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IAEA-D4.20.16-CR-2
LIMITED DISTRIBUTION
SECOND RESEARCH CO-ORDINATION MEETING
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
Co-ordinated Research Programme on
Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by
Genetic, Transgenic or Symbiont-Based Technologies
Scientific Secretary: Kostas Bourtzis
Holiday Inn, Panama City, Panama
27-31 March 2017
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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Scientific Secretary: Kostas Bourtzis 1
Selected References 20
Explanation / Justification 30
Participation of Agency’s laboratories 30
Other Resources required 30
Assumptions 30
Related TC projects 31
Narrative Summary 32
Specific Objectives 33
Outcomes 33
Outputs 35
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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 codling moth, the
pink bollworm, the new world screwworm, as well as disease transmitting mosquitoes. This
CRP will focus on comparing the performance of strains developed or improved by classical
genetic, transgenic and symbiont-based approaches to a level where a decision can be made
as to their suitability to produce high-quality sterile males for use in large scale SIT
programmes. Major beneficiaries will be operational AW-IPM programmes in MS that apply
the SIT against these major insect pests. By the end of the CRP several strains, including
strains for new target species, producing high quality sterile males will be available with the
following tangible benefits for pest control programmes in MS using SIT:
1.) As only the males are needed for the SIT, the production, handling and release costs
can be reduced significantly if sexing strains 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.
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.
Technique (SIT) has been a major objective of past and present CRPs. These activities have
resulted in significant progress in the development of SIT technologies, but gaps in
knowledge remain and refinement of current approaches is still needed. Moreover, the
introduction of measures that make the SIT more effective and cost efficient would be highly
desirable.
The SIT has also been used to mitigate the problem of introduction and establishment of
invasive species in the Americas, Europe, Africa, Australia and Asia where several dipteran
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,
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release technology, quality control, etc., even when operational programmes are already
being implemented
(http://nucleus.iaea.org/sites/naipc/dirsit/SitePages/All%20Facilities.aspx). However, one
critical area identified by programme managers, where important advances can be made
concerns the improvement of strains that are being reared and released. One example of how
strain improvement can significantly enhance SIT applicability and efficiency has been the
development and the use of Genetic Sexing Strains (GSS) of the Mediterranean fruit fly,
Ceratitis capitata, and the Mexican fruit fly, Anastrepha ludens in area-wide integrated pest
management (AW-IPM) programmes. These technologies were mainly developed through
the Agency’s CRP programme with support from the FAO/IAEA Agriculture and
Biotechnology Laboratory in Seibersdorf.
There are currently SIT programmes being implemented for several important dipteran and
lepidopteran species where the development of improved strains would lead to major
increases in applicability and efficiency of the SIT approach. Innovative methods to control
agricultural, veterinary and human pest related problems were developed during the CRP
entitled: “Development and evaluation of improved strains of insect pests for SIT”. These
methods for pest control include the development of several new GSS using biotechnologies
(i.e. genetic manipulation). This new CRP builds on the knowledge gained from that CRP
and the availability of newly developed strains to a next phase of comparative assessment and
validation of the performance of sterile males produced by classical genetic, transgenic or
symbiont-based approaches and their suitability for integration into control programs. In
addition to the comparative evaluation, refinement and validation of available and newly
developed strains should be assessed for the potential of horizontal transfer phenomena.
Overall, SIT programs would benefit from the:
1. Comparative evaluation of the performance and genetic stability of sterile males
produced by classical genetic, transgenic or symbiont-based technologies
2. Refinement of existing technologies for the development and field application of
strains for the control of agricultural pests and disease vectors
3. Assessment of potential horizontal transfer phenomena resulting from the use of
strains developed by transgenic or symbiont-based approaches for SIT applications.
The major outcome of these activities will be the availability of strains producing high
quality males allowing efficient implementation of SIT and other related control strategies in
area-wide programmes against some of the major insect pest populations of economic and
medical importance (Table 1).
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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.
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
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
Australia
and
Oceania
B. tryoni
B. aquilonis
B. jarvisi
C. capitata
D. suzukii
C. pomonella
G. molesta
Ae. aegpyti
Ae. albopictus
Lucilia cuprina
Europe B. oleae
C. capitata,
D. suzukii
C. pomonella
G. molesta
E. ceroniae
Ae. albopictus
Phlebotomus perniciosus
Classical Genetic Approaches
The Mediterranean fruit fly, Ceratitis capitata, is a classic example of the sophisticated
application of standard (non-transgenic) genetic manipulation for the development of GSSs
and successful integration of these strains into operational programmes. For this species, a
temperature-sensitive lethal based series of genetic sexing strains were developed by means
of irradiation and classical genetic approaches. Several of these strains (Vienna-7 and
Vienna-8) have been thoroughly evaluated and are currently being used in mass rearing
facilities for large scale AW-IPM programmes that include an SIT component.
In the Mexican fruit fly, Anastrepha ludens, a genetic sexing strain has been developed that is
based on an autosomal black pupae (bp) colour mutation and a translocation Y-A based
genetic sexing system, in which females homozygous for the recessive mutation have black
pupae, while genetically heterozygous males have brown pupae due to the wild-type allele
being translocated onto the Y chromosome. These characteristics allow the sex separation in
the pupal stage using mechanical means, followed by male-only irradiation and release. Since
2012 a large-scale production has been initiated in Mexico reaching a production of 15
millions of male pupae per week by 2014, field evaluation showed a detriment in the quality
of male flies in the field reason why it considers necessary the integration of a refreshment
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with wild material to the mother colony of this a significant improvement in the quality
control parameters and mating competitive test was observed. Using the same sexing
mechanism a new Mexfly strain (Family 10) has been developed in Guatemala, evaluated,
and implemented at Petapa (Guatemala) mass rearing facility. New strains carrying recessive
temperature-sensitive lethal mutations have been developed using classical genetic
approaches, and these strains also need to be evaluated for their rearing properties and field
performance.
Y-chromosome-autosome translocations and recessive white colour mutations of the
puparium have also been used to construct several GSSs in the oriental fruit fly (Bactrocera
dorsalis), two related species, B. correcta and B. carambolae, as well as in B. cucurbitae.
Two of the strains, Salaya1 in B. dorsalis and Salaya5 in B. carambolae, have a high
reproductive capacity comparable to wild-type strains. In addition, males produced by these
strains showed satisfactory performance in small-scale field experiments. Characteristics of
these two strains in large mass rearing and performance of released males in large-scale field
experiments remain to be evaluated. Population genetic studies have been performed,
comparing Salaya5 with B. carambolae populations across species range in South East Asia
and Suriname.
There has been an interest in developing GSSs using classical genetic approaches in pest
moths (Lepidoptera). However, the sexing system developed in two model species, the
Mediterranean flour moth (Ephestia kuehniella) and silkworm (Bombyx mori), which is based
on balanced sex-linked recessive lethal mutations, was found to be only marginally
applicable in mass rearing. GSSs amenable to mass rearing conditions, such as those
constructed in the Mediterranean fruit fly, could not be developed in any lepidopteran species
owing to their sex chromosome system being the WZ/ZZ system, in which females are the
heterogametic sex.
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.
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From sex determination studies to transgenic sexing strains in flies, butterflies and
mosquitoes.
Genetic studies of sex determining genes in Ceratitis capitata led to the exploitation of
female-specific splicing of Cctra gene to obtain transgenic sexing strains having either
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 envisioned in the near future taking advantages
of both novel genomic sequencing technologies and reverse genetics tools, as well as of new
knowledge gained from fundamental genetic studies concerning genetic developmental
programs of sex determination and sexual differentiation.
Various primary signals controlling sex determination have been clarified in dipteran
insects, including Drosophila melanogaster (X:A ratio in XX/XY), the mosquitoes Aedes
aegypti (nix gene, tra-2 related splicing factor encoding gene; homomorphic chromosomes:
M/m, m/m) and Anopheles gambiae (Y-linked Yob gene; XX/XY) and hymenopteran species
including Apis mellifera (haploid/diploid: complementary sex determining locus CSD). The
Y-linked M factor in C. capitata, which represses early embryonic female-specific activation
of Cctra needs to be identified. This effort may be facilitated by the search of the reciprocal
M factors of other Tephritid species, such as the olive fly, Bactrocera oleae, through
comparative genomics.
Yob gene of Anopheles which resides on the Y chromosome and nix gene which resides
on one of the homomorphic sex chromosome of Aedes trigger male development during early
embryonic stages. These studies revealed that the Yob-containing region of the Y
chromosome and M-locus region tend to be highly repeat-rich and thus difficult to assemble.
As a result, Y- and M-linked genes are often absent from genome assemblies. A few methods
have been developed to identify Y- or M-linked genes. One such method is called
chromosomal quotient (CQ) and it was successfully used to identify Y chromosome genes in
Anopheles mosquitoes as well as M-linked genes in Ae. aegypti. The M-locus gene Nix, was
also discovered in Ae. aegypti using the CQ method.
A new search for male determining genes in relevant pest insects which can be targeted by
SIT, started recently by taking advantage of de novo sequencing technologies, cytogenetic
micro dissection of sex chromosomes, sexed embryonal transcriptomes, availability of
genome sequences from male and female sex of various species, and novel bioinformatic
tools including chromosome quotient, edgeR statistical analysis for differentially expressed
genes in the two sexes.
In the insect order Lepidoptera, chromosome mechanism of sex determination is of the
WZ type. However, the actual role of the W and Z chromosomes remains unknown except
for the silkworm, Bombyx mori. However, after years of fruitless search for a W-linked
protein-coding gene that could be the primary trigger of female development, only recently
Kiuchi et al. (2014) made a surprising discovery that the feminizing factor in B. mori is a W-
encoded small PIWI-interacting RNA named Fem piRNA. The authors also showed that the
Fem piRNA downregulates the expression of a Z-linked gene, Masculinizer (Masc), which
promotes male development at the absence of the W chromosome. In other words, the Fem
piRNA controls female-specific splicing of the B. mori doublesex (Bmdsx) gene by
downregulating expression of the Masc gene. However, it is not yet known (i) whether the
Fem piRNA-Masc sex-determining pathway is conserved in other lepidopteran species with
the W chromosome and (ii) whether Masc plays a role in species with a Z0/ZZ sex
chromosome system that are thought to have the Z-counting mechanism of sex determination.
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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 both
C. capitata and Anastrepha suspensa, is based on a driver component that uses a promoter
active during early embryogenesis to induce a lethal effector gene resulting in early
embryonic lethality. The development of this system for other insect pests has also been
improved by new methods to pre-evaluate newly isolated driver and effector components
using a cell culture assay for cellular lethality, in addition to quantitative PCR. This will save
considerable time and effort in the validation of these components and, importantly, their
ability to function together previous to laborious germ-line transformation experiments.
