoptotic

gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator (tTA). To cause

early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter.

During rearing of such strains, addition of tetracycline (TET) to the food keeps the system in the OFF state, as tetracycline

blocks the binding of tTA to its response element (TRE). For the release generation, tetracycline is absent in the food and

therefore the sexing system is ON: in males, the male specific splicing of the transformer intron (tra-I) - placed after the

translation start codon (ATG) of the effector gene - includes a small exon containing a TAA stop codon between the start

codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein

allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector

and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.

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 Ae. aegypti was reported, 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 An. gambiae a ́ sex distortion´ approach was developed, which

destroys X-bearing sperm and resulted in 95-97% male progeny, while in Ae. 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 female-specific

lethality system such as those established in tephritid species and NWS 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 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 AsProsβ21 (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 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 (see below).

Transgenic sexing based on sex reversion by female 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 sex-reversion line of C.

capitata that shows 98% conversion of XX individuals into fertile males, with 2% intersexes 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 non-transgenic 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

of developing insect transgenic technologies leading to non-transgenic male only progeny. Such

strategies may 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.

Embryo injections with Cas9 ribonucleoparticles were used to target the Cctra gene, leading to full

masculinization of XX individuals, although no DNA sequence changes were observed. An interference

CRISPR/Cas9 mechanism could underlie this unexpected phenomenon. Cas9 protein+sgRNA could

bind the targeted Cctra region (coding region of the first exon1) without cutting (possibly due to

suboptimal sgRNA) but possibly provoking transient transcriptional repression of the gene and leading

to stable female-to-male splicing pattern changes of Cctra in XX embryos. Hence masculinization of

XX individuals can be achieved also by exploiting CRISPR/Cas9, although more research is needed to

clarify this mechanism and optimize sgRNA selection to achieve stable and transmittable Cctra loss of

function alleles.

Sex reversion was also achieved for An. stephensi, expressing the Guy1 transgene under the control of

a Tet-off conditional system. Although a promising 100% female lethality was observed, different

concentrations of tetracycline were unable to suppress the Guy1 transgene expression. In Ae. aegypti, a

knock down of the female isoform of dsx by feeding E. coli-dsRNA-dsx in the larval stage was sufficient

to inhibit 96% the development of females. Alternatively, transient Nix expression in the females can

convert females into sterile males or confer female lethality.

Genetic tools for genome manipulation

Improvements on transposon-based insect transgenesis. Even in times of advanced site-

specific genome editing tools, the improvement of DNA transposases is still a priority in the field of

transgenesis: especially in emerging model systems where evaluated integrase landing sites or

recombinase-mediated cassette exchange (RMCE) sites have not yet been created and more importantly

in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence

information and genome annotations are still pending. In fact, random insertional mutagenesis is

essential to identify new genomic locations that are not influenced by position effects and thus can serve

as future stable transgene integration sites. In this respect, a hyperactive version of the most widely

used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is

currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a

mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of

transposition when expressed in mammalian in vitro and in vivo systems compared to the classical

PBase at similar protein levels. Members of this CRP could demonstrate that the usage of helper

plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of

successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red

flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster.

Genomic targeting of transgene insertions. While transposable elements are still widely used as

vectors for integrating transgenes into the genome of insects, 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 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 mosquitoes Ae.

aegypti, An. gambiae, and An. 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. Cre/loxP RMCE has

also been successfully achieved in Ae. aegypti, though this is the only species, thus far, for which a two-

step procedure, requiring recombinase treatment in a second generation, has been necessary to complete

the RMCE reaction. Both the integrase and recombinase 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 at the determination of gene function. This commonly involves the

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

associate with the RISC complex to 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 constitutively express 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 sterilization of male mosquitoes, fruit flies or other pest

insects by silencing spermatogenesis genes such as boule, zpg and dsxM, or genetic sexing as part of SIT

programs, by targeting female-specific transcripts during the developmental stages of the generation to

be released. The production of sterilize males by RNAi mediated knockdown of spermatogenesis genes

in B. dorsalis showed very good results after being released into wild populations. Testis-related genes

targeted by dsRNA negatively affected male reproduction of a tephritid fly, resulting in an

approximately 90% sterilized male population. Thus, targeting of spermatogenesis-related genes by

dsRNA has the potential to induce sterility in adult males and can be achieved in B. dorsalis by feeding

adult males double-stranded RNAs of one, or different combinations of, spermatogenesis genes for

improved SIT. 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.

Use of house keeping genes in Tephritids for qPCR. Real-time quantitative-PCR has been a

priceless tool for gene expression analyses. The reaction, however, needs proper normalization with the

use of housekeeping genes (HKGs), whose expression remains stable throughout the experimental

conditions. Often, the combination of several genes is required for accurate normalization. Most

importantly, there are no universal HKGs which can be used since their expression varies among

different organisms, tissues or experimental conditions. A recent study evaluated nine common HKGs

(RPL19, tbp, ubx, GAPDH, α-TUB, β-TUB, 14-3-3zeta, RPE and actin3) in thirteen different body parts,

developmental stages and reproductive and olfactory tissues in the medfly and the olive fly (Sagri et

al., 2017). The study provided a useful consensus key for the choice of the best HKG combination in

these two insects. While this key is very useful for the two insects and a good starting point for other

relatives in the family, it should not be taken for granted in use in other tissues and/or time points or

other relatives of the family.

CRISPR/Cas9 gene-editing. 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 D. melanogaster and B. mori and has since been successfully used to modify the genomes

of numerous insect pest species. The Cas9 knock-in strategy based on homology directed repair (HDR)

developed in Drosophila took advantage of a loss of function mutation in the ligase 4, a gene required

for non-homologous end joining (NHEJ) DNA repair. Homozygous lig4 flies showed a 5-7 fold

increase in HDR. 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 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).

A CRISPR/Cas9 method based on embryo injections of recombinant his-tagged CAS9 expressed

in bacteria + in vitro transcribed sgRNAs was established in the medfly, olive fly, Queensland fruit fly,

and Musca domestica. In the medfly, gene targeting of the white eye and of the paired segmentation

genes showed high rates of biallelic somatic mutations (visible in up to 50% of the progeny) and of

transmission of mutations (up to 99% of germ cells). In Musca domestica, gene targeting of the

Drosophila yellow orthologue led to observe high rate transmission of germ line mutations (up to 58%).

CRISPR/Cas9 has also been used to test NHEJ-based gene-editing in A. suspensa, and has been used

to create an HDR temperature-sensitive mutation in the transformer-2 sex determination gene (Dstra-

2ts2) in the spotted-wing Drosophila, D. suzukii. At permissive temperatures of 20°C and below, both

XX and XY mutant flies develop as morphologically normal fertile adults. However, at restrictive

temperatures of 26°C and above, XY males develop normally but are sterile, and XX females develop

as intersexual adults, which have a predominantly male morphology, and are sterile. This provides a

model for development of a conditional temperature-dependent system for a sterile males-only

population for SIT.

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 insects 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 trans-infections 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. 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.

Since Wolbachia was first introduced into the primary dengue vector Ae. 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 Ae. albopictus by using the newly developed Ae. albopictus

HC strain with triple Wolbachia infection (wAlbA, wAlbB and wPip). The minimum irradiation dose

for the sterilization of Ae. albopictus HC females escaped from sex separation has been established,

without affecting the mating performance of HC males. It is necessary to produce sufficient sterile

males for the combined approach to suppress the target populations. Thus, it’s important to develop

Standard Operation Procedures (SOPs) for mosquito mass rearing. SOPs have been developed for the

mass rearing of the HC during larval and adult stage, which have contributed to improve the mass

rearing efficiency for the combined approach. The largest mosquito facility has been established in

2015 in Guangzhou, China and the maximum HC strain male production of this facility has reached at

5 million per week in 2017.

Table 2: Stable trans-infected medfly, olive fly and mosquito lines with the potential to be used for

agricultural and public health 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

B. oleae [wCer2] B. oleae (Democritus strain)*

wCer2 C. capitata (Vienna

8-E88)

WB1 Ae. aegypti wAlbB

Ae. albopictus

(Hou strain)

PGYP1 and 2 Ae. aegypti wMelPop D. melanogaster

MGYP2 Ae. aegypti wMel D. melanogaster

wMelwAlbB Ae. aegypti-wAlbB

wMel and

wAlbB Ae. aegypti-wMel

wAu Ae. aegypti (Malaysia strain) wAu

D. simulans

(Australia strain)

wAlbA Ae. aegypti (Malaysia strain) wAlbA

Ae. albopictus

(Indonesia strain)

wAlbB Ae. aegypti (Malaysia strain) wAlbB

Ae. albopictus

(Indonesia strain)

wAuwAlbB

Ae. aegypti-wAlbB

(Malaysia strain)

wAu and

wAlbB Ae. aegypti-wAu

wPip Ae. aegypti wPip

Cx.

quinquefasciatus

HTB Ae. albopictus, aposymbiotic wAlbB

Ae. albopictus

(Hou strain)

HTR Ae. albopictus, aposymbiotic wRi D. simulans

ARwP Ae. albopictus, aposymbiotic wPip Cx. pipiens

HouR Ae. albopictus

wAlbA, wAlbB,

wRi D. simulans

HTM Ae. albopictus, aposymbiotic wMelPop D. melanogaster

Uju.wMel Ae. albopictus, aposymbiotic wMel D. melanogaster

HC Ae. albopictus wAlbA, wAlbB, Cx. pipiens

wPip

HM Ae. albopictus

wAlbA, wAlbB,

wMel Ae.albopictus MGYP2

LB1 An. stephensi wAlbB Ae. albopictus

* The strain was developed but subsequently lost.

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 anti-pathogen 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 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, Tephritidae fly like B. dorsalis and C. capitata

(using both culture dependent and culture independent high throughput approaches), there can be a

complex 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 has

been revealed. B. dorsalis uses the BdDuox-dependent immune response not only as a defence in the

gut, but also for regulating its intestinal microflora homeostasis. It was recently discovered that

fecundity of B. dorsalis females under restricted nitrogen source is significantly increased after

supplemental feeding with its intestinal probiotics Klebsiella oxytoca BD177. Removal of gut bacteria

by antibiotics treatment would result in a significant decrease in the reproductive capacity of adults and

the expression of its BdYP, BdInR, BdAkt and BdS6K genes, the key signal molecules of Insulin/TOR

signal pathways. Re-introduction of BD177 results in a significant increase and recovering in

oviposition performance of females under adequate or restricted nitrogen source. Especially under low

nitrogen condition, this was more significant suggesting that the level of nitrogen in food affects the

interaction between host and gut symbiotic bacteria. Providing K. oxytoca BD177 in the larval diet

significantly increased the B. dorsalis pupal weight, eclosion rate and reproduction but did not influence

the egg hatching rate and mating competitiveness of the adults.

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

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. Although, this approach provides an

overview of the bacterial communities present, characterization at genus or species level cannot be

achieved. Whole Genome Sequence typing could be considered as a way to characterize key bacterial

isolates from the Enterobacteriaceae family, which are present in Tephritidae species and mosquitoes,

at the genus, or even at the species level. 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 as D.

suzukii and different Tephritidae species such as C. capitata and B. dorsalis. 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, B. 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 mosquitoes Ae. aegypti, Ae. albopictus,

and An. 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. Importantly, the broadly applicable 3xP3 promoter used to regulate various

fluorescent protein genes in several orders of insects has been found to be, thus far, uniquely non-

functional in several tephritid fruit fly species.

