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IAEA-D4.20.16-CR-3
LIMITED DISTRIBUTION
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.
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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
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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
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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).
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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
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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.
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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.
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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.
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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
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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
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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.
Page 12
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
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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
Page 14
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.
Page 15
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
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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
Page 17
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
Page 18
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.
Page 19
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.
Page 20
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.
Page 21
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
Page 22
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
Page 23
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
Page 24
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
Page 38
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
Page 39
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
Page 40
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.
Page 41
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.
Page 42
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.
Facilities and resources available.
Facilities and resources available.
Page 43
Outputs
1. A collection of strains of
agricultural importance
produced by classical genetic,
transgenic or symbiont-based
technologies with the
performance of sterile males
compared / evaluated.
2. A collection of strains of
veterinary importance produced
by classical genetic, transgenic
or symbiont-based technologies
with the performance of sterile
males compared / evaluated.
3. A collection of strains of
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,
transgenic or symbiont-based
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.
Biological material is available. QC
protocols are available or can be
developed.
Biological material is available. QC
protocols are available or can be
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.
Page 44
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.
Page 45
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.
Page 46
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
15:30 – 16:00 COFFEE BREAK
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)
Page 47
TUESDAY, 19 JUNE 2018:
MahaSawasdee 1 meeting room in the Salaya Pavilion Hotel
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.
10:30 – 11:00 COFFEE BREAK
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.
12:30 – 14:00 LUNCH: MahaSawasdee 2 room in the Salaya Pavilion Hotel
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.
15:30 – 16:00 COFFEE BREAK
16:00 – 16:30 Open discussion on the presentations
Page 48
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:
MahaSawasdee 1 meeting room in the Salaya Pavilion Hotel
08:30 – 10:30 Working groups discussion
10:30 – 11:00 COFFEE BREAK
11:00 – 12:30 Working groups discussion
12:30 – 14:00 LUNCH: MahaSawasdee 2 room in the Salaya Pavilion Hotel
14:00 – 15:30 Working groups discussion
15:30 – 16:00 COFFEE BREAK
16:00 – 17:00 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
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Drafting meeting report
18:00 Group dinner / Social event
Page 49
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
10:30 – 11:00 COFFEE BREAK
11:00 – 12:30 Drafting RCM3 final report
12:30 – 14:00 LUNCH
14:00 – 15:30 Drafting RCM3 final report
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Presentation and approval of the Final Report - General discussion
17:30 End of the 3rd RCM
17:30 Closing.
Page 50
THIRD RESEARCH COORDINATION MEETING
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.
Page 51
THIRD RESEARCH COORDINATION MEETING
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 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.
Page 52
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER Abstract:
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.
Page 53
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER Abstract:
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.
Page 54
THIRD RESEARCH COORDINATION MEETING
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
SHORT SUMMARY OF PAPER
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.
Page 55
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER Abstract:
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.
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THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER Abstract:
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.
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THIRD RESEARCH COORDINATION MEETING
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: 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.
SHORT SUMMARY OF PAPER
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.
Page 58
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER
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.
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THIRD RESEARCH COORDINATION MEETING
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: 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.
SHORT SUMMARY OF PAPER
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.
Page 60
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER Abstract:
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.
Page 61
THIRD RESEARCH COORDINATION MEETING
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)
SHORT SUMMARY OF PAPER
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.
Page 62
THIRD RESEARCH COORDINATION MEETING
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.
SHORT SUMMARY OF PAPER
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.
Page 63
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER
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.
Page 64
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER
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.
Page 65
THIRD RESEARCH COORDINATION MEETING
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 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.
SHORT SUMMARY OF PAPER Abstract:
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).
Page 66
THIRD RESEARCH COORDINATION MEETING
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 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.
SHORT SUMMARY OF PAPER Abstract:
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.
Page 67
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER
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.
Page 68
THIRD RESEARCH COORDINATION MEETING
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 different strains of Anastrepha ludens
Loew and Ceratitis capitata Wied.
AUTHOR (S): Ramírez-Santos Edwin
ORGANIZATION: MOSCAMED PROGRAM/Guatemala
SHORT SUMMARY OF PAPER
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
Page 69
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.
Page 70
THIRD RESEARCH COORDINATION MEETING
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: 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
SHORT SUMMARY OF PAPER
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.