These cell culture assays have allowed not only the evaluation of promoters from embryonic
genes, such as serendipity alpha, together with the endogenous pro-apoptotic genes hid,
reaper, and grim, but also the determination of the most-efficient driver-effector cassette
combinations for use in A. suspensa transformants, resulting in two hybrid strains exhibiting
100% lethality. In C. capitata two strains with 100% lethality were developed and evaluated
at the IPCL in Seibersdorf with one strain showing good performance and character stability
under semi-mass rearing conditions. The isolation and in vitro validation of species-specific
promoters and lethal effector genes greatly improved the efficiency of creating high-
performance conditional lethality strains, which can be extended to other insect pest species.
Tetracycline-repressible female-lethal sexing strains. Another critical component for
enhancing SIT has been the development of transgenic sexing strains (TSS) in fruit flies and
livestock pests to eliminate the costs of mass-rearing females and to eliminate mating
competition when sterile females must be released with males. Moreover, male-only releases
are a prerequisite for mosquito-SIT programmes, as released females will increase the risk of
disease transmission. Unfortunately, a highly effective genetically-based (non-transgenic)
technology to eliminate females as early embryos is only available for SIT programmes
targeting the Mediterranean fruit fly, and it will be costly and take many years to replicate
this system for other insects. Thus, among the many pest species currently subject to SIT
programmes, or in the planning stages, Anastrepha species in Central and South America and
Bactrocera species in Asia, Europe, and Africa, could specifically benefit from the much
more rapid implementation of TSS technology, especially those based on tetracycline-
repressible (Tet-off) female-specific embryonic lethality. Although these species are much
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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.
Female-specific Tet-off pupal sexing systems due to tTA overexpression lethality (based on
RIDL) have been also developed in C. capitata, the New World screwworm Cochliomyia
hominivorax, the Australian sheep blowfly Lucilia cuprina, the diamondback moth (Plutella
xylostella), pink bollworm, and in the silkworm. The C. hominivorax pupal TSS produce
100% males when reared on diet that lacks tetracycline and most are comparable to the
current production strain in various fitness parameters that are important for production.
Further, male aggression and male competitiveness of some of the strains are comparable to
the production strain. These would be the first sexing strains available in the over 50 year
history of the highly successful screwworm SIT program. However, such late sexing systems
do not eliminate the costs for female larval rearing. Other TSSs have been successfully
evaluated under semi/mass rearing and field cage conditions with support from the
FAO/IAEA IPCL in Seibersdorf. These achievements will help to explore different options
for TSS in other important agricultural and livestock pest insects (see Table 1).
In mosquitoes, currently no GSSs are available that have the potential for use in SIT
programs. However, promising research results and technologies have been reported recently
that, with further research and development, could lead to the development of mosquito
TSSs. A "flightless female" transgenic strain of A. aegypti exists, which carries a transgene
that destroys the female flight muscles when raised without tetracycline added to the diet.
However, this strain had poor fitness characteristics in large open field cage trials in Mexico.
In A. gambiae a ´sex distortion´ approach was developed, which destroys X-bearing sperm
and resulted in 95-97% male progeny, while in A. aegypti, double-stranded RNA against the
female-specific variant of the doublesex gene was fed to larvae resulting in up to 97% adult
males (by death of females). Moreover, the development of an early embryonic 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.
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Transgenic sexing based on sex reversion by sex-determination gene repression.
An approach to generate a male-only population by sex reversion of females to males has
been successfully tested in C. capitata, which could result in doubling the mass production of
male-only progeny, and avoiding the need for female-specific lethality. A transgenic sex-
reversion line of C. capitata that shows 98% conversion of XX individuals into fertile males,
with 2% intersexes generated. In vivo RNAi against Cctra driven by a transgene can be very
effective if the parental female deposits dsRNA molecules into oocytes, as has been revealed
by an RNAi maternal effect. When a parental female carrying one transgene copy (+/-) is
crossed with a non-transgenic XY male (-/-), the Cctra-specific dsRNA maternally deposited
can act efficiently to switch off the Cctra gene in both transgenic (+/-) and non-transgenic
embryos (-/-). Hence, 50% of male only progeny is composed of XX and XY individuals,
which are non-transgenic. This is a first preliminary proof of principle for the possibility of
developing insect transgenic technologies leading to non-transgenic male only progeny. Such
strategies may be used for SIT in countries having restrictions on the use of GM insects in the
wild. However, a rather complex design will be required to obtain a similar transgenic sexing
system operational for a mass rearing. Another alternative would be to develop a transgenic
sexing strain bearing a maternally masculinizing Cctra-IR transgene, homozygous in both
sexes, under conditional Tet-off control.
Genetic tools for genome manipulation
Genomic targeting of transgene insertions. Transposable elements are widely used as
vectors for integrating transgenes into the genome of insects. However, the random nature of
transposon vector integrations often results in mutations and makes transgene expression
subject to variable genomic position effects. This makes reliable quantitative comparisons of
different transgenes difficult and development of highly fit transgenic strains laborious. Tools
for site-specific transgene genomic targeting are essential for functional genomic
comparisons and to develop the most advanced transgenic insect strains for applied use.
Improved genomic targeting systems for non-drosophilid insects were tested as integration
and recombinase-mediated cassette exchange (RMCE) systems based on phiC31-attP/B and
Cre/loxP, respectively.
For C. capitata, A. suspensa, and A. ludens, the phiC31-attP/B system was established and
used for the stabilization of transgenes in the genome of C. capitata as well as the generation
of target-site lines with high fitness in A. ludens. In addition, the system was proven to be
functional in the 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.
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RNAi for invertebrate pest control. In insects, as in other organisms, RNAi is a powerful
tool for experimental studies aiming to determine gene function. This commonly involves the
microinjection of dsRNA into the target organism, often directly into the target tissue. The
dsRNA is cut by endogenous Dicer proteins into a population of small interfering RNAs
(siRNAs), which in turn bind and degrade complementary mRNA sequences. Careful dsRNA
design can ensure highly specific silencing in terms of both individual gene targets and
species.
In plants and some invertebrates (eg. C. elegans), the efficacy of RNAi is improved through a
combination of signal amplification and systemic spread, such that the entry of one dsRNA or
siRNA molecule into a single cell can lead to effective silencing of the target gene throughout
the target organism. In some insects, RNAi appears to be cell-autonomous, with no
amplification or cell to cell communication of the gene silencing signal.Insect pest control
methods are being developed through dsRNA oral delivery. The efficacy varies depending on
the insect species and genes. Some examples of delivery include paper soaked in dsRNA for
termites, plants coated with or expressing dsRNA and bacteria expressing dsRNA, fed with
the insect diet. In the case of vectors that transmit diseases, triatomines can be fed live
symbiotic bacteria that 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 by silencing
spermatogenesis genes or genetic sexing as part of SIT programs by targeting female-specific
transcripts during the developmental stages of the generation to be released. This application
of RNAi offers a greater level of control of delivery than other RNAi applications, but unlike
other applications demands near 100% efficacy. Depending on the target organism, oral
and/or topical delivery is possible.
CRISPR/Cas9 gene-editing (Clustered Regularly Interspersed Short Palindromic
Repeats. A wide variety of bacteria and archaea have a surprisingly complex adaptive
immune system based on clustered regularly interspaced palindromic repeats (CRISPR) and
CRISPR-associated protein 9 nuclease (Cas9) genes. The bacterial type II CRISPR-Cas9
system was very recently adapted as a genome-engineering tool in many different organisms,
including various insect species, and in vitro preparations, dramatically expanding the
possibility to modify, at single nucleotide level, specific genes in the genomes.
CRISPR/Cas9 genome editing in insects was first reported in D. melanogaster and B. mori
and has since been successfully used to modify the genomes of numerous insect pest species.
The Cas9 knock-in homologous DNA repair (HDR) strategy developed in Drosophila took
advantage also of a loss of function mutation in the ligase 4, a gene required for DNA non-
homologous repair. Homozygous lig4 flies showed a 5-7 fold increase in HDR. The high
precision and accuracy of gene editing technologies enables the creation and assembly of
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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).
Symbiont-based approaches
One aspect currently being explored is the potential role that insect microbiota may play in
insect reproduction, physiology, fitness and their ability to transmit pathogens. For instance,
it is now well established that Wolbachia, an intracellular bacterium that infects a large
variety of insects, has the ability to induce reproductive abnormalities like cytoplasmic
incompatibility (a kind of male sterility) as a strong inhibitory effect on the ability of
mosquitoes to transmit human pathogenic viruses (e.g. dengue, chikungunya) and other
important pathogens (e.g. Plasmodium sp.).
“Incompatible insect technique” (IIT) is referred to as population suppression and entails
the release of male mosquitoes infected with Wolbachia, resulting in sterile matings and a
reduction in the insect population. Wolbachia transinfection (or transfer of Wolbachia
between different insect host species through embryonic microinjection) to generate a stable
novel Wolbachia infection in the target pest species is the first step in developing a
Wolbachia-based IIT for the control of both agriculturally and medically important insect
pests. In C. capitata and B. oleae, stable Wolbachia 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. 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. The minimum
irradiation dose for the sterilization of Ae. albopictus females escaped from sex separation
has been established, without affecting the male mating performance. In addition, the
presence of different strains of Wolbachia in laboratory strains and natural populations of the
A. fraterculus species complex is currently being characterized and evaluated. Ongoing
analysis points to the presence of different Wolbachia strains in this species complex. The
characterization of the phenotype induced by Wolbachia in its host is also under study.
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Table 2: Stable transinfected 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
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, wPip Cx. pipiens
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
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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 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 is
currently being investigated. The hypothesis that the addition of probiotics to the larval diet
could increase pupal weight in mass rearing and consequently the size of sterile adult of B.
dorsalis and C. capitata males, as well as their dispersal, longevity, mating competitiveness
and sperm transfer to females is being studied.
The incorporation of probiotic supplements in the mass rearing protocols should be clearly
described and taken into account when comparing strains’ efficiency. Standardization of such
approaches would help in the replication of experiments among different groups. The
utilization of live bacteria, either as effective components of control (such as Wolbachia) or
as beneficiary supplements in diet, raises the concern of horizontal transfer events. In this
direction: a) the documented transfer of parts (or even the whole) Wolbachia genome in the
host genome (as evident at least in Drosophila and tsetse species), b) horizontal transfer of
symbionts among different species and, c) the naturally occurring hybridization of different
species, are issues that should be also taken into consideration.
Evaluation Technologies Technological advances are also allowing for more efficient evaluation of strains produced
for SIT, and for improved monitoring before and after release. The application of these
technologies as part of SIT programs should provide valuable information to improve rearing
and release practices against other species.