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 of the potential genetic breakdown of transgenic systems for sterility and

sexing

All genetically-based population control systems are subject to spontaneous mutations that can disrupt

the efficacy and, possibly the stability of the system. In Drosophila, such mutations, and the frequency

at which they occur, have been evaluated over many years. Mutations that include, primarily, deletions,

small insertions, and larger transposon insertions at a general frequency of 1 to 5 x 10-6 / locus/

generation, and point mutations that occur at lower frequencies. In an effort to quantify the frequency

of spontaneous mutations that disrupt lethality in the Tet-off embryonic lethality system, that would

result in transgenic survivors in the field in the absence of tetracycline, a modified version of the original

lethality system in D. melanogaster (Horn & Wimmer, 2003) was tested for lethality revertants under

mass rearing. In an initial phase test approximately 660,000 adults were tested for survival on

tetracycline-free media, resulting in an independent inheritable lethal reversion frequency of ~16.7 x

10-6. Four of these individuals had primary site deletions within or including the tTA transactivator gene

or the hidAla5 cell death lethal effector gene, at a frequency of ~6.1 x 10-6. The other 7 individuals had a

second-site lethality suppression effect currently under evaluation. This is the first evaluation of

potential genetic breakdown of a Tet-suppressible lethality system, that is relevant to prospective uni-

sex and female-specific embryonic lethality systems, and the RIDL pupal lethality system currently

under field-release evaluation ion Aedes aegypti. In an effort to suppress the survival of these lethality

revertants, redundant lethality systems, such as DTS7 pupal lethality or a second completely

independent food-controllable binary expression system - the inducible Q system,

(Eckermann et al., 2014; Handler, 2016), will be tested in combination with Tet-off lethality to suppress

survivorship from the systems when used individually.

Evaluation guidelines for the creation and analysis of transgenic or symbiont-infected

strains for eventual contained field release applications

Investigators should be aware of requisite information relevant to the genetic modification (or

transinfection with symbiont) 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

remobilization 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.

7) Use of irradiation to ensure the complete sterility of any accidentally released

Wolbachia-infected female mosquitoes thus preventing the potential spread and establishment

of Wolbachia into wild populations.

Evaluation guidelines - Quality control of insect strains

Evaluation of strains for use in SIT-based 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). For the transinfected with Wolbachia mosquito strains, the strength of cytoplasmic

incompatibility, the capacity of virus resistance and the fitness cost, after the introduction of the

Wolbachia infection, should be assessed.

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%. For the transinfected with Wolbachia mosquito strains, the

infection status should be monitored in every batch in both colony maintenance and male release. The

infection frequency of Wolbachia should be higher than 99%.

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, and as mentioned above, the infection titre and type should be regularly verified through

molecular techniques.

Table 2. Production parameters.

PRODUCTION PARAMETERS (medfly

tsl strain)

CURRENT MINIMUM

TRANSGENI

C Fluorescence

marker scoring

efficiency

Quality control

Manual V.6

2014 reference

(page number)

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 strain)

CURRENT MINIMUM

TRANSGENIC

Quality

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

Egg to pupae recovery (male only) 25% > 40% NA

Table 3. Quality control parameters

The recommended values for the Quality control (QC) analysis, based on the Mediterranean fruit fly

model, are:

QUALITY CONTROL

PARAMETERS (medfly tsl strain)

CURRENT MINIMUM

TRANSGENIC

Quality

control

Manual V.6

2014

reference

(page

number)

Mean acceptable pupal weight (mg) 7.84 ± 0.25 > 7.5 23

Mean % flight ability, post-irradiation 81 ± 4 (65-70) > 65 26

Sex ratio (% male)

● Control 50

● Permissive conditions 65 60 Not available

● Restrictive conditions (tsl heat-

treated; transgenic without

tetracycline)

99.8 ± 0.4 95

Recommendation: include emergence, survival under stress, timing emergence and other post-

irradiation parameters (when applied) that are included in Quality Control Manual Version 6.0 May

2014

QUALITY CONTROL

PARAMETERS (Mexfly Tap-7

strain)

Brown pupa

(Male)

Black pupa

(Female)

Percent pupation at 24hr 93.2 ± 3.7

Mean acceptable pupal weight (mg) 17.5 ± 1.3 19.3 ± 1.5

Mean % emergence 93.7 ± 4.3 93.0 ± 3.8

Mean % fliers 92.2 ± 5.0 90.8 ± 4.4

Sex ratio (male/female) 1.3 ± 0.2

Survival under stress 83.8 ± 7.1 78.4±7.1

Mean % emergence post-irradiation

Mean % fliers post-irradiation

Regarding the triple Wolbachia-infected Ae. albopictus HC strain, the recommended values

for the Quality Control (QC) analysis are:

QC parameters Measurement value

Colony maintenance Wolbachia infection of HC females 99%-100%

Female fecundity 60-80

Egg hatch rate 70-90%

Male mating competitiveness index >0.7

Male release Female pupae contamination rate <1.0%

Female adult contamination rate <0.5%

Wolbachia infection status of the

released HC males

95%-100%

Male weight 0.9-1.1 gram = 1000 male

adults

Field surveillance Release ratio of sterile males to wild

type males

5:1-20:1

Infection status of Wolbachia in the

captured females

The captured number of

Wolbachia-infected females

should be excluded from data

analysis.

Infection status of Wolbachia in the

ovitraps or breeding sites

The positive ovitraps or

breeding sites should not be

continuously found in the

same monitored positions.

For the evaluation of field performance of the strains, the FAO/IAEA/USDA product quality control

and shipping procedures for sterile mass reared Tephritid fruit flies manual is used as reference (pages

75-97), although in some instances additional protocols for the parameter evaluation have to be

prepared and discussed among researchers involved in the evaluations.

Table 4. Field Performance

FIELD PERFORMANCE PARAMETERS

(medfly tsl strain)

CURRENT MINIMUM

TRANSGENIC

Mating performance post-irradiation (or equivalent)

● Laboratory mating (% males mating) 80 ± 6 80

● Field cages (sterility index) 0.3 - 0.4 > 0.3

Longevity in the field, post-irradiation LT50 (days) 4 > 4

Dispersal in the field, post-irradiation (m) 100 > 100

Egg hatch of wild female x TSL male, post-irradiation 0.01%

Egg hatch of wild female x transgenic male (with and

without irradiation)

0.01%

Fluorescence marker persistence (monitoring) >3 weeks > 2 weeks

Fluorescence marker scoring efficiency 110-150

flies/hr/person

90 - 110 flies/hr/person

Fluorescence marker scoring accuracy 91% primary

9% secondary

< 1% tertiary

91% primary

9% secondary

< 1% tertiary

In respect to the strains developed by transgenic and / or symbiont-based approaches, the potential of

horizontal transfer of the transgene(s) and / or symbionts in other strains and / or species should be

evaluated using standard molecular biology and genetic approaches.

Recommendation: the assessment of additional specific test must be analysed case by case.

Evaluation guidelines - Domestication

Domestication is an important consideration both for the efficiency and evaluation of strains used in

SIT. Different studies using molecular markers (such as microsatellites and recently genome wide

sequencing markers) point to a non-uniform adaptation process among different species. Other recent

studies in symbiotic level (using, for example, culture dependent and/or culture independent high

throughput NGS approaches) also suggest that this adaptation severely influences the structure of the

symbiotic communities of the populations entering the lab. Although there are no standardized protocols

yet, the genetic, symbiotic and physiological characterization of strains used in SIT and populations

introduced for comparative studies should be studied. Development and application of universal

approaches across species will be beneficial for the comparison of the efficiency of different strains.

Following the understanding that symbiotic communities are important in parameters such as fitness

and mating competitiveness, different studies have recently focused on the characterization of symbiotic

communities of insects of economic and medical importance. Approaches still can be quite varying.

There can be differences either in the technology used (such as morphological/ biochemical

examination of colonies, 16S rRNA sequencing using classical Sanger sequencing or utilizing different

forms of the emerging NGS technologies) or in the samples analyzed (natural vs wild populations,

different developmental stages, whole adults vs tissue specific and more). These methodological

differences can create confusion and make the conduction of robust conclusions a difficult task. Recent

data that point to the dynamic nature of the symbiotic communities during development make the

characterization even more difficult. At the same time, probiotic diets are being developed and used for

different insect-targets of SIT, aiming to enhance its effectiveness through increasing important

parameters such as fitness and mating behavior. However, results can vary, since the domestication

process can be different within/between species. SIT important strains may have different properties,

attributed either to genetic or symbiotic factors because of differences in the wild material originally

introduced in the lab and/or differences in the rearing practices followed in the different laboratories

and facilities. The monitoring of wild populations during the laboratory domestication in different levels

(genetic, symbiotic and physiological), could provide insight in the changes happening during this

process. Documenting and monitoring the genetic and symbiotic profile with universally accepted

protocols could a) facilitate implementation and evaluation of enrichment procedures with wild

genomic background, b) enable the comparison of results of probiotic experiments performed in

different labs and, c) point to characters that are common or diverse during laboratory adaptation.

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Nuclear Component

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 any evaluated, refined and / or newly developed strains

produced in this CRP. As radiation induces random dominant mutations, there are no possibilities of

resistance development to this physical process. This possibility cannot be excluded with molecular

approaches that involve genomic insertions.

Explanation / Justification

Publication of results: Activities and final findings of the CRP will be published in a Special Issue of a

peer-reviewed open-access journal.

Participation of Agency’s laboratories

As few institutions are applying irradiation and classical genetics for the development of GSS in

agricultural pests and disease vectors, and given the need of a neutral body for the quality control

analysis of sterile males, 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 2.1.4.1. This R and D will focus on comparing the performance of

sterile males produced by genetic, transgenic or symbiont-based technologies.

Other Resources required

None.

Assumptions

Member States continue to recognize the benefits of developing the SIT package and other genetic and

environment-friendly methods for sustainable control of insect pests of agricultural, veterinary and

medical importance in AW-IPM programmes and continue to request improved technology and high-

quality SIT strains in order to maximise benefit/cost projections.

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

BKF5012 – Collecting baseline data and implementing fruit fly suppression in mango fruit.

CPR5020 - Integrating the Sterile Insect Technique (SIT) for Area-Wide Integrated Pest Management

of Tephritid Fruit Flies.

GUA5017 - Using the Sterile Insect Technique (SIT) to Establish Fruit Fly Low Prevalence Pilot Areas

and to Assess it as an Alternative for the Control of the Sugarcane Borer in Pilot Areas

HON5006 - Using Sterile Insect Technique (SIT) to Obtain Recognition as a Mediterranean Fruit Fly

Free Area in the Aguan River Valley

ISR5019 – Supporting a feasibility study for the implementation of leafminer (Liriomyza spp) sterile

insect technique combined with biological control under greenhouse conditions.

LIB5011 – Enhancing area-wide integrated management of fruit flies.

MAG5021 - Implementing the Sterile Insect Technique (SIT) in Integrated Fruity Fly Control for High

Quality Fruit Production.

MAR5022 – Reducing insecticide use and losses to melon fly (Bactrocera cucurbitae) trhough

environment-friendly techniques to increase production in different areas, Phase II.