Page 71
*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).
Page 72
LIST OF PARTICIPANTS
ARGENTINA
1. Ms Silvia Beatriz LANZAVECCHIA
Instituto Nacional de Tecnología Agropecuaria
Rivadavia 1439
1033 AAE BUENOS AIRES
ARGENTINA
Email: [email protected]
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
Email: [email protected]
CHINA
4. Mr Hongyu ZHANG
Huazhong Agricultural University
College of Plant Science and Technology
Shizishan Street 1
430070 WUHAN HONGSHAN
CHINA
Email: [email protected]
Page 73
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
Email: [email protected]
GERMANY
6. Ms Roswitha AUMANN
FraunhoferInstitute for Molecular Biology and Applied Ecology (IME)
Project Group "Bioresources"
Winchesterstrasse 2
35394 GIESSEN
GERMANY
Email: [email protected]
7. Mr A. Ernst WIMMER
Georg August University Göttingen
Institut für Zoologie, Anthropologie und Entwicklungsbiologie
Justus-von-Liebig-Weg 11
37077 GÖTTINGEN
GERMANY
Email: [email protected]
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
Email: [email protected]
Page 74
GUATEMALA
10. Ms Pamela PENNINGTON
Universidad del Valle de Guatemala
18 Avenida 11-95
Zona 15, Vista Hermosa III
01015 CIUDAD DE GUATEMALA
GUATEMALA
Email: [email protected]
11. Mr Edwin Mauricio RAMÍREZ SANTOS
Medfly Program Guatemala
16 Calle 3-38 Zona 10
01010 GUATEMALA
GUATEMALA
Email: [email protected]
ITALY
12. Mr Giuseppe SACCONE
Università Degli Studi Di Napoli
Federico II
Dipartimento Di Biologia
Corso Umberto I
80138 NAPOLI
ITALY
Email: [email protected]
MEXICO
13. Mr Jose Salvador MEZA HERNANDEZ
Subdirector de Sexado Génetico
Programa Moscafrut
Acuerdo SAGARPA-IICA
MEXICO
Email: [email protected]
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
Email: [email protected]
Page 75
THAILAND
15. Mr Sujinda THANAPHUM
Department of Biotechnology, Faculty of Science, Mahidol University
272 Rama VI Road
10400 BANGKOK, RATCHATHEWE
THAILAND
Email: [email protected]
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
Email: [email protected]
17. Mr Maxwell SCOTT
North Carolina State University
2701 Sullivan Drive
P.O.Box CB 7514
27695-7614 RALEIGH, NC
UNITED STATES
Email: [email protected]
OBSERVERS
AUSTRALIA
18. Ms Simon WADE BAXTER
ARC Future Fellow
School of Biological Sciences
The University of Adelaide
ADELAIDE, SA 5005
AUSTRALIA
Email: [email protected]
Page 76
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
Email: [email protected]
20. Mr Marc SCHETELIG
FraunhoferInstitute for Molecular Biology and Applied Ecology (IME)
Project Group "Bioresources"
Winchesterstrasse 2
35394 GIESSEN
GERMANY
Email: [email protected]
ITALY
21. Mr Giuliano GASPERI
Department of Biology and Biotechnology
University of Pavia
Via Ferrata 9
27100 PAVIA
ITALY
Email: [email protected]
SWITZERLAND
22. Mr Daniel BOPP
Institute of Molecular Life Sciences
University of Zürich
Winterthurerstrasse 190
CH-8057 ZÜRICH
SWITZERLAND
Email: [email protected]
THAILAND
23. Ms Nidchaya AKETARAWONG
Department of Biotechnology
Faculty of Science
Mahidol University
272 Rama VI, Ratchathewee
BANGKOK 10400
THAILAND
Email: [email protected]
Page 77
24. Ms Siriwan ISSAWIN
Department of Biotechnology
Faculty of Science
Mahidol University
272 Rama VI, Ratchathewee
BANGKOK 10400
THAILAND
Email: [email protected]
25. Ms Kamoltip LAOHAKIEAT
Department of Biotechnology
Faculty of Science
Mahidol University
272 Rama VI, Ratchathewee
BANGKOK 10400
THAILAND
Email: [email protected]
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
Email: [email protected]
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
Email: [email protected]