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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. 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 Drosophila suzukii and different Tephritidae species such as C. capitata and B.
dorsalis. Moreover, draft genomes have recently been completed for the melon fly (B.
cucurbitae), olive fly (B. oleae), Qfly (B. tryoni) and the Australian sheep blowfly (L.
cuprina). A reference genome sequence does not contain the full genetic diversity of a
species, which can be better captured by sequencing individuals from various populations or
strains. Inexpensive Illumina sequencing methods can now be employed to quantify this
genetic variation at a genome-wide scale, which can be used to examine variation that exists
among wild populations, or changes that occur during the domestication or release processes
as part of an SIT program. Genome-wide markers are also extremely useful to generate high
density linkage maps in target species. In addition to their value in improving genome
assemblies by joining and ordering scaffolds into chromosomes, these markers can also be
associated with phenotypes of relevance to SIT – and hence can be used both to understand
and to monitor the fitness of strains used in SIT programs.
Varying levels of transcriptome data accompany these genomes, and with the greater
accessibility of RNAseq methodologies to laboratories worldwide, the quantity and quality of
transcriptome data is likely to increase for all target species. As such, transcriptome
assessments may become standard practice as part of strain evaluation procedures in the near
future.
Genetic-based marking
Genetic-based marking is also a critical component of the SIT providing the ability to
monitor released males to distinguish them from the field population when collected in traps,
and to monitor the frequency of sterile male matings to females in the field. In addition,
markers are important tools for the production of high quality insects in mass rearing
facilities. While phenotypic markers have been isolated as visible mutations useful for SIT,
their identification has often been serendipitous, they are species-specific, and optimal
markers can take years to be developed, if at all, for some species. In contrast, transgenic
fluorescent protein markers have been shown to be widely applicable, with the same genetic
constructs functional in many species, including green or red fluorescent proteins introduced
into A. suspensa, A. ludens, Bactrocera dorsalis, B. oleae, B. tryoni, C. capitata, D. suzukii,
C. hominivorax, L. cuprina and several mosquito species. Through the use of different tissue-
specific promoters and transgene integration sites, hundreds of transgenic lines with different
tissues expressing the fluorescent protein could be established. In particular, tissue-specific
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sperm or Y-linked markers were developed for C. capitata, A. suspensa, A. ludens, B. tryoni,
B. mori, and the mosquitoes Ae. aegypti and An. stephensi. These markers may be used for
sexing in some species (using fluorescence-based sorters), and allow identification of females
that have mated with released males based on the spermathecal storage of fluorescent sperm.
Moreover, they have been proven to be successful in the medfly for tracing differential sperm
use in presence of multiple mating, which is particularly relevant to SIT applications.
Importantly, the broadly applicable 3xP3 promoter used to regulate various fluorescent
protein genes in several orders of insects has been found to be, thus far, uniquely non-
functional in several tephritid fruit fly species.
Horizontal transfer - transposon- and/or symbiont mediated
A critical concern for transposon vector-based and symbiont-based strain manipulations is
the potential for horizontal interspecies transfer (HT) of the transposon vector or symbiont
(or symbiont-mediated transfer of a transformation vector). This presents a critical ecological
safety concern for associated insect and non-insect species within a field release site, and
especially for beneficial species that might be negatively impacted. Symbionts may have a
non-specific, if not a broad host range, and autonomous transposons are thought to utilize HT
as a natural mechanism for their maintenance and proliferation. Non-autonomous transposon
vectors are normally incapable of self-mobilization (in the absence of functional transposase),
but the unintended or unrecognized presence of the same or cross-mobilizing transposase
may allow their transmission directly into closely associated predators and symbionts, or
through indirect transmission via symbionts or viral systems. However, establishment of
stable horizontal transfer requires the introduction into the germ line. Moreover, most of the
considered constructs are probably evolutionary neutral or even have negative selection
characteristics (lethality, sterility), which would not favour the selection of rare horizontal
transfer events. Nevertheless, it should be a high priority to evaluate potential HT between
transgenic and/or symbiont infected host species and closely associated predatory (e.g.
parasitoid) organisms or natural symbiont populations.
Evaluation guidelines for the creations and analysis of transgenic strains for eventual
contained field release applications
Investigators should be aware of requisite information relevant to the genetic modification
protocols that may be necessary for eventual applications for the contained field release of
their organisms, especially as it relates to risk assessment. In particular is the required
information outlined in the NAPPO agreement in the following sections: 2.1.2.3 Description
of the genetic construct; 2.1.2.4 Characterization of the transgene inserted into the transgenic
arthropod; and 2.1.2.5 Description of the phenotype of the transgenic arthropod. Investigators
should also be aware of potential risk issues associated with the modification of particular
insect species modified with particular genetic elements released into particular ecological
environments, and modifications that could diminish risk, and in some cases be prerequisites
for approved release. These considerations should include:
1) Robust and stable genetic marking systems that allow identification of released transgenics
after field trapping, both by visible inspection and sensitive molecular tests.
2) Use of post-integration vector immobilization systems integrated into the vector, or a
means to evaluate potential cross-mobilization within a host genome, to assess and mitigate
potential 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.
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3) Use of robust species-specific intracellular lethality systems to ensure that survivors do not
occur normally, that lethality is confined to the host organism leaving predatory organisms
unaffected, and is also confined to the released species.
4) Use of genomic targeting systems where possible to avoid potential genomic site-specific
effects including insertional mutations and modification of transgene expression.
5) Specific use of cassette exchange systems (i.e. RMCE) for both primary and secondary
transgene integrations to avoid introduction of plasmid DNA including antibiotic resistance
genes.
6) Potential use of dual redundant lethality systems to ensure that genetic breakdown of either
system does not result in lethal revertant survivors in the field.
Evaluation guidelines - Quality control of insect strains Evaluation of strains for use in SIT programmes should be conducted by documenting the
two most important parameters: (a) rearing performance (production and quality control) and
(b) field performance (field cage or open field).
Rearing performance of a strain: Before any strain is used in small or large scale applications,
any new strain should be evaluated and, ideally, be compared with currently used strains, if
available. During the rearing process, there are several relevant performance parameters that
need to be evaluated using as a reference the classical genetics Mediterranean fruit fly TSL
strain (see below tables with production parameters).
Stability of the strains: This parameter measures the number of aberrant insects that appear
during the rearing process. In order to ¨clean¨ a strain from aberrant/recombinant insects, a
filter rearing system has been designed. This process allows documenting the number of
recombinant flies in the initial colony, their removal and starting a new colony free of
aberrant insects. The suggested value is: recombinants < 2% for classical genetic sexing
strains like that of the Mediterranean fruit fly, while for transgenic strains it is expected to be
< 1%.
Production Parameters: The initial and most important parameter for the rearing process is
the evaluation/comparison of strain fertility, fecundity including the pre-ovipository and
ovipository phase. These filter systems should be extended to accommodate transgenic
strains. Regarding symbiont-based strains, the infection titre and type should be regularly
verified through molecular techniques.
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
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The recommended values of the additional parameters for comparing the rearing process,
based on the Mediterranean fruit fly model, are:
PRODUCTION PARAMETERS
(medfly tsl strain)
CURRENT MINIMUM
TRANSGENIC
Quality
control
Manual V.6
2014
reference
(page
number)
Egg to pupae recovery (male only) 25% > 40% NA
Liters of Pupae/kg larval diet 0.18 ± 0.01 > 0.18 ± 0.01 NA
LARVAL DEVELOPMENT PERIOD
Colony larval development time at 25°C
(days)
10 ≤ 10 NA
PUPAE DEVELOPMENT
Percent pupation at 24hr 90 90 NA
Egg to pupae recovery (male only) 25% > 40% NA
Table 3. Quality control parameters
The recommended values for the Quality control (QC) analysis, based on the Mediterranean
fruit fly model, are:
QUALITY CONTROL
PARAMETERS ( medfly tsl strain)
CURRENT MINIMUM
TRANSGENIC
Quality
control
Manual V.6
2014
reference
(page
number)
Mean acceptable pupal weight (mg) 7.84 ± 0.25 > 7.5 23
Mean % flight ability, post-irradiation 81 ± 4 (65-70) > 65 26
Sex ratio (% male)
● Control 50
● Permissive conditions 65 60 Not available
● Restrictive conditions (tsl
heat-treated; transgenic
without tetracycline)
99.8 ± 0.4 95
Recommendation: include emergence, survival under stress, timing emergence and other
post-irradiation parameters (when applied) that are included in Quality Control
ManualVersion 6.0 May 2014
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.
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Table 4. Field Performance
FIELD PERFORMANCE PARAMETERS (
medfly tsl strain)
CURRENT MINIMUM
TRANSGENIC
Mating performance post-irradiation (or
equivalent)
● Laboratory mating (% males mating) 80 ± 6 80
● Field cages (sterility index) 0.3 - 0.4 > 0.3
Longevity in the field, post-irradiation LT50
(days)
4 > 4
Dispersal in the field, post-irradiation (m) 100 > 100
Egg hatch of wild female x TSL male, post-
irradiation
0.01%
Egg hatch of wild female x transgenic male (with
and without irradiation)
0.01%
Fluorescence marker persistence (monitoring) >3 weeks > 2 weeks
Fluorescence marker scoring efficiency 110-150
flies/hr/person
90 - 110
flies/hr/person
Fluorescence marker scoring accuracy 91% primary
9% secondary
< 1% tertiary
91% primary
9% secondary
< 1% tertiary
In respect to the strains developed by transgenic and / or symbiont-based approaches, the
potential of horizontal transfer of the transgene(s) and / or symbionts in other strains and / or
species should be evaluated using standard molecular biology and genetic approaches.
Recommendation: the assessment of additional specific test must be analysed case by case
Evaluation guidelines - Domestication Domestication is an important consideration both for the efficiency and evaluation of strains
used in SIT. Different studies using molecular markers (such as microsatellites and recently
genome wide sequencing markers) point to a non-uniform adaptation process among different
species. Other recent studies in symbiotic level (using, for example, culture dependent and/or
culture independent high throughput NGS approaches) also suggest that this adaptation
severely influences the structure of the symbiotic communities of the populations entering the
lab. Although there are no standardized protocols yet, the genetic, symbiotic and
physiological characterization of strains used in SIT and populations introduced for
comparative studies should be studied. Development and application of universal approaches
across species will be beneficial for the comparison of the efficiency of different strains.
Following the understanding that symbiotic communities are important in parameters such as
fitness and mating competitiveness, different studies have recently focused on the
characterization of symbiotic communities of insects of economic and medical importance.
Approaches still can be quite varying. There can be differences either in the technology used
(such as morphological/ biochemical examination of colonies, 16S rRNA sequencing using
classical Sanger sequencing or utilizing different forms of the emerging NGS technologies)
or in the samples analyzed (natural vs wild populations, different developmental stages,
whole adults vs tissue specific and more). These methodological differences can create
confusion and make the conduction of robust conclusions a difficult task. Recent data that
point to the dynamic nature of the symbiotic communities during development make the
characterization even more difficult. At the same time, probiotic diets are being developed
and used for different insect-targets of SIT, aiming to enhance its effectiveness through
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increasing important parameters such as fitness and mating behavior. However, results can
vary, since the domestication process can be different within/between species. SIT important
strains may have different properties, attributed either to genetic or symbiotic factors because
of differences in the wild material originally introduced in the lab and/or differences in the
rearing practices followed in the different laboratories and facilities. The monitoring of wild
populations during the laboratory domestication in different levels (genetic, symbiotic and
physiological), could provide insight in the changes happening during this process.