MOR5032 - Supporting Control of the Mediterranean fruit fly Using the Sterile Insect Technique for

Citrus Fruits and Early Fruits and Vegetables to Establish Low Mediterranean fruit fly Prevalence Zones.

OMA5002 – Assessing the suitability of sterile insect technique (SIT) and related techniques for

combating date palm insect pests.

PAN5020 – Strengthening technical capacity to control Mediterranean fruit fly using the sterile insect

technique (SIT).

PLW5001 – Improving the quality of fruits and vegetables through an area-wide integrated pest

management of Bactrocera fruit flies in production areas of Palau.

SEY5005 – Enhancing the melon fruit fly area-wide integrated pest management programme using the

sterile insect technique (SIT) to implement national food security.

THA5052 – Developing sustainable management of fruit flies integrating sterile insect technique with

other suppression methods.

VIE5017 – Supporting area-wide integrated pest management to improve the quality of fruit for export.

RAF5061 - Supporting Capacity Building and a Feasibility Study on Control of Fruit Flies of Economic

Significance in West Africa.

RAF5062 - Preventing the Introduction of Exotic Fruit Fly Species and Implementing the Control of

Existing Species with the Sterile Insect Technique and Other Suppression Methods.

RAS5059 - Supporting Area-Wide Integrated Pest Control of Native and Exotic Flies in the Middle

East Subregion Incorporating the Sterile Insect Technique (SIT).

RAS5067 – Integrating sterile insect technique for better cost-effectiveness of area-wide fruit fly pest

management programmes in Southeast Asia.

RER5020 - Controlling Fruit Flies in the Balkans and the Eastern Mediterranean.

INT5151 - Sharing Knowledge on the Use of the Sterile Insect and Related Techniques for Integrated

Area-Wide Management of Insect Pests

LOGICAL FRAMEWORK:

Narrative Summary Objective

Verifiable

Indicators

Means of

Verification

Important Assumptions

Overall Objective

to compare the performance of

sterile males produced by classical

genetic, transgenic or symbiont-

based technologies to address the

increasing demand for

environment-friendly and

sustainable integrated pest

management approaches for insect

pests of agricultural, veterinary or

medical importance

N/A

N/A

Requests by Member States in the

area of insect pest and disease vector

control using the SIT are increasing.

To make this nuclear technology

available to Member States for

several insect species, the evaluation

of the rearing efficiency and male

mating competitiveness of sterile

male strains, refinement of

technologies and the assessment of

genetic breakdown, stability, and

horizontal transfer phenomena are an

essential precondition prior to the use

of these strains in operational

programs. Biological material is

available.

Specific Objectives

1. To comparatively evaluate the

performance of sterile males

produced by classical genetic,

transgenic or symbiont-based

technologies

2. To refine, if necessary, existing

technologies and/or adopt new

ones for the development and

application of strains for the

control of agricultural pests and

disease vectors

3. To assess potential genetic

instability, genetic breakdown,

and/or horizontal transfer

phenomena towards the use of

strains developed by classical

genetic, transgenic or

symbiont-based approaches for

SIT applications

Sterile male

strains of at least

one target species

produced by each

one of the three

technologies

evaluated.

At least one

technology refined

and/or newly

adopted.

Potential genetic

instability, genetic

breakdown and/or

horizontal transfer

phenomena in at

least one strain per

system assessed.

Reports and

published

papers.

Reports and

published

papers.

Reports and

or published

papers.

Quality control tests can be applied

for the sterile male strains produced

by the three technologies or can be

developed.

Refinement and/or adoption of new

technologies is possible.

Tools for the assessment of genetic

instability, genetic breakdown,

and/or horizontal transfer phenomena

are available or can be developed.

Outcomes

1. Sterile male strains

produced by classical

genetic, transgenic or

symbiont-based

technologies

comparatively evaluated

2. Existing and/or new

technologies for the

development and

application of sterile male

strains produced by

classical genetic,

transgenic or symbiont-

based approaches refined

or adopted

3. Genetic instability, genetic

breakdown, and/or

potential horizontal

transfer phenomena

towards the use of strains

developed by transgenic or

symbiont-based

approaches for SIT

applications assessed

Protocols and

approaches

determined

Tools and

protocols for the

refinement of

existing and / or

newly adopted

technologies

developed

Tools and

protocols

developed

Data

collected and

feasibility

analysis

Data

collected and

feasibility

analysis

Data

collected and

feasibility

analysis

Facilities and resources available.



Outputs

1. A collection of strains of

agricultural importance

produced by classical genetic,


technologies with the

performance of sterile males

compared / evaluated.


veterinary importance produced

by classical genetic, transgenic

or symbiont-based technologies

with the performance of sterile

males compared / evaluated.


medical/public health

importance produced by

classical genetic, transgenic or

symbiont-based technologies

with the performance of sterile

males compared / evaluated.

4. A set of SIT strains with robust

marking properties for field use.

5. Refined strains with improved

characteristics such as sexing,

mass rearing, mating

competitiveness, sterility for

SIT applications.

6. Stable classical genetic,


strains as assessed by long-term

and large scale production.

7. Strains for new target species of

agricultural, veterinary or

medical importance generated

by the use of available

technologies.

8. Information on the frequency of

genetic instability, genetic

breakdown, and/or potential

horizontal transfer phenomena

in SIT strains.

At least three

strains of

agricultural

importance

compared /

evaluated.

At least three

strains of

veterinary

importance

compared /

evaluated.

At least three

strains of

medical/public

health importance

compared /

evaluated.

At least three SIT

strains with robust

marking properties

for field use

developed.

At least three

refined strains

identified

At least three

stable SIT strains

developed

Strains for at least

one new target

species generated

At least three

strains assessed

Reports and

or published

papers.

Reports and

or published

papers.

Reports and

or published

papers.

Reports and

or published

papers.

Reports and

or published

papers

Evaluation

data

collected and

published

Reports and

published

papers

Reports and

or published

papers

Biological material is available. QC

protocols are available or can be

developed.



developed.



developed.

Tools are available.

Biological material and tools are

available. QC protocols are available

or can be developed.

Testing is feasible.

Biological material and tools are

available.

Tools are available.

9. Mass rearing and semi-field

validation of at least three new

strains developed by either

classical genetic, transgenic or

symbiont-based technologies.

10. Publication of results in a peer

reviewed journal.

Three new SIT

strains validated

Papers drafted and

submitted.

Reports and

published

papers

Journal issue

with

published

scientific

papers.

Testing and validation is possible.

Data for publication available.

Activities

1. Form a network of research

collaborators

2. Organise 1st RCM to refine the

logical framework and plan the

overall activities of the CRP.

3. Organise 2nd RCM to analyse

progress in delivering research

outputs and plan the next phase of

the project.

4. Organise 3rd RCM to analyse

progress in delivering the research

outputs and plan the final phase of

the project.

5. Organise final RCM to assess the

success of the CRP in reaching its

objectives and review the final

publication.

6. Publish the results of the CRP in

a special issue of an

international journal.

Proposals

evaluated and 9

Research

Contracts, 9

Research

Agreements and 1

Technical

Contract awarded.

1st RCM held

2015.

2nd RCM to be

held 2017.

3rd RCM to be

held 2018.

4th RCM to be

held 2019.

Signed

contracts and

agreements.

Participants’

activities and

logical

framework

revised.

Participants

and RCM

Progress

Reports.

Participants

and RCM

Progress

Reports.

Participants

and RCM

Final

Reports

Scientific

publication.

Suitable proposals submitted,

funding available and approval of

Contracts and Agreements by

CCRA-NA committee.

Contracts and Agreements signed by

counterpart organisations.

Progress satisfactory.

Progress satisfactory and mid-CRP

evaluation approved by CCRA-NA

committee.

Final reports are submitted to the

Agency.

Consensus can be found on

appropriate international journal and

acceptance by journal obtained.

THIRD FAO/IAEA RESEARCH COORDINATION MEETING ON

“Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced

by Genetic, Transgenic or Symbiont-based Technologies”

18-22 June 2018

Regional R&D Training Center for Insect Biotechnology (RCIB),

Department of Biotechnology, Faculty of Science,

and The Salaya Pavilion Hotel

Mahidol University at Salaya Campus, Thailand

AGENDA

MONDAY, 18 JUNE 2018:

MahaSawasdee 1 meeting room in the Salaya Pavilion Hotel

09:00 – 09:05 Kostas Bourtzis (Scientific Secretary, FAO/IAEA):

Welcome statement.

09:05 – 09:15 Associate Prosessor Sittiwat Lertsiri (Dean: MUSC): Welcome statement.

09:15 – 09:30 Introduction of participants, administrative announcements.

09:30 – 10:00 Rebecca J. Davis, Esther J. Belikoff, Elizabeth H. Scholl, Fang Li and Maxwell J.

Scott: no blokes is essential for male viability and X chromosome gene expression in

the Australian sheep blowfly.

10:00 – 10:30 Daniel Paulo, Alex Arp, Agustin Sagel, Steven Skoda, Adalberto De-Leon, Ana

Azeredo-Espin, Owen McMillan, Max Scott and Carolina Concha: Evaluation of an

early lethal sexing strain of the New World Screwworm fly, Cochliomyia hominivorax

(Diptera: Calliphoridae) and development of CRISPR/Cas9 genome editing technology

for functional genomics.

10:30 – 10:40 GROUP PHOTO

10:40 – 11:00 COFFEE BREAK

11:00 – 11:30 Amanda Choo, Peter Crisp, Isabel Chen, Polychronis Rempoulakis, Philip Taylor,

Owain Edwards, Louise O’Keefe, Robert Saint and Simon Baxter: Developing a male

only strain of Bactrocera tryoni using CRISPR/Cas.

11:30 – 12:00 Amanda Choo, John Sved, Isabel Y. Chen, Deborah Shearman, Peter Crisp and Simon

W. Baxter: Identification of Y chromosome scaffolds and a predicted protein coding

gene in male Bactrocera tryoni fruit flies.

12:00 – 12:30 Roswitha Aumann, Irina Häcker, Alfred M. Handler and Marc F. Schetelig:

Evaluation of FRT/Flp recombinase-mediated cassette exchange in Ceratitis capitata.

12:30 – 13:00 Zhao, Y. and Handler, A.M.: Conditional lethal tephritid strains for improved SIT and

genetic stability.

13:00 – 14:00 LUNCH: MahaSawasdee 2 room in the Salaya Pavilion Hotel

14:00 – 14:30 Hassan M. M. Ahmed, Kolja N. Eckermann, Ingrid M. Curril, Musa D. Isah and

Ernst A. Wimmer: CRISPR/Cas9 based multifactorial reproductive sterility for SIT

approaches and transcriptomics analysis on reproductive biology of Ceratitis capitata.

14:30 – 15:00 Claudia Paiz, Pamela Flores, Gabriela Lara, Paula Villatoro and Pamela Pennington:

Modifying mosquito gut microbiota to induce male sterility through RNA interference:

optimization of dsRNA production and encapsulation for gene silencing.

15:00 – 15:30 Svenia Heinze, Akash Sharma, Tea Kohlbrenner, Yanli Wu, Luca Lenzi, Anja Bösch,

Louis van de Zande, Ernst Wimmer, Mark Robinson, Leo Beukeboom and Daniel Bopp:

Sex determination in the common housefly: a tale of intraspecific variations at the

instructive level


16:00 – 16:30 P. Primo1, A. Meccariello, M. Gucciardino, F. Forlenza, M. Perrotta, S. Monti, M.

Buonanno, A. Gravina, A. Ruggiero, P. Papathanos, M. Salvemini, L. Vitagliano, E.

Giordano and G. Saccone: Gene targeting of the female determining transformer gene

in the major agricultural pest Ceratitis capitata, the Mediterranean fruit fly: an

unexpected CRISPR/Cas9 interference effect.

16:30 – 17:00 KT Tsoumani, M-E Gregoriou, E. Zorbas and Kostas D. Mathiopoulos: New

olfactory and reproductive targets for alternative olive fly control.

17:00 – 17:30 Alessandro Di Cosimo, Nidchaya Aketarawong, Mariconti M, Mosè Manni, Sujinda

Thanaphum, Francesca Scolari, Ludvik M Gomulski, Anna R Malacrida and Giuliano

Gasperi: Comparative evaluation of the chemioreception and reproduction

performance of different populations and strains of sanitary and agricultural

importance.

19:00 IAEA reception (start traveling by vans at 18:30 from the Salaya Pavillion)

TUESDAY, 19 JUNE 2018:


09:00 – 09:30 Panagiota Koskinioti, Antonios Augustinos, Danilo Carvalho, Misbah Ul Haq, Gulizar

Pillwax and Kostas Bourtzis: Development and evaluation of genetic sexing strains for

the population suppression of Aedes mosquito vector species using SIT approaches.

09:30 – 10:00 Margareth Lara Capurro Guimarães, Danilo O. Carvalho and Jair F. Virginio:

“Evaluation of transgenic lines for the population control of Ceratitis capitata and

Aedes aegypti”.

10:00 – 10:30 Dongjing Zhang and Zhiyong Xi: Mass rearing of the triple Wolbachia-infected Aedes

albopictus HC strain.


11:00 – 11:30 Sujinda Thanaphum, Nidchaya Aketarawong, Siriwan Isasawin and Kamoltip

Laohakieat: Comparing rearing efficiency and competitiveness of males from Salaya

genetic sexing strains (Bactrocera spp.) including the refinement and transfer of

existing technology for the improvement and application of strains for area-wide

integrated pest management.

11:30 – 12:00 D. H. Orozco Davila, J. S. Meza, M. Roblero Roblero, V. García Martínez, J. Ibañez

Palacios, S. Aguirre, M. F. Ruiz Pérez: Evaluation and improvement of Anastrepha

ludens strains for SIT: a) Transgenic and b) Tapachula-7 under mass rearing conditions.

12:30 – 12:30 Lanzavecchia Silvia, Claudia Conte, Giardini Cecilia, Scannapieco Alejandra, Milla

Fabián, German Crippa, Segura Diego and Jorge Cladera: Evaluation of genetic and

biotechnological tools towards the development of an Anastrepha fraterculus sexing

strain.


14:00 – 14:30 Antonios A. Augustinos, Georgia Gouvi, George Kyritsis, Katerina Nikolouli, Carlos

Caceres, Anastasios Mintzas, George Tsiamis and Kostas Bourtzis: Symbiotic and

genetic analysis evaluation of strains used in SIT.

14:30 – 15:00 Edwin Ramírez-Santos: Evaluation of different strains of Anastrepha ludens Loew and

Ceratitis capitata Wied.

15:00 – 15:30 Yichen Wang, Yushan Li, Zhaohui Cai, Zheng Zhao, Zhichao Yao, Ping Zhang, Shuai

Bai, Muhammad Fahim Raza and Hongyu Zhang: Gut bacteria improve the fitness of

Bactrocera dorsalis: influence on development and reproduction.


16:00 – 16:30 Open discussion on the presentations

16:30 – 17:00 Discussion on the CRP Special Issue

17:45 – 18:00 Scenic walk to the Music Square Reception room at the college of music

18:00 Mahidol University reception

WEDNESDAY, 20 JUNE 2018:


08:30 – 10:30 Working groups discussion







18:00 Group dinner / Social event

THURSDAY, 21 JUNE 2018

Meeting rooms at the SC1 building of the Faculty of Science

08:30 – 09:00 Travel to the field site

09:00 – 12:00 Field trip activities

12:00 – 12:20 Travel to a Thai restaurant

12:30 – 13:45 LUNCH OUTSIDE

13:45 – 14:00 Travel back to meeting rooms at the SC1 building of the Faculty of Science

14:00 – 15:30 Drafting meeting report


16:00 – 17:30 Drafting meeting report

18:00 Group dinner / Social event

FRIDAY, 22 JUNE 2018:

meeting rooms in the SC1 building of the Faculty of Science

08:30 – 10:30 Reports of Working Groups and Revision of Logical Framework


11:00 – 12:30 Drafting RCM3 final report

12:30 – 14:00 LUNCH

14:00 – 15:30 Drafting RCM3 final report


16:00 – 17:30 Presentation and approval of the Final Report - General discussion

17:30 End of the 3rd RCM

17:30 Closing.


On “Comparing Rearing Efficiency and Competitiveness of Sterile Male

Strains Produced by Genetic, Transgenic or Symbiont-based Technologies”

Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: no blokes is essential for male viability and X chromosome

gene expression in the Australian sheep blowfly AUTHOR (S): Rebecca J. Davis, Esther J. Belikoff, Elizabeth H. Scholl, Fang Li, and Maxwell

J. Scott ORGANIZATION: North Carolina State University

SHORT SUMMARY OF PAPER Abstract:

It has been hypothesized that the Drosophila fourth chromosome is derived from an ancient X

chromosome. In the Australian sheep blowfly, Lucilia cuprina, the heterochromatic X chromosome

contains few active genes and orthologs of Drosophila X-linked genes are autosomal. Of 8 X-linked

genes identified previously in L. cuprina, 6 were orthologs of Drosophila fourth chromosome genes.

The X-linked genes were expressed equally in males and females. We have identified an additional 51

X-linked genes and show that most are dosage compensated. Orthologs of 49 of the 59 X-linked genes

are on the fourth chromosome in D. melanogaster. As painting of fourth (Pof) is important for

expression of Drosophila fourth chromosome genes, we used Cas9 to make a loss-of-function knock-

in mutation in a L. cuprina Pof ortholog we call no blokes (nbl). Homozygous nbl males derived from

homozygous nbl mothers die at the late pupal stage. Homozygous nbl females are viable, fertile and

live longer than heterozygous nbl females. RNA expression of most X-linked genes was reduced in

homozygous nbl male pupae and to a lesser extent in nbl females compared to heterozygous siblings.

The results suggest that NBL could be important for X chromosome dosage compensation in L. cuprina.

NBL may also facilitate gene expression in the heterochromatic environment of the X chromosome in

both sexes. This study supports the hypothesis on the origin of the Drosophila fourth chromosome and

that a POF-like protein was required for normal gene expression on the ancient X chromosome. If

further studies confirm that loss of nbl function has little effect on female fitness, a Cas9-based gene

drive targeting nbl in the female germline could be an effective means for genetic control of this major

pest of sheep in Australia.

In addition, we will give an update on our work on L. cuprina transgenic embryonic sexing strains

presented at the previous RCM in Panama.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation of an early lethal sexing strain of the New

World Screwworm fly, Cochliomyia hominivorax (Diptera: Calliphoridae) and

development of CRISPR/Cas9 genome editing technology for functional genomics.

AUTHOR (S): Daniel Paulo, Alex Arp, Agustin Sagel, Steven Skoda, Adalberto De-Leon,

Ana Azeredo-Espin, Owen McMillan, Max Scott and Carolina Concha

ORGANIZATION: UNICAMP, Brazil. COPEG-USDA, Panama; North Carolina State

University, USA; USDA-ARS, USA. Smithsonian Tropical Research Institute, Panama.

SHORT SUMMARY OF PAPER

Abstract:

The New World Screwworm fly (NWS), Cochliomyia hominivorax, is the only obligatory parasitic

blowfly in the Neotropical region. Their larvae infest warm-blooded vertebrates and feed on host’s live

tissues, resulting in severe livestock losses. We have developed an embryonic female lethal strain for

an improved SIT control program in Panama. This strain contains a two-component system consisting

of the gene promoter L. cuprina bottleneck driving expression of the tTA gene and a tetO-Lchid gene

effector, both in the same insertion site in the genome. The addition of the sex-specifically spliced intron

from the transformer gene within the Lchid gene ensures that only females die when insects are reared

in the absence of tetracycline. This strain has been evaluated for fitness characteristics that are relevant

for its performance in a mass rearing facility as well as mating characteristics relevant to their potential

success in the field, showing promise for use in a future control program.

CRISPR/Cas9-based genome editing has been successfully used for functional genomic studies in many

organisms, and has recently emerged as a potential tool for insect pest control. Here, we report for the

first time the successful establishment of an efficient CRISPR protocol for the generation of inheritable

mutations in the NWS. In order to demonstrate its potential, we targeted two C. hominivorax loci: yellow,

which determines the recessive unpigmented cuticle phenotype in adult flies and the olfactory co-

receptor Orco, that forms a complex with all the Odorant-selective Receptors and is required for the

transduction of the odor stimuli into behavioral responses. Our results show we successfully induced a

high rate of somatic biallelic mutations in 46 to 68% of the surviving G0 flies. Notably, 90% of these

mosaic flies transmitted their mutated alleles to the next generation (G1), resulting in mutant progenies

at percentages ranging from 14 to 88%. Medium (300bp) to large (2.2Kb) genomic deletions were also

induced by simultaneously microinjecting two sgRNAs targeting these genes, demonstrating that large

DNA motifs or even entire exons can be removed from the NWS genome. Using diverse crossings and

genotyping strategies, we found the first CRISPR-mediated homozygous mutant strains of NWS, named

ChoYellow-/- and ChoOR83b-/-, currently being maintained at COPEG. We next used the developed

protocol to target the female determining transformer locus (tra), aiming the reprograming of all

progeny toward to males. Following the Cas9-RNPs microinjections, 60% of the surviving females

developed partially to fully transformed ovipositors into male genitalia and abnormal reproductive

tissue. Most probably, the male-like structures in these mosaic females are due to somatic mutations

induced in one or both of alleles of tra in these cells. These results stand as a proof-of-concept that

genome-editing strategies based on Cas9-mediated mutagenesis could be an effective mean for

controlling Screwworm natural populations.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Developing a male only strain of Bactrocera tryoni using

CRISPr/Cas

AUTHOR (S): Amanda Choo1, Peter Crisp2,3, Isabel Chen1,2, Polychronis Rempoulakis4,

Philip Taylor4, Owain Edwards5, Louise O’Keefe1, Robert Saint1,6, Simon Baxter1

ORGANIZATION: 1School of Biological Sciences, University of Adelaide, South Australia, Australia. 2South Australian Research and Development Institute (SARDI), South Australia, Australia 3School of Agricultural, Food and Wine, University of Adelaide, South Australia, Australia 4Flinders University, South Australia, Australia 5Macquarie University, New South Wales, Australia 6Commonwealth Scientific and Industrial Research Organisation, Australia


Bactrocera tryoni, otherwise known as the Queensland fruit fly (Qfly), is among one of the most

devastating horticultural pests in eastern Australia with the propensity to spread to other areas of

Australia where it has not yet been established. Sterile Insect Technique (SIT) is used as one of the

methods to eradicate the outbreak populations, with the current SIT program in Australia using bi-sex

release of sterile flies. SIT is an ideal control strategy as it is species-specific and environmentally

friendly, however a male-only strain is required to improve the effectiveness and cost-efficiency of the

Qfly SIT program.