Documenting and monitoring the genetic and symbiotic profile with universally accepted
protocols could a) facilitate implementation and evaluation of enrichment procedures with
wild genomic background, b) enable the comparison of results of probiotic experiments
performed in different labs and, c) point to characters that are common or diverse during
laboratory adaptation.
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Nuclear Component:
The SIT relies on the use of ionizing radiation to sterilize large numbers of insects and
currently there is no alternative that could replace radiation. However, there are developments
taking place which intend to use molecular methods for generating lethality in field
populations. These approaches are not included in this new CRP as their non-confined use
would create significant concerns relating to biosafety and long-term effectiveness.
Radiation-induced sterility provides a very high level of biosafety and can be used in
combination with any evaluated, refined and / or newly developed strains produced in this
CRP. As radiation induces random dominant mutations, there are no possibilities of
resistance development to this physical process. This possibility cannot be excluded with
molecular approaches that involve genomic insertions.
Explanation / Justification
Publication of results: Activities and final findings of the CRP will be published in a Special
Issue of a peer-reviewed open-access journal.
Participation of Agency’s laboratories
As few institutions are applying irradiation and classical genetics for the development of
GSS in agricultural pests and disease vectors, and given the need of a neutral body for the
quality control analysis of sterile males, the CRP needs therefore to be supported through
adaptive research and development carried out at the IPCL, FAO/IAEA Agriculture and
Biotechnology Laboratories, Seibersdorf as part of Project 2.1.4.1. This R and D will
focus on comparing the performance of sterile males produced by genetic, transgenic or
symbiont-based technologies.
Other Resources required
None.
Assumptions
Member States continue to recognize the benefits of developing the SIT package and other
genetic and environment-friendly methods for sustainable control of insect pests of
agricultural, veterinary and medical importance in AW-IPM programmes and continue to
request improved technology and high quality SIT strains in order to maximise benefit/cost
projections.
The demand for area-wide integrated insect pest management approaches, including SIT and
augmentative biological control as non-polluting suppression/eradication components,
continues to increase, mandating expansion and improvement in cost-effectiveness of these
environment-friendly, sustainable approaches.
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31
Related TC projects
BKF5012 – Collecting baseline data and implementing fruit fly suppression in mango fruit.
CPR5020 - Integrating the Sterile Insect Technique (SIT) for Area-Wide Integrated Pest
Management of Tephritid Fruit Flies.
GUA5017 - Using the Sterile Insect Technique (SIT) to Establish Fruit Fly Low Prevalence
Pilot Areas and to Assess it as an Alternative for the Control of the Sugarcane Borer in Pilot
Areas
HON5006 - Using Sterile Insect Technique (SIT) to Obtain Recognition as a Mediterranean
Fruit Fly Free Area in the Aguan River Valley
ISR5019 – Supporting a feasibility study for the implementation of leafminer (Liriomyza
spp) sterile insect technique combined with biological control under greenhouse conditions.
LIB5011 – Enhancing area-wide integrated management of fruit flies.
MAG5021 - Implementing the Sterile Insect Technique (SIT) in Integrated Fruity Fly Control
for High Quality Fruit Production.
MAR5022 – Reducing insecticide use and losses to melon fly (Bactrocera cucurbitae)
trhough environment-friendly techniques to increase production in different areas, Phase II.
MOR5032 - Supporting Control of the Mediterranean fruit fly Using the Sterile Insect
Technique for Citrus Fruits and Early Fruits and Vegetables to Establish Low Mediterranean
fruit fly Prevalence Zones.
OMA5002 – Assessing the suitability of sterile insect technique (SIT) and related techniques
for combating date palm insect pests.
PAN5020 – Strengthening technical capacity to control Mediterranenan 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.
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RAF5062 - Preventing the Introduction of Exotic Fruit Fly Species and Implementing the
Control of Existing Species with the Sterile Insect Technique and Other Suppression
Methods.
RAS5059 - Supporting Area-Wide Integrated Pest Control of Native and Exotic Flies in the
Middle East Subregion Incorporating the Sterile Insect Technique (SIT).
RAS5067 – Integrating sterile insect technique for better cost-effectiveness of area-wide fruit
fly pest management programmes in Southeast Asia.
RER5020 - Controlling Fruit Flies in the Balkans and the Eastern Mediterranean.
INT5151 - Sharing Knowledge on the Use of the Sterile Insect and Related Techniques for
Integrated Area-Wide Management of Insect Pests
LOGICAL FRAMEWORK:
Narrative Summary Objective
Verifiable
Indicators
Means of
Verification
Important Assumptions
Overall Objective
to compare the performance of
sterile males produced by
classical genetic, transgenic or
symbiont-based technologies to
address the increasing demand
for environment-friendly and
sustainable integrated pest
management approaches for
insect pests of agricultural,
veterinary or medical
importance
N/A
N/A
Requests by Member States in the
area of insect pest and disease
vector control using the SIT are
increasing. To make this nuclear
technology available to Member
States for several insect species,
the evaluation of the rearing
efficiency and male mating
competitiveness of sterile male
strains, refinement of
technologies and the assessment
of horizontal transfer phenomena
are an essential precondition prior
to the use of these strains in
operational programs. Biological
material is available.
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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 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 and/or
horizontal
transfer
phenomena in at
least three strains
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
technologies is possible.
Tools for the assessment of
genetic instability and/or
horizontal transfer phenomena are
available or can be developed.
Outcomes
1. Sterile male strains
produced by classical
genetic, transgenic or
symbiont-based
technologies
comparatively evaluated
2. Existing and/or new
technologies for the
development and
application of sterile
male strains produced by
classical genetic,
transgenic or symbiont-
based approaches
refined or adopted
3. Genetic instability
and/or potential
horizontal transfer
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
Facilities and resources available.
Facilities and resources available.
Facilities and resources available.
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34
phenomena towards the
use of strains developed
by transgenic or
symbiont-based
approaches for SIT
applications assessed
collected
and
feasibility
analysis
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35
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, redundant lethality,
pathogen interference for
SIT applications.
6. Stable classical genetic,
transgenic or symbiont-
based strains as assessed by
long-term and large scale
production.
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
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
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.
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36
7. Strains for new target
species of agricultural,
veterinary and medical
importance generated by the
use of available
technologies.
8. Information on the
frequency of genetic
instability and/or potential
horizontal transfer
phenomena in SIT strains.
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.
least one new
target species
generated
At least three
strains assessed
Three new SIT
strains validated
Papers drafted
and submitted.
Reports and
published
papers
Reports and
or
published
papers
Reports and
published
papers
Journal
issue with
published
scientific
papers.
Biological material and tools are
available.
Tools are available.
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
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
Signed
contracts
and
agreements.
Participants
’ activities
and logical
framework
revised.
Participants
and RCM
Progress
Reports.
Participants
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-
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37
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.
held 2018.
4th
RCM to be
held 2020.
and RCM
Progress
Reports.
Participants
and RCM
Final
Reports
Scientific
publication.
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.
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38
SECOND FAO/IAEA RESEARCH COORDINATION MEETING ON
“Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced
by Genetic, Transgenic or Symbiont-based Technologies”
27-31 March 2017
Holiday Inn, Panama City, Panama
AGENDA
MONDAY, 27 MARCH 2017
09:00 – 09:05 Kostas Bourtzis (Scientific Secretary, FAO/IAEA):
Welcome statement.
09:05 – 09:25 Matthew Larsen (Director, STRI): Welcome statement.
09:25 – 09:45 HE Mr Eduardo Enrique Carlés, Minister of Agriculture:
Welcome statement.
09:45 – 10:00 Introduction of participants, administrative announcements.
10:00 – 10:30 Carolina Concha, Ying Yan, Evelin Quilarque, Agustin Sagel,
Guillermo Fadul, Steve Skoda and Max Scott: Development and
evaluation of a transgenic sexing strain of the New World Screwworm,
Cochliomyia hominivorax, carrying an embryonic female lethal system.
10:30 – 11:00 COFFEE BREAK
11:00 – 11:30 Ying Yan, Megan Williamson, Katherine Knudsen and Max Scott:
Transgenic sexing strains: evaluation of the influence genetic
background in Drosophila and new Lucilia cuprina gene promoters for
driving tTA expression.
11:30 – 12:00 Alfred M. Handler*, J. Li*, Marc F. Schetelig, Asya Targovska, Jose
Salvador Meza, and Kostas Bourtzis: Conditional lethal tephritid
strains for improved SIT and genetic stability.
12:00 – 12:30 Marc F. Schetelig and Irina Haecker: Systems for unbiased
comparison of transgenic sexing systems in Aedes aegypti, Ceratitis
capitata, and Anastrepha ludens.
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12:30 – 13:30 LUNCH
14:00 – 14:30 Kolja N. Eckermann, Mohammad KaramiNejadRanjbar, Hassan
M. M. Ahmed, Stefan Dippel and Ernst A. Wimmer: Instant
induction of resistance evolution against CRISPR/Cas9 gene drive and
CRISPR/Cas9 based multifactorial reproductive sterility for SIT
approaches.
14:30 – 15:00 Pamela Pennington, Claudia Paiz, Mabel Taracena: Tool
development for silencing of spermatogenesis genes in Anopheles
albimanus to produce sterile males.
15:00 – 15:30 COFFEE BREAK
15:30 – 16:00 Angela Meccariello, Marco Salvemini, Simona Monti, Maria Grazia
Inghilterra, Pasquale Primo, Giuseppe Del Corsano, Andrea
Gravina, Rita Colonna, Philippos Papathanos, Valeria Petrella,
Kallare Arunkumar, Elena Chiavacci, Christian Mosimann, Svenia
D. Heinze, Daniel Bopp, Stephan Schmeing, Mark Robinson, and
Giuseppe Saccone: CRISPR-Cas9 functional genomics of medfly sex
determination and the hunting for the Y-linked M factor: a first list of
putative Y-linked M candidates.
16:00 – 16:30 KT Tsoumani, M-E Gregoriou and Kostas D. Mathiopoulos: RNAi
silencing of loci implicated in olfactory and reproductive processes, as
potential targets to improve the olive fruit fly population control
techniques.
16:30 – 17:00 Giuliano Gasperi, Paolo Gabrieli, Ludvik M. Gomulski, Francesca
Scolari, Alessandro Di Cosimo, Anna R. Malacrida: Exploring the
male competitiveness in Ceratitis capitata strains.