The CRISPR/Cas genome editing technology is a new genetic tool that allows the introduction of

specific genetic changes into genomes without generating transgenic organisms. We have successfully

established the CRISPR/Cas technology in Qfly and generated two different strains that we are now

further developing for the purpose of SIT application. We have firstly generated a white-eye mutant

strain in which flies lack colour pigments required for normal eye colour. Fitness of these white-eyed

flies is currently being assessed. These flies are visually impaired due to their lack of eye colour pigment

and have reduced fitness for survival in the wild. There could hence be a potential for developing a

genetic-sexing, “functionally male-only” strain, in which the females are white-eyed and will die in the

wild soon after a bi-sex SIT release whilst males have the normal eye colour and will be able to mate

with wild females effectively.

Temperature sensitive lethal (tsl) genetic sexing strains have been shown to be most effective and cost-

efficient for SIT, as seen in another tephritid pest, Ceratitis capitata. The mutation in the C. capitata

Vienna-8 tsl strain however has not yet been identified, thus preventing targeting of the orthologous

gene to generate a similar strain in Qfly. We have instead selected a tsl mutation in the shibire gene that

results in temperature sensitivity in Drosophila melanogaster and successfully introduced that single

base change into Qfly shibire. Homozygous shibire tsl mutants will be assessed for embryonic lethality

at high temperatures to determine if the mutation has a temperature sensitive effect in Qfly.

Translocation of a wildtype shibire allele to the Y chromosome will then be carried out to generate a tsl

genetic sexing strain in which female embryos can be eliminated by heat treatment, leaving only males

that will be made sterile and released for SIT.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Identification of Y chromosome scaffolds and a

predicted protein coding gene in male Bactrocera tryoni fruit flies

AUTHOR (S): Amanda Choo1, John Sved2, Isabel Y. Chen1, 3, Deborah Shearman2, Peter

Crisp3, 4 and Simon W. Baxter1

ORGANIZATION: 1School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia. 2Evolution and Ecology Research Centre, University of New South Wales, Sydney, NSW,

Australia 3South Australian Research and Development Institute (SARDI), Adelaide, SA, Australia


Sequencing the Y chromosome of heterogametic male insects can reveal genes involved with male

fertility and sex determination, plus provide candidate regions for transgene insertion. However, male

limited Y chromosomes are generally gene poor, highly repetitive and can lack any interspecific

similarity between related species, which make them difficult to identify and validate. Here we report

a series of Y chromosome scaffolds from Bactrocera tryoni (Queensland fruit fly, Qfly), a significant

horticultural pest in Australia. Genotype-by-Sequencing (GBS) data from three independent crosses

identified 73 scaffolds with a combined length of 1.3 Mb, which were only present in male progeny.

Re-sequenced genomes of pooled B. tryoni males or females were then aligned to these scaffolds to

further assess which were likely to be genuine Y chromosome regions. Robust male specific PCR assays

were designed and validated for eight scaffolds using DNA from a laboratory strain and field collections,

and these regions may be useful for future male-only transgene insertions. Only one scaffold contained

a predicted protein coding gene, with five exons a 1725 base pair open reading frame, that we name Bt-

Y1. PCR analysis confirmed Bt-Y1 is expressed in early embryos plus male flies and the gene evolved

sometime after Bactrocera split from Ceratitis, probably through gene duplication and subsequent

degeneration, although its function remains unknown.


On “Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains

Produced by Genetic, Transgenic or Symbiont-based Technologies”

Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation of FRT/Flp recombinase-mediated cassette

exchange in Ceratitis capitata

AUTHOR (S): Roswitha Aumann1, Irina Häcker1,2, Alfred M. Handler3, Marc F. Schetelig1,2

ORGANIZATION: 1 Justus-Liebig-University Gießen, Institute for Insect Biotechnology, Department of Insect

Biotechnology in Plant Protection, Winchesterstr. 2, 35394 Gießen, Germany 2 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Divison of

Bioresources, Department of Insect Pest and Vector Control, 35394 Gießen, Germany 3 Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service,

US Department of Agriculture, Gainesville, Florida, USA


Abstract:

The genomic position of a transgene strongly influences its expression and functionality. Therefore, it

is highly desirable to use a positively evaluated landing site for various transgenes. Furthermore, a

comparison of the potential of different transgenes is only possible if they are integrated at the very

same genomic position.

One way to site-specifically modify previously integrated transgenes is via the phiC31-mediated

integration system, which was establish in C. capitata (Medfly) previously. However, recombination

sites (attP and attB) are not preserved after phiC31-integrase recombination, as they are converted to

incompatible attL or attR sites. Thus, they can be used only once.

Multiple successive recombinations events are possible when using a recombinase-mediated-cassette

exchange (RMCE) system like FRT/Flp. RMCE in such system is based on double-recombination

between two pairs of heterospecific recombination sites (FRT), which are flanking the gene cassettes

and are restored after recombination via the site-specific recombinase Flp (‘flipase’).

To establish FRT-RMCE in Medfly we injected embryos of two homozygous FRT landing site lines

with different concentrations of donor plasmid and Flp-recombinase expressing plasmid to mediate the

exchange of DsRed to EGFP or vice versa. Results will be presented and discussed.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Conditional Lethal Tephritid Strains for Improved SIT

and Genetic Stability

AUTHOR (S): Zhao, Y. and Handler, A.M.

ORGANIZATION: Center for Medical, Agricultural, and Veterinary Entomology, USDA,

ARS, 1700 SW 23rd Drive, Gainesville, FL, 32608 USA


Despite increased efficiency and cost effectiveness, genetic approaches to pest population control based

on conditional lethality are subject to genetic breakdown due to mutations (and other genetic aberrations)

affecting the lethality system. Such breakdown will diminish, if not eliminate, the effectiveness of the

population control system and allow survival of transgenic populations that might be refractory to

further control by the same or similar lethality system. To evaluate the frequency of genetic breakdown,

and to potentially mitigate lethal revertant survival, two independent conditional lethal systems are

being tested in D. melanogaster. These include the Tet-off embryonic lethality system (Horn &

Wimmer, 2003, Nat Biotechnol, 21:64), where embryonic expression of the hid cell death gene is

suppressed by tetracycline, and a dominant temperature-sensitive (DTS) pupal lethality system, based

on the Prosβ21 DTS mutation (Nirmala et al., 2009, Insect Mol Biol, 18:333). Both systems will be

reared under large-scale conditions to quantify the frequency of F1 survival under non-permissive

conditions, and then tested as a combined redundant dual-lethality system to evaluate its ability to more

effectively eliminate embryonic lethal survivors. Currently, an initial phase of large scale rearing for

the Tet-off embryonic lethality system has been completed, with >660,000 zygotes heterozygous for

the lethality system screened for survival to adulthood on Tet-free diet. F1 adults survived at a frequency

of 0.008%, comparable to F1 survival in small-scale tests in the original strain (0.01%). Heritable

survival was tested by F1 backcrosses which yielded F2 survival in 11 lines at a frequency of ~17 x 10-

6, with an additional F2 backcross suggesting that second-site maternal effect suppression of lethality

may have been selected for in 7 of the lines. Three of the remaining four survivor lines were analyzed

for primary genetic aberrations in the driver and lethal effector cassettes, revealing deletions of the hid

gene in two lines, and a small knock-out deletion in the tTA driver cassette in one line. If the fourth line

also has a primary site defect, these aberrations will have occurred at a 6.1 x 10-6 frequency. This study

has important implications for understanding the frequency and molecular basis of genetic breakdown

in Tet-off conditional lethal strains. Future tests will determine whether use of redundant DTS pupal

lethality will effectively suppress the survival of embryonic lethal survivors.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: CRISPR/Cas9 based multifactorial reproductive sterility

for SIT approaches and transcriptomics analysis on reproductive biology of Ceratitis

capitata

AUTHOR (S): Hassan M. M. Ahmed, Kolja N. Eckermann, Ingrid M. Curril, Musa D. Isah &

Ernst A. Wimmer

ORGANIZATION: Georg-August-University Goettingen, Dept. of Developmental Biology,

Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, GZMB,

Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany


Our aim is to apply the CRISPR/Cas9 system in a non-gene-drive scenario for causing multifactorial

reproductive sterility in SIT. Classic SIT based on sterilization by irradiation is an exception in the

resistance development context, as the radiation-induced breaks of the chromosomes are random and

vary among all individuals thus providing built-in redundancy. Ideally a transgenic reproductive

sterility system should itself also be highly redundant to cause many different lethal mutations. To

achieve this, we develop conditional chromosome shredding based on transgene combinations. By

transgenic expression of several short guide RNAs, it has been shown that the Cas9 endonuclease can

be aimed at several diverse targets, which can lead to a mutagenesis rate of up to 100%. By using the

spermatogenesis-specific b2tub promoter, Cas9 will be restricted and expose only the sperm

chromosomes. To cause chromosome shredding, guideRNAs can be employed to direct Cas9 to several

repetitive sequences. The induced double strand breaks will lead to large chromosomal aberrations

causing aneuploidies that will mediate multifactorial reproductive sterility. Targeting many

chromosomal locations will thus provide the intended redundancy bringing the transgene-induced

reproductive sterility a step closer to the built-in redundancy of radiation-induced sterility. In contrast

to radiation, however, the sperm-restriction will save somatic tissues and make the production of

otherwise healthy, fit, and competitive males possible.

To transfer such systems to the emerging fruit pest, the cherry vinegar fly Drosophila suzukii, we have

started to evaluate both classical transgenic approaches by transposases, site-specific recombination,

and the CRISPR/Cas9 genome editing system. In addition to isolate respective genes – such as b2tub,

vasa, nanos, sryα, hsp70, U6, from D suzukii, – we have already started to develop sperm-marked, attP-

docking, and embryonic driver strains. In respect to CRISPR/Cas9, we have identified the endogenous

U6 and hsp70 promoters to be efficient for expression of gRNAs and Cas9, respectively.

In addition, we have performed a detailed transcriptomics analysis based on RNAseq of male and

female reproductive organs of Mediterranean fruit fly Ceratitis capitata: testes and male accessory

glands versus spermathecae, fertilization chamber, and female accessory glands of virgin and mated

females. In this approach, we could identify many sequences encoding secreted peptides in the male as

well as copulation-induced enzymes in the female.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Modifying mosquito gut microbiota to induce male

sterility through RNA interference: optimization of dsRNA production and

encapsulation for gene silencing.

AUTHOR (S): Claudia Paiz, Pamela Flores, Gabriela Lara, Paula Villatoro, Pamela

Pennington

ORGANIZATION: Center for Biotechnology Studies and Biochemistry Department,

Universidad del Valle de Guatemala.


Abstract:

Introduction. In Guatemala, Anopheles albimanus is the primary vector of malaria. Data suggest the

emergence of insecticide resistance, threatening the malaria elimination goal in the region. The sterile

insect technique is a potential control method that could be applied in areas where insecticides are no

longer effective. We propose to produce sterile male mosquitoes by feeding larvae with dsRNA specific

for speramatogenesis genes. We also intend to reduce female development to improve the production

process, by silencing a female-biased actin gene and female-specific doublesex.