17:45 Group dinner at Mi Ranchito Restaurant, Amador Causeway.
TUESDAY, 28 MARCH 2017
08:30 – 09:00 Phil W. Taylor, Lee R, Yeap HL, Lynch K and John Oakeshott:
‘Quality’: What is it, and how do we know when we have it?
09:00 – 09:30 Sujinda Thanaphum, Nidchaya Aketarawong, Siriwan Isasawin and
Kamoltip Laohakieat: Comparing rearing efficiency and
competitiveness of males from Salaya genetic sexing strains (Bactrocera
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spp) including the refinement and transfer of existing technology for the
improvement and application of strains for area-wide integrated pest
management.
09:30 – 10:00 D. H. Orozco Davila, J. S. Meza, M. Roblero Roblero, V. García
Martínez, J. Ibañez Palacios, S. Aguirre, M. F. Ruiz Perez:
Evaluation and improvement of Anastrepha ludens strains for SIT: a)
Transgenic and b) GSS Tapachula-7 under mass rearing conditions.
10:00 – 10:30 COFFEE BREAK
10:30 – 11:00 Jair F. Virginio: Transgenic lines competitiveness evaluation for
Ceratitis capitata and Aedes aegypti to be used in insect control.
11:00 – 11:30 Dongjing Zhang and Zhiyong Xi: Mass rearing of triple Wolbachia-
infected Aedes albopictus and its field pilot application in China.
11:30 – 12:00 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.
12:00 – 12:30 Ramírez-Santos Edwin. Performance of a Genetically Modified Strain
of the Mediterranean Fruit Fly and the effect of irradiation doses on its
sterility and biological security. Evaluation and implementation of GSS
“family 10”2:30 – 13:30 LUNCH
13:30 – 14:00 Fleur Ponton, Tahereh Moadeli, Bishwo Mainali and Phillip W.
Taylor: A new gel-based diet formulation to improve rearing efficiency
of Qfly.
14:00 – 14:30 Antonios A. Augustinos, 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 Hongyu Zhang, Chaohui Cai, Zhichao Yao, Yushan Li, Bruno
Lemaitre, Zhiyong Xi and Kostas Bourtzis: Intestinal probiotics
restore Bactrocera dorsalis’ ecological fitness damaged by Co60
irradiation and the homeostasis regulation of gut bacterial community.
15:00 – 15:30 COFFEE BREAK
15:30 – 16:30 Open discussion on the presentations.
16:30 – 17:30 Working Group Discussions.
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WEDNESDAY, 29 MARCH 2017
07:00 – 08:00 Transportation to the COPEG plant
08:00 – 12:00 Visit to the COPEG plant
12:00 – 13:00 Transportation to the hotel
13:00 – 14:30 LUNCH
14:30 – 15:00 Working Group Discussions
15:00 – 15:30 COFFEE BREAK
15:30 – 17:30 Working Group Discussions
THURSDAY, 30 MARCH 2017
08:30 – 10:30 Working Group Discussions.
10:30 – 11:00 COFFEE BREAK
11:00 – 12:30 Working Group Discussions.
12:30 – 14:00 LUNCH
14:00 – 15:30 Drafting Report.
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Drafting Report.
18:00 Group dinner at Miraflores Restaurant, Panama Canal.
FRIDAY, 31 MARCH 2017
08:30 – 10:30 Reports of Working Groups and Revision of Logical Framework.
10:30 – 11:00 COFFEE BREAK
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11:00 – 12:30 Drafting of Final Report.
12:30 – 14:00 LUNCH
14:00 – 15:30 Drafting of Final Report.
15:30 – 16:00 COFFEE BREAK
16:00 – 17:30 Presentation and approval of the Final Report - General discussion.
17:30 Closing.
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PARTICIPANT ABSTRACTS
SECOND RESEARCH COORDINATION MEETING
On “Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains
Produced by Genetic, Transgenic or Symbiont-Based Technologies”
Panama City, Panama
27 to 31 March 2017
TITLE OF WORKING PAPER: “Development and evaluation of a transgenic sexing strain
of the New World Screwworm, Cochliomyia hominivorax, carrying an embryonic female
lethal system”.
AUTHORS: Carolina Concha, Ying Yan, Evelin Quilarque, Agustin Sagel, Steve Skoda
and Max Scott
ORGANIZATION: COPEG-USDA, Panama; North Carolina State University, USA; USDA-
ARS, USA
SHORT SUMMARY OF PAPER
Abstract:
The New World Screwworm fly (NWS), Cochliomyia hominivorax, is a parasite of warm-
blooded animals and a major pest of livestock in tropical regions of South America. This
insect pest was eradicated from North and Central America using the Sterile Insect Technique
(SIT) and, currently, a control program managed by the governments of The United States
and Panama, releases millions of sterilized insects in the border between Panama and
Colombia to maintain a barrier that prevents its introduction from South America. The
control program releases both sterile males and females in the field but it has long been a
major goal for it to develop a male-only strain of the NWS to improve their operations.
Recently, our research team developed a sexing strain based on a single component
tetracycline repressible female lethal system. Although the transgenic system works very
efficiently and can introduce significant savings in production costs for mass rearing, further
savings can be achieved by the development of an embryonic lethal strain that would reduce
larval diet costs. Here, we report the development of embryonic sexing strains of NWS
carrying a two-component system consisting of the gene promoters L. cuprina bottleneck and
C. macellaria CG14427 driving expression of the tTA gene and a tetO-Lchid gene effector.
The insertion of the sex-specifically spliced intron from the transformer gene with the Lchid
gene ensures that only females die when insects are reared in the absence of tetracycline.
Female lethality occurs at the embryonic and first instar larval stages. Several transgenic lines
have been reared to homozygosity and studied for fitness characteristics that are relevant for
mass rearing. Our results suggest that some of these transgenic lines may be adequate for use
in the NWS control program and will be further tested in mass rearing conditions.
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TITLE OF WORKING PAPER: Transgenic sexing strains: evaluation of the influence
genetic background in Drosophila and new Lucilia cuprina gene promoters for driving tTA
expression.
AUTHOR (S): Ying Yan, Megan Williamson, Katherine Knudsen and Max Scott
ORGANIZATION: North Carolina State University
SHORT SUMMARY OF PAPER
Abstract:
We previously reported the development of single and two component transgenic sexing
strains (TSS) for the Australian sheep blow fly Lucilia cuprina. The two component TSS
contain two transgenes, a tTA effector and a tTA-regulated tetO-Lshid effector. As the Lshid
gene contained the sex-specific intron from the transformer gene only females produced
LsHID protein. tTA expression was driven by the Lucilia bottleneck or spitting image gene
promoters. As the promoters had some activity in adult females, we found it necessary to add
some tetracycline to the adult diet to obtain eggs. Here we report TSS assembled using the
Lucilia nullo and CG14427 gene promoters. With these drivers tTA is expressed mostly in
the embryo. We find that the TSS assembled with the new drivers do not need to be fed a diet
with tetracycline. Staged lethality tests are currently being performed but it appears that
female lethality occurs at the embryo stage.
Although radiation sterilization is a hallmark of SIT, the step could potentially be avoided for
a single component TSS that is fully dominant female lethal. Modelling suggests this strategy
could be particularly effective if the TSS carried a conditional female lethal gene on each
chromosome. However, this approach would fail if the lethal mechanism was not effective in
variety of genetic backgrounds. To test this possibility we generated transgenic Drosophila
melanogaster that carry a single component TSS. We used an isogenic strain for the
transgenesis. The strain is 100% female lethal when homozygous but 80-90% lethal in
heterozygotes. The strain was then crossed to each of the 201 highly inbred DGRP lines. We
found significant variation in dominant female lethality. As the genome sequences for all
DGRP lines are known, we performed a GWAS analysis and identified nucleotide
polymorphisms that associate with low or high female lethality. Our results suggest that
single component TSS are very sensitive to genetic background variation and would suggest
caution for using this approach for genetic control of pest species.
TITLE OF WORKING PAPER: Conditional Lethal Tephritid Strains for Improved SIT and
Genetic Stability.
AUTHOR (S): Handler, A.M.*, Li, J.*, Schetelig, M.F., Targovska, A., Meza, J.S., and
Bourtzis, K.
ORGANIZATION: *Center for Medical, Agricultural, and Veterinary Entomology, USDA,
ARS, 1700 SW 23rd Drive, Gainesville, FL, 32608 USA
SHORT SUMMARY OF PAPER
Abstract:
Current sterile insect technique (SIT) programs are often inefficient due to the inability to
eliminate females previous to male release, and the negative effects of radiation used to
sterilize males. To develop new genetic modification strategies for conditional sexing and
male sterility in fruit fly pests, we tested the E. coli tetracycline-suppressible (Tet-off) system
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for conditional female-specific embryonic lethality for sexing in Anastrepha ludens (Mexican
fruit fly). Tet-off embryonic serendipity-a promoter-tTA driver constructs and hid cell death
gene lethal effector constructs, having a sex-specific intron splicing cassette, were created
and transformed into A. ludens. Parental adults double-homozygous for the driver/effector
vector cassettes were expected to yield male-only progeny due to female-specific lethality
when reared on Tet-free diet, but a complete lack of oviposited eggs resulted in the three
hybrid strains tested. Dissection revealed non-vitellogenic oocytes in all strains, which was
reversible by feeding females tetracycline for 5 days after eclosion resulting in male-only
progeny in one strain. Presumably the sry-α promoter exhibits pre-zygotic maternal
expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off
sterility maternal effect in addition to female-specific embryonic lethality. Another approach
to developing sterile male-only strains is based on the Drosophila melanogaster transformer-
2 (tra-2) temperature-sensitive mutation, Dm-tra-2ts2
, that results in phenotypically normal
male and female fertile adults when reared at the permissive temperature of 160C. But when
reared at the restrictive temperature of 290C, chromosomal XX females develop as sterile,
phenotypic males (or pseudomales), while XY chromosomal males develop as normal, but
sterile, adults. Thus, a normal breeding population could be reared at 160
-200
C, whose
progeny reared at 290C would all develop as sterile males for release. To test this system in
D. suzukii, CRISPR-Cas9 gene-editing was used to create the analogous Ds-tra-2ts2
mutation
by Ds-tra-2 mutated sequence replacement and the addition of a DsRed marker gene. Putative
Ds-tra-2ts2
mutant lines were tested at 16-200C where homozygous males and females
developed as normal fertile adults, but at 290C it was discovered that D. suzukii adults failed
to eclose. Survival was possible at 260C where XY mutant males developed normally but
were sterile, while XX chromosomal females were intersexual and sterile, but primarily
expressed a male phenotypic morphology. XX; Ds-tra-2ts2
pseudomale adults survived at
29°C, and Ds-Tra-2 function could be tested in adults by assessing female-specific Yolk
protein 1 transcription, that was down-regulated at 29°C and up-regulated when shifted to
16°C. Together, these studies confirmed the temperature-dependent function of the Ds-tra-
2ts2
gene-edited mutation, and provided proof of principle for the creation of temperature-
sensitive tra-2 alleles by gene-editing in other species having the orthologous sex-
determination gene, and subject to SIT.
TITLE OF WORKING PAPER: Systems for unbiased comparison of transgenic sexing
systems in Aedes aegypti, Ceratitis capitata, and Anastrepha ludens.
AUTHOR (S): Marc F. Schetelig & Irina Haecker
ORGANIZATION: Justus-Liebig-University Giessen, Institute for Insect Biotechnology,
Winchesterstr. 2, 35394 Giessen, Germany
SHORT SUMMARY OF PAPER
Abstract:
Sexing systems are urgently needed for control programs using the sterile insect technique
(SIT). Male-only releases not only increase the efficiency of SIT programs, but are also a
prerequisite for mosquito SIT. Release of female mosquitoes, even if sterilized, poses a risk
to human health, as they will still bite and transmit disease. In the case of agricultural pests,
released females will still cause fruit damage. Safe sexing systems to produce male-only
strains for release programs are therefore an important research focus.
An RMCE system has been established in the vector Aedes aegypti in our group, and another
one is being tested in the agricultural pest Ceratitis capitata (Medfly). RMCE allows to easily
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and quickly add distinct biotechnologically engineered features including markers, lethality
and sexing systems, into pre-evaluated genomic target sites of so-called landing site lines.
Thus, different systems can be compared at the same genomic location without the influence
of position effects that interfere with the comparison at random genomic insertion sites as
obtained by transposon-mediated integration. Thereby, we will be able to identify the most
effective system(s) with regard to efficacy of sexing, rearing efficiency and mating
competitiveness. In Ae. aegypti, endogenous candidate genes and promoters for sexing
systems could be identified and isolated. The functionality of these genes has been
successfully tested in cell culture and we currently create transgenic mosquito lines with
these elements to test their suitability for sexing systems.
In medfly, the FRT/Flp-mediated RMCE system is tested and donor vectors have been
injected into pre-blastodermal embryos of a landing site medfly line. In case the
recombination is not observed, several other landing site lines will be used for the RMCE
assays. Once a functional RMCE system is established in medfly, we can start comparing the
available sterile male systems for medfly without the bias of position effects.
A similar approach has been successfully conducted in Anastrepha ludens, showing different
expression levels of fluorescent markers when additional markers were added at the landing
site. To build a sexing system in A. ludens, the transgenic embryonic sexing system (TESS)
developed in A. suspensa has been transferred and rearing procedures adapted to the mexfly.
Effects of the system on development have been observed and are further analysed.
TITLE OF WORKING PAPER: Instant induction of resistance evolution against
CRISPR/Cas9 gene drive and CRISPR/Cas9 based multifactorial reproductive sterility for
SIT approaches.
AUTHOR (S): Kolja N. Eckermann, Mohammad KaramiNejadRanjbar, Hassan M. M.
Ahmed, Stefan Dippel & 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:
The recent demonstration of efficient CRISPR/Cas9 Gene Drives (CGD) in flies and
mosquitos has sparked a discussion about the risks of CGDs not only for release but even for
laboratory use leading to recommendations by the US National Academies. Nonetheless, the
method-intrinsic induction of resistance development already observed in an HIV-1 therapy
approach has been experimentally neglected and so far only been modelled. Here we
demonstrate the instant emergence of gene drive-resistant alleles in the first generation of a
suppression gene drive system with a specifically high selection pressure. We targeted the
transformer locus involved in Drosophila melanogaster sex determination to potentially
transform all progeny to males, which should finally lead to a population collapse. In this
system, we could immediately identify and isolate non-homologous end joining-derived
resistant alleles indicating the increased rate of mutagenesis at the target site due to error
prone double-strand break repair. In our population collapse experiments, we observed –
despite an initial increase of males to up to 84% – that the population rebounded to about
50% males within 15 generations. This instantly induced resistance evolution thus suggests
that CGD designs need to be much more complex than previously proposed, but fortunately
serves as a system-inherent feature that provides a valuable natural safety control mechanism.
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Nevertheless, CRISPR/Cas9 can be applied 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.
TITLE OF WORKING PAPER: Tool development for silencing of spermatogenesis genes in
Anopheles albimanus to produce sterile males.
AUTHOR (S): Pamela Pennington, Claudia Paiz, Mabel Taracena
ORGANIZATION: Universidad del Valle de Guatemala
SHORT SUMMARY OF PAPER
Abstract:
Abstract: The control of malaria and other mosquito-borne diseases poses a significant
challenge in developing countries around the world. The sterile insect technique is an
alternative vector control method designed to suppress insect populations. In the 1970s, a
successful evaluation of a chemosterilization method was performed in El Salvador for
Anopheles albimanus, the primary vector of malaria in Central America. We propose to
develop a new generation of mosquito sterilization tools based on the oral delivery of bacteria
expressing dsRNA specific for spermatogenesis genes expressed during larval development.
We selected the An. albimanus homologues of boule and zpg, for their importance in meiosis
and sperm development. We studied their expression during the life cycle of the mosquito.
We designed a protocol for oral delivery of dsRNA produced by bacteria. We also developed
a protocol to evaluate male sterility in the laboratory. After testing several gene fragments,
we obtained stable inducible inserts of ~200 bp for both genes. We are currently designing
long dsRNA and hairpin-dsRNA, to test silencing efficiencies with alternative bacterial
expression strategies. We will use the tools and protocols thus far developed to feed larvae
with bacteria expressing dsRNA and evaluate the silencing effects on fecundity.
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TITLE OF WORKING PAPER: CRISPR-Cas9 functional genomics of medfly sex
determination and the hunting for the Y-linked M factor: a first list of putative Y-linked M
candidates.
AUTHORS: Angela Meccariello1, Marco Salvemini
1, Simona Monti
5, Maria Grazia
Inghilterra1, Pasquale Primo
1, Giuseppe Del Corsano
1, Andrea Gravina
1, Rita Colonna
1,
Philippos Papathanos3, Valeria Petrella
1, Kallare Arunkumar
4, Elena Chiavacci
2, Christian
Mosimann2, Svenia D. Heinze
2, Daniel Bopp
2 Stephan Schmeing
2, Mark Robinson
2, and
Giuseppe Saccone1
ORGANIZATION: 1Department of Biology, University of Naples Federico II, Napoli 80126,
Italy; 2Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland;
3Institute Section of Genomics and Genetics, University of Perugia, Perugia 06132 , Italy;
4Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India.;
5Institute of
Biostructure and Bioimaging (IBB-CNR), Naples, Italy
SHORT SUMMARY OF PAPER
Abstract:
CRISPR/Cas9-based genome engineering has been successfully used in few insect species for
gene function studies. Here, we developed an efficient DNA-free Cas9 gene editing protocol
for the generation of somatic and heritable germline mutations for the first time in a major
invasive agricultural pest species: the Mediterranean fruit fly Ceratitis capitata. We targeted
two Ceratitis genes by injecting into early Ceratitis embryos in vitro pre-assembled Cas9
protein-single-guide RNAs ribonucleoproteins. This resulted in high rate biallelic somatic
mutations among the G0 individuals (up to 19%) and germline transmission of mutated
alleles for more than 70% of tested flies. This CRISPR/Cas9 DNA-free mutagenesis protocol
for the medfly, together with the recent availability of its genome sequence and
transcriptomes, will significantly speed up the study of this species and facilitate the
development of new strategies for control of this pest of agricultural importance. We recently
started to target by Cas9 the female determining transformer gene and sexual transformations
toward maleness were observed.
A combination of differential expression analysis by XX and XX/XY embryonal
transcriptomics and FAM18 genomics (PacBio sequencing) led us to define a first list of
putative Y-linked M candidates which are under structural (PCR) and functional analysis (by
CRISPR and RNAi). However, none of the list is encoding a splicing factor-like protein, and
most of transcripts/genes are either encoding proteins with low similarity to known DNA
binding or enzymatic domains or showing no similarity with NCBI protein database. Hence
we suspect that more collaborative efforts are needed to expand the list of putative M factors
of medfly.
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TITLE OF WORKING PAPER: RNAi silencing of loci implicated in olfactory and
reproductive processes, as potential targets to improve the olive fruit fly population control
techniques.
AUTHOR (S): Tsoumani KT, Gregoriou M-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) represents the most significant enemy of the olive tree
worldwide causing significant production losses and olive fruit impoverishment. The need to
diminish conventional chemical approaches and develop efficient and more specific control
methods on the olive fruit fly leads without doubt in the study of biological systems related to
its harmful effect. In this context, the analysis of the olfactory and reproductive systems
through molecular approaches will demonstrate the respective molecular targets that
determine species-specific features on reproductive and sexual behavior. Genes implicated in
premating sexual communication and reproductive behaviour as well as reproduction have
been already identified through transcriptomics analysis.
In order to investigate their functional role we performed transient gene expression silencing
of selected loci through RNAi, either by injecting RNA duplexes (dsRNA) directly in
hemolymph of the insect or by bacterial feeding. The gene expression was evaluated by
quantitative Real-Time PCR demonstrating the effectiveness of the RNAi machinery via both
approaches in this species. Furthermore, the effect of silencing was evaluated during the
mating and oviposition processes. We observed the copulation rates and explored the effect
of the olfactory co-receptor Orco gene-silencing in female olive fruit flies during the
oviposition. Moreover silencing of two reproductive genes, yellow and sex peptide receptor
in male and female insects respectively, affected egg production capacity and mortality rate.
The resulted phenotypes suggest that the examined genes play crucial roles in the
reproductive behavior of the olive fly, since pre- and/or post- mating processes were affected.
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.
The project is implemented under the "ARISTEIA" Action of the "OPERATIONAL
PROGRAMME EDUCATION AND LIFELONG LEARNING" and is co-funded by the
European Social Fund (ESF) and National Resources.
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TITLE OF WORKING PAPER: Exploring the male competitiveness in Ceratitis capitata
strains.
AUTHOR (S): Giuliano Gasperi, Paolo Gabrieli, Ludvik M. Gomulski, Francesca Scolari,
Alessandro Di Cosimo, Anna R. Malacrida
ORGANIZATION: Dept. of Biology & Biotechnology, Univ. of Pavia, 27100 Pavia, Italy
SHORT SUMMARY OF PAPER
Abstract:
In comparing the performance of Medfly strains, one topic which deserves particular
attention is the analysis of male competitiveness that we are approaching at different levels.