Methods. We have determined the expression profiles of bol and zpg in all stages and sexes through

qPCR. We have cloned stable constructs for the spermatogenesis genes (bol and zpg), a control gene

and a female-biased actin gene, identified through 3´ RACE. We optimized the production of bol and

zpg dsRNA for oral delivery in larval stages. We characterized microscopically chitosan encapsulation

of dsRNA for oral delivery, determining particle size and physicochemical characteristics. We are

currently evaluating the phenotypes and silencing levels after feeding larvae with unencapsulated

dsRNA of bol, zpg and female-biased actin.

Results. We found that bol and zpg are expressed at similar levels in all larval stages and in both sexes

in pupae. We identified a female-biased actin gene that is expressed at higher levels in female pupae

and adults. In adults, bol is equally expressed in both sexes, however, zpg is more highly expressed in

females. Chitosan dsRNA particles are a combination of micro and nanoparticles with amorphous shape

and viscous consistency.

Conclusions. We have optimized the production of zpg and bol dsRNA from E. coli and the

measurement of gene expression in all stages. We are now in the process of evaluating silencing

phenotypes. We are pending the development of a method to silence genes involved in female

development to improve the mosquito production methods.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Sex determination in the common housefly: a tale of

intraspecific variations at the instructive level

AUTHOR (S): Svenia Heinze, Akash Sharma, Tea Kohlbrenner, Yanli Wu, Luca Lenzi, Anja

Bösch, Louis van de Zande, Ernst Wimmer, Mark Robinson, Leo Beukeboom and Daniel

Bopp

ORGANIZATION: University of Zurich, Institute of Molecular Life Sciences


Abstract:

Despite sex determination being a fundamental developmental process in bisexual species, its genetic

basis is remarkably variable and evolutionary labile. This is particularly true for the common housefly,

Musca domestica, which co-opted at least four different sex determining systems and hence presents

itself as a remarkable example of sex determination plasticity. Our studies indicate that these variations

center around the regulation of the conserved switch gene transformer (tra) and involve changes in the

use of upstream regulators such as the recently identified male determiner, Mdmd. This male determiner

arose from a duplication of the generic spliceosomal factor CWC22, also referred to as nucampholin

(ncm). Mdmd is located on the Y chromosome in XY strains but can also be found on autosomal

chromosomes in natural populations. Organization of the genomic region around Mdmd in these strains

suggest that a large part of the original region has translocated to the new site where it reassumed its

role as a male determiner. Factually, these Mdmd bearing autosomes have become proto-Y

chromosomes. But not only translocations of a pre-existing M factor may have contributed to create this

diversity in houseflies, we have now also evidence for the existence of a male determiner on

chromosome I different from Mdmd. Furthermore, we are investigating a maternal determiner which

renders females arrhenogenic. Females carrying this dominant Ag mutation produce only sons because

they are devoid of maternal TRA, a pre-requisite to activate tra and the female program in the zygote.

Several findings suggest that Ag is derived from a male determiner which lost its somatic but retained

its germline activity to repress tra. Finally, we even identified a dominant female determiner in the

housefly which appears to be a gain-of-function allele of tra, traD. This traD chromosome has effectively

become a proto-W chromosome which rapidly dispersed on all continents. Altogether our studies show

that seemingly different sex determination systems (Y-linked versus autosomal male determiners,

maternal determiner, female determiner) can arise from subtle changes in an otherwise well conserved

pathway.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Gene targeting of the female determining transformer gene

in the major agricultural pest Ceratitis capitata, the mediterranean fruitfly: an

unexpected CRISPR/Cas9 interference effect.

AUTHOR (S): Primo1, P., Meccariello1, A., Gucciardino1, M., Forlenza1, F., Perrotta1, M.,

Monti1, S., Buonanno2, M., Gravina1, A. Ruggiero2, A., Papathanos3, P., Salvemini1, M.,

Vitagliano2, L., Giordano1, E., and Saccone1, G.

ORGANIZATION: 1Department of Biology, University of Naples “Federico II”, Napoli, Italy. 2Institute of Biostructures and Bioimaging (IBB), CNR, Naples, Italy. 3Department of Experimental Medicine, University of Perugia, Italy.


Abstract:

The Mediterranean fruitfly Ceratitis capitata (medfly) is an agricultural pests of high economic impact.

We targeted medfly transformer, the key female determining gene with the ability to autoregulate, in

a way similar to Sex-lethal in Drosophila. We injected Cas9-sgRNA RNPs into Ceratitis female-only

XX embryos which developed into G0 adult XX flies, with up to 50% presenting a complete

masculinized phenotype and exclusively a male-specific Cctra splicing pattern. However, Cctra DNA

sequence analysis revealed a lack of gene editing events in both G0 and G1 progenies. We reasoned

that Cctra transcription was transiently suppressed by an unplanned CRISPR interference (CRISPRi),

usually achieved by a defective Cas9 variant (dCas9) unable to cut DNA. This unexpected CRISPRi

seems to have caused a masculinization of XX embryos by a transient biallelic gene transcriptional

silencing rather than biallelic DNA mutations. We propose that, similarly to dCas9, a wild type Cas9

protein can cause CRISPR interference in autoregulated genes that are required very early during

embryogenesis. We propose that also a wild type Cas9 protein, similarly to a defective Cas9 version

(dCas9), can cause CRISPR interference, in genes able to positively autoregulate.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: New olfactory and reproductive targets for alternative

olive fly control

AUTHOR (S): Tsoumani KT, Gregoriou M-E, Zorbas E and Mathiopoulos KD

ORGANIZATION: Department of Biochemistry and Biotechnology, University of Thessaly,

Greece


The olive fruit fly, Bactrocera oleae, is the major threat of the olives worldwide. Alternative methods

of biological control are needed in order to replace the principal chemical suppression of its populations.

A manipulation of two different “harmlessness” systems, olfactory and reproductive, that are involved

in mating behavior, reproduction and food localization can be a promising improvement of such

strategies.

The analysis of head transcriptomes revealed genes that are implicated directly or indirectly to

premating sexual communication and reproductive behavior. Likewise, transcriptomics analysis of

female and male pre- and post-mated reproductive tissues resulted in genes with potential role in

reproduction. We functionally analyzed two olfactory (the olfactory co-receptor Orco and the Sensory

Neuron Membrane Protein, SNMP) and four reproductive genes (sex peptide receptor, yellow, lingerer

and troponin-C) through dsRNA-mediated silencing and phenotype investigation. Our analysis suggests

that the examined genes play crucial roles in the reproductive behavior of the olive fly, since pre- and/or

post- mating processes were affected. Furthermore, the observed behavioral changes render these genes

potential targets for the improvement and specialization of the olive fruit fly population control

techniques in order to be more efficient and environmentally friendly.

Regarding the molecular manipulation of the sex in this fly, the necessary transformation constructs

were developed for the engineering of female-specific conditional lethal strains so that females can be

eliminated at will very early during embryogenesis. The tetracycline suppressible (Tet-off) regulatory

system was selected to control lethal effector expression. For the driver construct a ~1kb region of the

embryonically active B. oleae serendipity α (Βο-sry-α) promoter was isolated and cloned into a

piggyBac transformation vector to drive the Tet-transactivator (tTA) expression. We further analyzed

the pro-apoptotic cell death gene head involution defective (hid) of B. oleae which was already used as

a lethality factor in other species of the Tephritid family. Its sequence shows very high conservation

with already used hid sequences, retaining 100% homology at the essential regions of A. ludens hid

which has already been used in transgenes for A. ludens and A. suspense, indicating the potential

applicability of the same transgene for the olive fruit fly. Successive crossings between the driver and

the effector lines will ultimately establish the engineered embryonic sexing system (EESS) in B. oleae

allowing the elimination of females prior to the larval stage, avoiding the high cost of rearing.


On “Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies”

Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Comparative evaluation of the chemioreception and

reproduction performance of different populations and strains of sanitary and

agricultural importance

AUTHOR (S): Alessandro Di Cosimo1, Nidchaya Aketarawong2, Mariconti M1, Mosè

Manni1,3, Sujinda Thanaphum2, Francesca Scolari1, Ludvik M Gomulski1, Anna R

Malacrida1, Giuliano Gasperi1

ORGANIZATION: 1Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy 2Department of Biotechnology, Mahidol University, Bangkok, Thailand 3Department of Genetic Medicine and Development, University of Geneva Medical School

and Swiss Institute of Bioinformatics, Geneva, Switzerland (present address)


Abstract:

Our research topics have been addressed to:

a) Characterization of Y chromosomes in different tepritid species. Apart from elucidating

the nature of the male determining factor, understanding the evolutionary history of sex

chromosomes within the Tephritidae species may help for the generation of GSS strains for

control purposes. On this background, after the characterization of Y sequences from the medfly

and olive fly, we are now examining the Y chromosome of Bactrocera dorsalis. Four sequences

have been obtained and characterized in lab strains and in several samples from different

geographic origins. The identified sequences provide 1) entry points to explore the nature of Y

cromosome in B. dorsalis and to infer its evolutionary history; 2) male specific markers for

sexing embryos in very early developmental stages; 3) Y specific markers for tracing the

inheritance of this chromosome in different crosses/populations and in monitoring the stability

of Y/traslocated strains.

b) Structural and functional characterization of OBPs in Aedes albopictus. The

characterization of chemioreception is an important step for interpreting the performance of

males and females of different strains and populations and in mass rearing facilities. Three OBPs

which display differential expression in different populations of Aedes albopictus have been

considered. They have been characterized for their 1) tissue expression and developmental

patterns; 2) binding affinities and structural features. Two of them pertain to the Plus-C OBP

subfamily, so that this study will be important to clarify the role of OBPs not only in the olfaction

but also in other biological processes where they are found to participate.


On “Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies”

Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation of transgenic lines for the population control of

Ceratitis capitata and Aedes aegypti.

AUTHOR (S): Margareth Lara Capurro Guimarães1; Danilo O. Carvalho2; Jair F. Virginio3

ORGANIZATION: (1) São Paulo University; (2) FAO/IAEA Joint Division IPCL; (3)

Biofábrica Moscamed Brasil.


Abstract:

The increasing number of cases of infection by different arboviruses, for instance dengue, chikungunya

and zika, it is necessary to develop new techniques for controlling the transmission of these pathogens.

The genetic manipulation allows the obtention of genetically modified mosquitoes that are capable of

suppressing the wild population or prevent the transmission of etiological agents causing diseases. The

study aimed to establish genetically sterile strains of Aedes aegypti. The genetic construct is designed

to provide conditional sterility to males in the presence or absence of antibiotic in the environment

where these mosquitoes develop during the larval stage. Six transgenic lines were obtained, where five

have amplification of the desired fragment of cDNA. Among these strains only two showed a significant

difference in the challenge of sterility with reduced fertility of 38.7% and 62.3% in the absence of the

antibiotic doxycycline. Thus, without the need to use radiation for sterile insects, it is possible to

improve the quality of released adult males and increase the competitiveness of the same to compete

with wild females and additionally generate the desired frame population suppression. Regarding the

activities about the fruit fly, Ceratitis capitata, all bureaucratic import processes continued to be carried

out to bring pupae of the C. capitata FSEL#32 strain. News documents had to be prepared and submitted

for analysis and approval by the Department of Plant Health, Ministry of Agriculture. In December

2017, the "Import License" for FSEL # 32 transgenic strain was granted by the Ministry of Agriculture.