At the core of male reproductive success is the male ejaculate transferred to the females
during mating process. Our aim is to disentangle the effect of seminal fluid vs sperm in
modulating Medfly female post-mating behaviour. We were able to demonstrate that sperm
transfer is not necessary to reduce female sexual receptivity, and to increase oviposition and
fecundity. The seminal components play a key role in modulating female post-mating
responses. This result highlights the importance of further investigations on the effect of
irradiation, currently used to produce sterile males, on the male accessory glands and the
activity and integrity of seminal fluid proteins (SFPs).
Concerning the chemoreception in Medfly, our interest is focused on the characterization of
components (OBPs and ORs) which are involved with sexual behaviour and attraction. We
are analyzing the genes, the proteins and their specific ligands in different strains. These
components play a key role in male competitiveness. Moreover we are exploring the impact
of different larval diets on the production of volatile components emitted by male and
females which can impact mating interaction. This represents a key point to be explored in
mass-reared conditions of different strains, given that different volatile blend affect male and
female reproductive potential.
TITLE OF WORKING PAPER: ‘Quality’: What is it, and how do we know when we have
it?
AUTHOR (S): Taylor PW, Lee R, Yeap HL, Lynch K, Oakeshott J
ORGANIZATION: Macquarie University
SHORT SUMMARY OF PAPER
Abstract:
The Queensland fruit fly, Bactrocera tryoni Froggatt, or ‘Q-fly’, is Australia’s most
damaging fruit fly pest. Over the past decade, Q-fly has substantially expanded its range
southward to increasingly encroach upon areas that until recently enjoyed the marketing
advantages of fruit fly free status. Adjacent regions that remain fruit fly free face increasing
threat. As a pro-active response, Australia has, through the ‘SITplus’ consortium of
governmental and university researchers, committed to establishing a substantial new SIT
program that will protect Australia’s major southern growing regions. Diverse approaches are
being used to establish genetic sexing strains as this remains the most significant remaining
hurdle. Under the SITplus umbrella, research teams aree currently developing strains using
RNAi, CRISPR and pupal colour selection.
Research aligned with the development of strains has focused closely on identifying the
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nature of ‘quality’, how quality varies across strains and through the domestication process,
and how quality can be preserved and promoted. While the standard Quality Control (QC)
assays provide simple, effective and economical tools to probe for perturbations in
production systems they are perhaps not the right tools to define quality in terms of the
biological tasks required of released flies. In biological terms we can think of quality as
relating to (1) the ability of released males to secure copulations with wild type females,
including competitiveness (‘sexual quality’), and (2) the ability of released males to disperse,
survive and develop (‘ecological quality’). Studies of SIT have well-established QC assays
for sexual quality. However, we contend that the importance of ecological quality has not
received due recognition and that significant advances in SIT performance might be achieved
by greater consideration of the environment in which released flies must perform. In
Australia, high temperatures combined with low humidity present some of the greatest
challenges to the ecological performance of released flies and we have developed a series of
effective and repeatable assays to probe these important parameters. Current research
investigates regional variation in thermal and desiccation tolerances of Q-fly, and the effects
of domestication. Detailed studies of physiology and genetics will then seek to reveal the
underlying mechanisms. This knowledge will enable the development of strains that have
superior development, dispersal and longevity under often-hostile field conditions, thereby
increasing the operational overflooding ratio for a given release of flies.
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:
A Regional R&D Training Center for Insect Biotechnology has been established. A new mass
rearing laboratory (ca. 697 m2) has been designed and constructed based on an expert mission
from the International Atomic Energy Agency. Adults- and pupae- mass-rearing cages, and
automatic pupae colour sorter machine were attained. It was expected that this new mass-
rearing facility can produce up to 3 million male pupae per week. The Salaya1 genetic sexing
strain (based on brown-white pupae), Bactrocera dorsalis, filter rearing was transferred from
a modular mass-rearing prototype unit to actual mass-rearing environment. A filter rearing
system has been conducted from a clean stream to an initiation stream. Comparing rearing
efficiency and competitiveness of the Salaya1 males from these streams was investigated to
optimize and refine the mass-rearing methods.
Progress in the development and technology transfer of molecular-based tools to genetic
sexing strains (in supporting of SIT programs against the Bactrocera species) will be
reported.
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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 Perez.
ORGANIZATION: National Program Fruit Flies. SAGARPA-SENASICA.
SHORT SUMMARY OF PAPER
Abstract:
The genetic sexing strain “Tapachula-7” (Tap-7) of the Mexican fruit fly (Mexfly),
Anastrepha ludens was developed to provide the possibility of male-only releases in the
sterile insect technique (SIT) suppression program in Mexico. In early 2012 the mass
production of Tap-7 at the Moscafrut facility was 5 millions male pupae per week. In 2013
was 7.5 millions and in 2014 reached 15 millions. In 2015 the production was increased to
43.5 millions per week during a short period, and then production was reduced to 25 million,
mainly because of poor quality detected in sterile males mating performance.
The Tap-7 integrity in the mass rearing is maintained through a filter rearing system (FRS)
and recently a mother colony (MC) was implemented. The main objective of MC is to
maintain the biological attributes required for successful SIT application, which usually are
diminished or lost during the domestication process. This is achieved through stress reduction
of the rearing regime, enriching environment, directed selection and hybridation with wild
populations (refreshing the genetic background). In order to improve the quality of Tap-7, the
first refreshing was carried out in the MC, using three different and simultaneous hybridation
with a wild population from Chiapas. After refreshing, production and quality control
parameters were significantly improved. Mating performance and sterility induction were
significant increased compared with males without refreshment. The old Tap-7 MC was fully
replaced in 2016at its 66th
generation.
In collaboration with IPCL in Seiberdorf a new marker named “slow development larvae”
(swl) from Anastrepha ludens was isolated. The swl allele was linked to the “black pupa”
(bp) allele, and was introduced into the Tap-7 through backcrosses. This allows the
production of normal pupae (males) faster than black pupae (females). In this new version,
named Tap-7/slow, the females are homozygous for both mutations (bp-swl) and the larvae
reach its pupation process two days after normal males, which allow the female self-
separation from the majority of males during the pupation process. A small remaining
proportion of pupae have a blend of colors, and males still can be separate by using the
sorting machine. The plan will be to test it under semi-mass rearing conditions.
Transgenic Sexing Strains (TSS). Three TSS were evaluated (Tap-7/E3, T(Ygfp
/bp+)-3,
P_M6y), using as control the Tap-7 and wild strains. The TSS P_M6y showed higher fitness
than the others TSS and similar to the normal type. This strain is now in maximization to
validate its potential use in SIT. Next step will be to verify the feasibility of sex separation at
the pupal stage through a fluorescent sorting machine (only the male pupae show red
fluorescent). The males will be tested for mating performance against wild flies, under field
cage condition. Additionally male mating performance of the transgenic embryonic sexing
strain (D-419-17-E-443-1) under field cage condition is ongoing at IPCL.
Thermal Sensibility Lethal induction (tsl). Two different protocols of crosses were
implemented in order to isolated tsl recessive mutations, currently 12 and 4 families from
each protocols had been isolated as possible tsl strains, their confirmation is in progress.
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TITLE OF WORKING PAPER: Transgenic lines competitiviness evaluation for Ceratitis
capitata and Aedes aegypti to be used in insect control.
AUTHOR (S): J. F. Virginio
ORGANIZATION: Universidade de Sao Paulo, Instituto de Ciencias Biomedicas, Sao Paulo,
Brazil
SHORT SUMMARY OF PAPER
Abstract:
This project has two major goals: (1) test and to compare the effectiveness of the classic SIT
and transgenic strains of Ceratitis. capitata in order to enhance the quality of flies produced
for SIT purposes; and (2) test Aedes aegypti transgenic lines that are sterile under the
regulation of TET-OFF system and evaluate their effectiveness in field conditions. During
2016 all bureaucratic import processes continued to be carried out in order 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 Permit” for the transgenic medfly FSEL#32 strain was
granted by the Ministry of Agriculture. In parallel to the import process, all experimental
methodology and procedures were discussed and defined so that when the material arrives, all
practices and tests could be executed. Regarding mosquito activities, six Sterile Conditional
Construction (SCC) for A. aegypti males were developed. To obtain these strains, it was
made a selection of elements to promote (β2-tubulin), regulate (tTAV-tTO system) and
sterility effector molecule (endonuclease, CviAII, and an inhibitor apoptosis antagonist,
Michelob_x). Up to now, two candidate lines (MFDX and MPD02) presented statistical
difference in the number of larvae/female (39% and 62% of sterility in heterozygosis at
laboratorial conditions) but no difference in the number of eggs/female, as expected. The
preliminary results show that those lines can induce sterility in wild population. To finish the
characterization of this line, more additional challenges are needed. However, these lines
need to achieve the homozygous colony status, so sterility challenges can be repeated and
present a better and more realistic status of each line sterility. Only then, tests in semi-field or
open-field conditions will be developed. Also it was carry out the evaluation of the
conditional sterility of transgenic lines by subjecting SCC mosquitoes to absence and
presence of tetracycline at different concentrations. The number of eggs and the number of
larvae was recorded and evaluated statistically. The presence or absence of tetracycline for
the Higgs line (control) did not present statistical difference (p value <0.05). Identical to the
control strain, MEFA07, MPC03, MFDX and MPD02 lines also did not present any
statistical difference between the values observed for the number of eggs and the number of
larvae. In parallel, the ratio of the number of larvae to the number of eggs per female was
calculated and the control strain (Higgs) did not present statistically significant difference
using analysis of variance. The same is observed among the lines MFA07, MFDX and
MPD02, where there is no statistical difference between the groups with absence or presence
of tetracycline. The strains which shows differences in the hatch rate were MPC03 and
MPC02 (p <0.05). The data points to an expression of the gene which conditionality
(presence/absence) is not present. This implies the activation of the sterility promoter gene all
the time, and it is not possible to inactivate it, as it was initially conceptualized. In the attempt
to deactivate the SCC transgene, we assembled two protocols of successive addition and
maintenance of tetracycline in different concentrations due to the growth during
developmental larval stage. This attempt was made due to the inability to deactivate the gene
in MPD02 when supplemented with tetracycline, thus increasing the dose, or keeping it at
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constant levels, the gene could be deactivated/regulated. The first experimental design used
100 larvae with the following lines: Higgs (non-transgenic and used as control), MFDX (SCC
strain), MPD02 (SCC strain) and OX513A (line dependent of tetracycline, used as a positive
control). The standard tetracycline concentration used in OX513A line is 30 μg/ml. However,
in the attempt to inactivate the SCC gene a limit of 50 μg/ml was established based on the
concentration dose for Wolbachia elimination of infected mosquitoes with this symbiont. A
second experimental design was established including a new concentration of 100 μg/ml. In
both experimental designs the concentration change have occurred every 24 and the limit
concentration was maintained until the insects reached the adult stage. The result presented
that, regardless of the concentration used and even with the continuous water change of the
tetracycline-containing larvae, there was no statistically significant difference between the
lines regarding the number of eggs and numbers of larvae counted for the two experiments.