However, this license only authorizes the importation of biological material and its maintenance under

containment regime.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Mass rearing of the triple Wolbachia-infected Aedes

albopictus HC strain

AUTHOR (S): Dongjing Zhang & Zhiyong Xi

ORGANIZATION: Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou,

Guangdong 510080, China


Abstract:

The triple Wolbachia-infected (wAlbA, wAlbB and wPip) of Aedes albopictus HC strain has been

developed by transferring the wPip from Culex mosquito to the natural double Wolbachia-infected

(wAlbA and wAlbB) Ae. albopictus. The HC strain induces both CI toward the wild type mosquito and

resistance to dengue/zika virus. In addition, minor fitness cost is observed after wPip forms symbiosis

with the host. Thus, the HC strain has been considered to be suitable for controlling the wild Ae.

albopictus population by using the combined SIT/IIT strategy. Mass production is an important part of

SIT/IIT strategy when apply in area-wide. To evaluate the rearing efficiency of HC strain, we compared

two larval rearing units (Wol-unit and IAEA-unit) for their capacity of producing male pupae

production. In addition, we tested three types of adult maintenance cages for the capacity of producing

egg production. The average male pupae production was 0.89 × 105 for the Wol-unit and 3.16 × 105 for

the IAEA-unit. No significant difference was observed on the male mating competitiveness index of

HC males, regardless of whether they were reared in the Wol-unit or IAEA-unit. An adult rearing unit

for HC strain on the basis of big cage structure (30 × 30 × 90 cm) has been developed with the capacity

of producing 10 million eggs within 15 days. Our results indicate that the HC strain, either in their larval

or adult stages, has a high mass rearing efficiency with our developed standard mass rearing methods,

which is useful for the establishment of a mosquito mass-rearing facility.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Comparing Rearing Efficiency and Competitiveness of

Males from Salaya Genetic Sexing Strains (Bactrocera spp.) Including the Refinement

and Transfer of Existing Technology for the Improvement and Application of Strains

for Area-Wide Integrated Pest Management.

AUTHOR (S): Sujinda Thanaphum, Nidchaya Aketarawong, Siriwan Isasawin,

and Kamoltip Laohakieat

ORGANIZATION: Regional R&D Training Center for Insect Biotechnology (RCIB),

Department of Biotechnology, Faculty of Science, Mahidol University, Thailand


Abstract:

The Bactrocera dorsalis Salaya1 genetic sexing strain; from a modular mass-rearing laboratory (clean

colony); has been filtered, quality assured and controlled, and monthly transferred to the mass-rearing

facility of the Regional R&D Training Center for Insect Biotechnology (RCIB). This is to start up an

initiation colony management system in the mass production facility. Basic fitness parameters of the

clean and initiation colonies have been continuously monitored for the optimization of rearing

efficiency during the year 2017-2018. Rearing methods were optimized. The level of egg hatching,

pupae recovery, and adult emergence of the Salaya1 clean and initiation colonies are similar. In addition,

the productivity rates of the two type of colony management (the clean and the initiation) are

comparable after mass-rearing adjustment. This infers that the fruit fly rearing management is stable

and consistent in the modular mass-rearing unit and the mass-rearing facility.

The pupae color sorter machine was applied to separate brown and white pupae. It was found that the

pupae survival rate from the sorter is lower than the manual separation. Nonetheless, the rate of

abnormal pupae development is similar in both of the sorting systems.

The research work is in the process of asking licences for using and/or producing fruit fly for scientific

purposes. There is a new law on animal (including all insects) for scientific purposes followed the

‘Animal for Scientific Purpose ACT, B.E. 2558’ in Thailand. However, Animal Biosafety Level (ABSL)

1 and 2 facilities have been constructed, audited, certified, and registered to be complied with the new

law.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation and improvement of Anastrepha ludens

strains for SIT: a) Transgenic and b) Tapachula-7 under mass rearing conditions.

AUTHOR (S): D. H. Orozco Davila, J. S. Meza, M. Roblero Roblero, V. García Martínez, J.

Ibañez Palacios, S. Aguirre, M. F. Ruiz Pérez.

ORGANIZATION: National Program Fruit Flies. SAGARPA-SENASICA.


Currently 60 million pupae per week of the genetic sexing strain (GSS) "Tapachula-7" of the Mexican

fruit fly, Anastrepha ludens, are being produced at the MOSCAFRUT facilities in Mexico, which means

that around 30 Millions of sterile males per week, are being released in different states of the country,

for sterile insect technique application. The integrity of the sexing system is maintained through a filter

rearing system (FRS), where the recombinants are removed from an initial small colony. However

despite of the FRS application a gradual increasing of recombinant individuals has been observed,

through the generations. Thus, in order to improve the stability in this sexing system another two

translocation were analyzed (Tap-4 GSS and Guate-10 GSS) in comparison with the Tapachula-7 GSS.

The breakdown of the 3 GSS were monitored and a cytogenetic analysis on mitotic chromosome were

performed. During the 10 generations observed, there was a tendential increase of females emerging

from the normal brown pupa in Tap-7 and Guate-10, while males emerging from black pupa (bp) were

found at very low frequency with not tendency to increase. In contrast, an increase of males emerging

from black pupa was observed in Tap-4, and very few females emerging from normal brown pupa. The

mitotic chromosome analysis shows different sizes of the translocated fragments between GSS s; In

Tap- 4, the translocated chromosome (2Y) was found reduced in 2.51% respect to its normal

homologous chromosome 2, Tap-7 shows a reduction of 2.30% and Guate-10 only a reduction of 0.77%.

Which mean that Guate-10 carry the smallest translocated fragment and also shows the best fitness in

mass rearing conditions.

The last year the slow larva mutant (sl) was incorporated into the Tapachula-7 GSS to observe the

viability of a self-sexing during the puparation phase, this new GSS was named Tapachula/slow-7. Its

population was increased to semi-mass rearing condition and it was determined that after 10 days of

larval development and at 21 h of puparation time, it is possible to separate the 55.82% of pupa from

the rest of larva (the larva will be tested for the rearing of parasitoid). In this percentage of pupa, was

found the 80.10% and 19.89 % of male and female respectively of the total produced. Additionally, the

recombination occurrences was analyzed and it was determinates that the bp allele is located in the

translocated fragment (to a distant of 0.01 cM from the breakpoint), while the sl allele was kept in the

chromosome (to a distant of 0.16 cM from the breakpoint).




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation of genetic and biotechnological tools towards

the development of an Anastrepha fraterculus sexing strain

AUTHOR (S): Lanzavecchia Silvia, Conte Claudia, Giardini Cecilia, Scannapieco Alejandra,

Milla Fabián, German Crippa, Segura Diego and Jorge Cladera

ORGANIZATION: Instituto de Genética (IGEAF), Instituto Nacional de Tecnología

Agropecuaria (INTA). Argentina.


Anastrepha fraterculus is currently described as a complex of cryptic species. Multidisciplinary efforts

to elucidate genetic entities in the complex have demonstrated the existence of at least 8 morphotypes

distributed in the American continent. After the analysis of wild and laboratory populations of

Argentina, only one morphotype was registered (named A. fraterculus sp1 o Brazilian 1 morphotype).

Taking this into account, the development of control strategies against A. fraterculus, as the Sterile

Insect Technique (SIT), must be applied at a regional level considering mating compatibility among

morphotypes. In particular, the main goal of our project is to contribute with genetic and

biotechnological tools supporting the development of an A. fraterculus sp1 genetic sexing strain (GSS)

useful for SIT implementation. During the last year of the project, we continued working on the gene

expression analysis of A. fraterculus transcriptome, considering mature adults (females vs. males) and

an immature stage (72 h embryo). This approachment has provided valuable information of genes

potentially involved in molecular mechanisms of interest (e.g. early sex-determination pathways). and

genes used as main components of constructs for germ line transformation. Regarding this last issue,

important knowledge was acquired using A. suspensa as a model to introduce the use of egg

microinjection in the obtaining of transformed strains of A. fraterculus sp. 1. In addition, we continued

working in the cytogenetic characterization of wild populations to differentiate them from the

established laboratory colonies.



Produced by Genetic, Transgenic or Symbiont-Based Technologies”

Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Symbiotic and genetic analysis evaluation of strains used

in SIT

AUTHOR (S): Augustinos AA1,2, Gouvi, G.3, Kyritsis G1,4, Nikolouli K2, Caceres C2, Mintzas

A1, Tsiamis G3, Bourtzis K2

ORGANIZATION: 1Department of Biology, University of Patras, Greece; 2Insect Pest Control

Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture,

Seibersdorf, Vienna, Austria; 3Department of Environmental and Natural Resources

Management, University of Patras, Agrinio, Greece; 4Laboratory of Entomology and

Agricultural Zoology, Department of Agriculture Crop Production and Rural Environment,

University of Thessaly, N. Ionia Magnisia, Greece


Abstract:

Laboratory adaptation can be a severe process that may impact the quality and fitness of strains that a) are being used or are candidates for SIT applications and, b) are being used as ‘wildish’ material in mating compatibility/competitiveness experiments for the evaluation of SIT important strains. In the genetic level, domestication can drastically reduce genetic diversity in the very few first generations, depending on founding population size and rearing practices. In the symbiotic level, although not fully resolved yet, domestication seems to reduce symbiotic diversity and is accompanied by loss of previously important symbiont, the increase of others that are present in low relative abundances or the ‘de novo’ emergence of novel symbionts. These conclusions derive from the indirect comparison of symbiotic communities of natural and laboratory populations of different species. The degree of domestication and rearing practices are expected to be important factors in the structuring of the gut symbiotic communities.

Using the Mediterranean fruit fly, Ceratitis capitata, as a model species, we tried to provide more direct evidence and a follow up of genetic and gut symbiotic structuring during lab adaptation. For this purpose, one population deriving from Greece was introduced in IPCL and was monitored for 10 generations. To gain insight in the effect of rearing practices on structuring, this population was divided in two and reared using two different approaches. Our results indicate as expected that the degree of domestication, the developmental stage sampled, and the rearing practices heavily influence the structure of the gut symbiotic communities. For the genetic analysis a set of 8 microsatellite markers has been selected and optimized against the F0 and F1 generations with the preliminary results from 30 individuals of F0 showing a rather limited genetic diversity of the original population that was colonized.

In order to shed light to the underlying differences that may influence the Quality Control parameters of important laboratory strains (such as the medfly VIENNA 7 and 8 GSS), the putative gut symbiotic contribution towards the exceptional properties of the Vienna 8 strain from Israel were examined using an Illumina amplicon sequence approach coupled with a culture-dependent.

In collaboration with IPCL, this study will expand to a) include the analysis of more, recently introduced laboratory populations from different species, geographic origins and hosts, b) provide standardized and universalized protocols for the monitoring of genetic and symbiotic changes, (c) identify putative bacterial markers that are tightly link with fitness characteristics, and, (d) explore the utilization of NGS based approaches for the monitoring of the laboratory adaptation process.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Evaluation of different strains of Anastrepha ludens

Loew and Ceratitis capitata Wied.

AUTHOR (S): Ramírez-Santos Edwin

ORGANIZATION: MOSCAMED PROGRAM/Guatemala


Abstract:

A series of experiments were conducted in order to develop and evaluate different strains of Anastrepha

ludens Loew and Ceratitis capitata (Wied.) with high efficiency in mass rearing and competitiveness

in the field. The main characteristics of the viable candidates for its use in SIT, AW-IPM programs are:

stability of the genetic sexing mechanism, presence of morphological or genetical markers, low

operational costs and adequate competitiveness of the sterile males. For these reasons, our approach has

been to obtain and select colonies of flies (for A. ludens and C. capitata) with an adequate performance

to have an opportunity to be used in the mass rearing.

For Anastrepha ludens Loew, the following results were registered: 1) two new strains, EP10a = Familiy

10 females x wild irradiated males, and EP254 = Tap-7 females x wild irradiated males, were obtained

from a selection procedure among 1000 lines coming from crosses between fertile females and low dose

irradiated wild males. When compared with familiy 10, these two new strains had higher egg fertility

(+17 %) and a higher egg to pupae conversion rate (+8 %), for both new strains.

2) when comparing the new strain Tap/slow-7 GSS against Family 10, it showed a slower female (black

colored pupae) development rate. In the first collection (24 hrs), only 20 % was black colored (females)

and 80% Brown (males); in Family 10, for the same collection 80 % black pupae (females) were

recovered. The slow development of the Tap/slow-7 GSS females might imply a higher efficiency in

mass rearing and the potential advantage of releasing 100 % males in the field; however, the Tap/slow-

7 GSS when compared with Family 10, showed a lower egg to pupae conversion rate (15 vs 39 %) and

lower number of eggs per female per day (21 vs 31). A porcess of crossing and line selection is under

development to find lines of strains with slow development, such as Tap/slow-7 GSS, but with higher

yield. 3) the search for morphological mutants by means of chemical mutagenesis (ethyl-methyl-

sulphonate EMS), geneated several lines of flies with differential colors in puparium and eyes. From

these lines, the E59-V100 with black puparium showed an adequate performance in small scale mass

rearing. Three additional lines showed, when thermal treatment was applied, a pattern of mortality in

the eggs instar. Currently, a protocol for crosses and selection is under development, exploring the

possibility of finding a selective thermal sensitivity for females.

For Ceratitis capitata (Wied.), the following results were obtained: 1) as shown in previous studies, the

genetically modified strain VIENNA 8 1260 is a strong, viable candidate for its use in SIT, AW-IPM

programs due to the high stability of the genetic sexing mechanism and the steady expression of

fluorescence through all the generations; however, this strain has a lower yield when compared with

the VIENNA 8 D53- strain, currently under mass rearing conditions. In order to increase their potential

use in mass rearing, a process was developed for the selection of high performance lines. 2) The

VIENNA 8 1260-181 was selected because it had a significantly higher yield (+40 %) as compared to

the original VIENNA 8 1260 strain. The use of the VIENNA 8 1260-181 strain opens the possibility of

establishing the mass rearing of transgenic strains with operational costs equivalent to those of other

strains under mass-rearing systems.




Bangkok, Thailand

18–22 June 2018

TITLE OF WORKING PAPER: Gut bacteria improve the fitness of Bactrocera dorsalis:

influence on development and reproduction

AUTHOR (S): Yichen Wang, Yushan Li, Zhaohui Cai, Zheng Zhao, Zhichao Yao, Ping

Zhang, Shuai Bai, Muhammad Fahim Raza, Hongyu Zhang*

ORGANIZATION: State Key Laboratory of Agricultural Microbiology, Key Laboratory of

Horticultural Plant Biology (MOE) and Institute of Urban and Horticultural Entomology,

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan,

430070, China


Abstract:

The symbiotic gut microbial community is generally known to have a strong impact on the fitness of

its host. However, the mechanisms by which impact of gut microbiota on the hosts’ fitness under

restricted environmental conditions such as poorly nutrient diet are less clear. We investigated

influences of the intestinal symbiosis Klebsiella oxytoca BD177 on development and reproduction of

B. dorsalis by adding BD177 in adult and larvae’s diets to get a better understanding of host-microbiota

interactions under different levels of nutrients availability.

In the experiment about influences on adults, the results showed that fecundity of restricted nitrogen

source females was significantly increased after supplemental feeding strain BD177, but there was no

significant fecundity increase in adequate nitrogen source females. Antibiotics treatment would result

in a significant decrease in reproductive capacity of fly adults and expression of its BdYP, BdInR, BdAkt

and BdS6K genes, the key signaling molecules of Insulin/TOR signaling pathways. Reinfecting strain

BD177 resulted in a significant increase and recovering in oviposition performance of both adequate

and restricted nitrogen source females. In addition, the recovery effect of BD177 under low nitrogen

condition was more significant than that of high nitrogen condition, suggesting that the level of nitrogen

in food affects the interaction between host and gut symbiotic bacteria.

Second, we evaluated effects of the intestinal probiotics K. oxytoca BD177 on development and

reproduction of B. dorsalis by adding BD177 into the larval diet. The results showed that supplementing

the intestinal probiotics K. oxytoca BD177 in the larval diet significantly increased the B. dorsalis pupal

weight, eclosion rate and reproduction but did not influence the egg hatching rate and mating

competitiveness of the adults.

In additional, to provides a basis for understanding the beneficial interactions between K. oxytoca

BD177 and host inscet, whole genome of the strain was sequenced and analyzed. In summary, our

results indicate that the intestinal probiotics K. oxytoca BD177 can be a potentially important factor in

maintaining host B. dorsalis fitness.

*Corresponding author. Tel.: +86 2787286962.

Email: [email protected]

Granted project: This work was supported by the International Atomic Energy Agency’s Coordinated

Research Project (No. D42016), the National Natural Science Foundation of China (No. 31572008), the

earmarked fund for the China Agricultural Research System (No. CARS-26).

LIST OF PARTICIPANTS

ARGENTINA

1. Ms Silvia Beatriz LANZAVECCHIA

Instituto Nacional de Tecnología Agropecuaria

Rivadavia 1439

1033 AAE BUENOS AIRES

ARGENTINA


AUSTRALIA

2. Mr Peter CRISP

Mr Peter Crisp

South Australian Research and Development Institute (SARDI)

Gate 2b, Hartley Grove

GPO Box 397

ADELAIDE, SA 5001

AUSTRALIA

Email [email protected]

BRAZIL

3. Ms Margareth De Lara CAPURRO-GUIMARAES

Universidade de Sao Paulo

Instituto de Ciencias Biomedicas

Av. Prof. Lineu Prestes, 2415

Butantan

05508-000 SAO PAULO

BRAZIL


CHINA

4. Mr Hongyu ZHANG

Huazhong Agricultural University

College of Plant Science and Technology

Shizishan Street 1

430070 WUHAN HONGSHAN

CHINA


mailto:[email protected]




5. Mr Dongjing ZHANG

Zhongshan School of Medicine, Sun Yatsen University

Zhongshan 2nd Road, No. 74

Microbiology Building, Floor 5, Room 602

510440 GUANGZHOU GUANGDONG

CHINA


GERMANY

6. Ms Roswitha AUMANN

FraunhoferInstitute for Molecular Biology and Applied Ecology (IME)

Project Group "Bioresources"

Winchesterstrasse 2

35394 GIESSEN

GERMANY


7. Mr A. Ernst WIMMER

Georg August University Göttingen

Institut für Zoologie, Anthropologie und Entwicklungsbiologie

Justus-von-Liebig-Weg 11

37077 GÖTTINGEN

GERMANY


GREECE

8. Mr George TSIAMIS

University of Patras

2 Seferi St.

30100 Agrinio

ETOLOAKARNANIA

GREECE

Email: [email protected]; [email protected]

9. Mr Kostas MATHIOPOULOS

Department of Biochemistry and Biotechnology

University of Thessaly

Ploutonos 26

LARISSA 41221

GREECE







GUATEMALA

10. Ms Pamela PENNINGTON

Universidad del Valle de Guatemala

18 Avenida 11-95

Zona 15, Vista Hermosa III

01015 CIUDAD DE GUATEMALA

GUATEMALA


11. Mr Edwin Mauricio RAMÍREZ SANTOS

Medfly Program Guatemala

16 Calle 3-38 Zona 10

01010 GUATEMALA

GUATEMALA


ITALY

12. Mr Giuseppe SACCONE

Università Degli Studi Di Napoli

Federico II

Dipartimento Di Biologia

Corso Umberto I

80138 NAPOLI

ITALY


MEXICO

13. Mr Jose Salvador MEZA HERNANDEZ

Subdirector de Sexado Génetico

Programa Moscafrut

Acuerdo SAGARPA-IICA

MEXICO


PANAMA

14. Mr Daniel Fernando PAULO

Short-term investigator at the Smithsonian Tropical Research Institute (STRI) and Comision

Panamá Estados Unidos para la Erradicacion y Prevencion del Gusano Barrenador del

Ganado (COPEG)

PANAMA







THAILAND

15. Mr Sujinda THANAPHUM

Department of Biotechnology, Faculty of Science, Mahidol University

272 Rama VI Road

10400 BANGKOK, RATCHATHEWE

THAILAND


UNITED STATES OF AMERICA

16. Mr Alfred HANDLER

US Department of Agriculture (USDA), Agricultural Research Service (ARS)

Center for Medical, Agricultural and Veterinary Entomology (CMAVE)

1700 SW 23rd Drive

GAINESVILLE FL 32608

UNITED STATES


17. Mr Maxwell SCOTT

North Carolina State University

2701 Sullivan Drive

P.O.Box CB 7514

27695-7614 RALEIGH, NC

UNITED STATES


OBSERVERS

AUSTRALIA

18. Ms Simon WADE BAXTER

ARC Future Fellow

School of Biological Sciences

The University of Adelaide

ADELAIDE, SA 5005

AUSTRALIA






GERMANY

19. Mr Hassan MUTASIM MOHAMMED AHMED

Georg August University Göttingen

Institut für Zoologie, Anthropologie und Entwicklungsbiologie

Justus-von-Liebig-Weg 11

37077 GÖTTINGEN

GERMANY


20. Mr Marc SCHETELIG

FraunhoferInstitute for Molecular Biology and Applied Ecology (IME)

Project Group "Bioresources"

Winchesterstrasse 2

35394 GIESSEN

GERMANY


ITALY

21. Mr Giuliano GASPERI

Department of Biology and Biotechnology

University of Pavia

Via Ferrata 9

27100 PAVIA

ITALY


SWITZERLAND

22. Mr Daniel BOPP

Institute of Molecular Life Sciences

University of Zürich

Winterthurerstrasse 190

CH-8057 ZÜRICH

SWITZERLAND


THAILAND

23. Ms Nidchaya AKETARAWONG

Department of Biotechnology

Faculty of Science

Mahidol University

272 Rama VI, Ratchathewee

BANGKOK 10400

THAILAND







24. Ms Siriwan ISSAWIN


Faculty of Science

Mahidol University


BANGKOK 10400

THAILAND


25. Ms Kamoltip LAOHAKIEAT


Faculty of Science

Mahidol University


BANGKOK 10400

THAILAND


26. Mr Worachart SIRAWARAPORN

Center of Excellence for Vectors and VectorBorne Diseases

2nd Floor, Science Building 2, Faculty of Science

Mahidol University at Salaya

999 Phutthamonthon 4 Road

NAKHON PATHOM 73170

THAILAND


27. Ms Pattamaporn KITTAYAPONG

Center of Excellence for Vectors and VectorBorne Diseases

Faculty of Science, Mahidol University at Salaya

Science Building 2

999 Phutthamonthon 4 Road

73170 NAKHON PATHOM

THAILAND




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