TITLE OF WORKING PAPER: Mass rearing of triple Wolbachia-infected Aedes albopictus
and its field pilot application in China.
AUTHOR (S): Dongjing Zhang, Zhiyong Xi
ORGANIZATION: Sun Yat-sen University-Michigan State University Joint Center of Vector
Control for Tropical Diseases, Guangzhou, Guangdong 510080, China
SHORT SUMMARY OF PAPER
Abstract:
The endosymbiotic bacterium Wolbachia is widely recognized for its ability to induce both a
reproductive abnormality known as cytoplasmic incompatibility (CI) and a resistance to
dengue virus in mosquitoes. Wolbachia-based population suppression strategy, referred to as
Incompatible Insect Technique (IIT), entails the release of male mosquitoes infected with
Wolbachia, resulting in sterile matings and a reduction in the mosquito population. Here, we
will report an ongoing field pilot trial to control dengue mosquito vectors through release
males to induce female sterility in Guangzhou China. The released Aedes albopictus HC
strain carry a novel Wolbachia from Culex pipiens mosquito, which induces both CI toward
the wild type mosquito and resistance to dengue virus. Mass rearing capacity has been
successfully developed with a production of >5 million HC males per week. An X-ray
irradiator has been developed for mass production to prevent from the potential population
replacement caused by the released females escaped from the sex separator. Significant
suppression of mosquito population has been accomplished in the release sites compared to
the control. We will discuss our results in relation to implementation of SIT/IIT for the area-
wide suppression of mosquito population for disease control.
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TITLE OF WORKING PAPER: Evaluation of genetic and biotechnological tools towards the
development of an Anastrepha fraterculus sexing strain
AUTHOR (S): Lanzavecchia Silvia, Claudia Conte, Giardini Cecilia, Scannapieco Alejandra,
Milla Fabián, German Crippa, Segura Diego and Jorge Cladera
ORGANIZATION: Instituto de Genética (IGEAF), Instituto Nacional de Tecnología
Agropecuaria (INTA)
SHORT SUMMARY OF PAPER
Abstract:
Genetics and genomics of the “South American fruit fly” Anastrepha fraterculus Wiedemann
(Diptera: Tephritidae) are currently being intensively investigated from different points of
view, including unravel genetic entities within the cryptic species complex by using SNPs
and transcriptomic approaches and, the study of candidate genes to evaluate molecular
pathways of interest. Our project is contributing with valuable information regarding the
development of an A. fraterculus sp1 GSS useful to the SIT implementation in Argentina.
During the past year we have worked to obtain the first results of A. fraterculus trancriptome,
including information of expressed genes in mature adults (females vs. males) and immature
stages (72 h embryo). This approach brings useful specific markers to differentiate sexes at
early develomental stages and information regarding molecular mechanisms of interest (e.g.
early sex-determination pathways). The information obtained so far will be used to initiate
studies on reproduction and olfaction, two of the most basal pathways involved in insect
performance and success in the field (host alocation, individual recognition and
comunication). In addition, the analysis of differential expressed genes between sexes will
contribute to the identification of candidate regions to be evaluated as estructural markers to
the cytogeneitc analysis of A. fraterculus sp. 1 strains, also providing useful information to
the characterization and selection of specific gene regions for transgenic experiments using A.
fraterculus strains.
Regarding the cytogenetic characterization of wild populations of Argentinean A. fraterculus,
we are curenlty involved in a new sampling effort in order to describe the current distribution
of karyotypes in our country. This information is important to develop purified A. fraterculus
strains carrying specific karyotypic variants.
TITLE OF WORKING PAPER: Performance of a Genetically Modified Strain of the
Mediterranean Fruit Fly and the effect of irradiation doses on its sterility and biological
security.
AUTHOR (S): Ramírez-Santos Edwin
ORGANIZATION: MOSCAMED PROGRAM/Guatemala
SHORT SUMMARY OF PAPER
Abstract:
A series of trials were conducted to determine the viability of the use of a Genetically
Modified Strain of Mediterranean Fruit Fly (VIENNA 8 1260). The VIENNA 8 1260 strain
had a lower yield than the control strains, VIENNA 8 with D53 inversion (VIENNA 8) and
without D53 inversion (VIENNA 8 D53-). At mass rearing scale, yield gradually increased in
three generations without reaching the control strains values. The VIENNA 8 1260 strain was
stable in the genetic sexing mechanism (>99.9 %) and expression of fluorescence (100 %).
In field cages the VIENNA 8 1260 males reduced the mating potential of wild males in the
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same magnitude as the VIENNA 8, when evaluated in independent cage tests. However, the
relative sterility index (RSI) and the strain male relative performance index (SnMRPI) of
VIENNA 8 1260 males were significantly lower than those of the VIENNA 8. There were no
significant differences in longevity of these strains. In spite of the performance differences
documented between the VIENNA 8 1260 strain and the VIENNA 8 D53- or VIENNA 8
strains, it is important to point out that these results suggest that the yield and quality are
subject to improvement (i.e. by modifying some of the rearing conditions, or by selection of
the best lines during the mass rearing process ) and that the VIENNA 8 1260 expresses
desirable traits, such as the stability in the fluorescence and genetic sexing mechanism (tsl
and wp genes), which makes it a strong candidate to be included in Mediterranean fruit fly
SIT, AW-IPM programs.
The general effect of irradiation on VIENNA 8 1260 follows the same patterns documented
for VIENNA 8 D53-. Doses of 80 Gy or greater reduced fertility and induced high levels of
sterility in wild females. Fecundity reduction was higher in VIENNA 8 1260 than in
VIENNA 8 D53- females. Vertical transmission of the fluorescence gene was confirmed up
to the F4 generation. Substerilization in the VIENNA 8 1260 could jeopardize the usefulness
of the transgenic strain due to the possible vertical transfer of the fluorescence transgene,
from the sterile to the wild flies. A biologically safe higher irradiation dose could result in
reduced competitiveness of the VIENNA 8 1260 strain. With this strain it is necessary to
consider the selection of an optimal dose (high enough to induce sterility, but without
reaching levels that compromise the quality, especially the sexual performance, of the
irradiated males), while at the same time establish the maximum level of biosafety control in
mass rearing laboratories, as well as in the field. Under no circumstances should
substerilizing doses be used with VIENNA 8 1260 males, in an attempt to increase their
mating performance in the field, as has been suggested for other, nontransgenic strains. Based
in our results, we recommend an irradiation dose of 140 or 145 Gy. The remating
experiments suggest: a) a relative precedence in the use of sperm by the Medfly females and
b) a lower fitness of the VIENNA 8 1260 sperm after mating, as compared with the non-
fluorescent sperm.
To assess the horizontal transfer risk of genetic sequences of the fluorescence transgene from
the VIENNA 8 1260 strain to its parasitoid Fopius ceratitivorus (Braconidae), a DNA
extraction and PCR amplification was performed using specific primers for the fluorescence
markers at four independent laboratories. No horizontal transfer of the transgene was detected
from the VIENNA 8 1260 to its parasitoid for 16 generations. The conclusions are consistent
when comparing the PCR results from four different laboratories.
TITLE OF WORKING PAPER: A new gel-based diet formulation to improve rearing
efficiency of Qfly.
AUTHOR (S): Fleur Ponton, Tahereh Moadeli, Bishwo Mainali, Phillip W. Taylor
ORGANIZATION: Department of Biological Sciences, Macquarie University, Sydney, NSW
2109, Australia
SHORT SUMMARY OF PAPER
Abstract:
New larval diets are needed for economical mass rearing of high quality insects for use in
SIT. The Queensland fruit fly or ‘Q-fly’, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae),
is Australia’s most economically damaging insect pest of horticulture, and the sterile insect
technique (SIT) has been adopted for management of Q-fly populations. The success of SIT
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is underpinned by efficient mass rearing and release of millions of sterile flies to curtail
reproduction of wild populations. Solid larval diets that incorporate biological bulking agents
have long been used for fruit fly mass rearing, but these present problems of substantial waste
and variable quality. Liquid larval diets have been successfully developed as an effective
alternative for small-scale rearing, but these also suffer drawbacks such as difficulty in
handling, fermentation and heterogeneity that have limited their adoption in large-scale
production. The present study considers whether some of the deficiencies of liquid larval diet
might be overcome by incorporating agar to create a semi-liquid or gel diet. Overall, gel diets
containing greater than 0.5% agar outperformed liquid diets and semi-liquid diets of identical
nutritional composition, especially in terms of development rate and overall productivity, and
compare very favorably to reported performance of traditional solid diets used for Q-fly. In
addition to developing a solid diet, we assessed the effects of different nutritional ingredients
such as yeast and fatty acids on Qfly performance. Our results show that common yeast
strains have good nutritional properties when added to the gel-based diet comparing to more
expansive blends. Indeed, diets made up with cheap yeast strains, such as Brewer’s yeast,
produced high quality flies. In parallel, experiments to assess the effects of fatty acids were
run. Results show that flight ability depends on unsaturated fatty acids whereas development
and recovery do not depend on specific fatty acids. With these experiments, larval diet
formulation was refined to improve rearing efficiency using a biological approach to better
understand the nutritional biology of Qfly.
TITLE OF WORKING PAPER: Symbiotic and genetic analysis evaluation of strains used in
SIT
AUTHOR (S): Augustinos AA1,2
, Kyritsis G2,3*
, Nikolouli K2, Caceres C
2, Mintzas A
1,
Tsiamis G4, Bourtzis K
2
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; 3Laboratory of Entomology and Agricultural
Zoology, Department of Agriculture Crop Production and Rural Environment, University of
Thessaly, N. Ionia Magnisia, Greece; 4Department of Environmental and Natural Resources
Management, University of Patras, Agrinio, Greece; *present address
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
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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 that the degree of domestication, the developmental stage sampled and
the rearing practices heavily influence the structure of the gut symbiotic communities.
Genetic analysis using microsatellite markers is ongoing, aiming to create the genetic and
symbiotic profile of these populations at the same generations.
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)
shed light to the underlying differences that influence the Quality Control parameters of
important laboratory strains (such as the medfly VIENNA 7 and 8 GSS), c) provide
standardized and universalized protocols for the monitoring of genetic and symbiotic changes
and, d) explore the utilization of NGS based approaches (such as Pool-seq) for the monitoring
of the laboratory adaptation process.
TITLE OF WORKING PAPER: Intestinal probiotics restore Bactrocera dorsalis’ ecological
fitness damaged by Co60
irradiation and the homeostasis regulation of gut bacterial
community.
AUTHOR (S): Hongyu Zhang1* Chaohui Cai
1 Zhichao Yao
1 Yushan Li
1 Bruno Lemaitre
2
Zhiyong Xi3 Kostas Bourtzis
4
ORGANIZATION: 1.Huazhong Agricultural University, China. 2.Global Health Institute,
Switzerland. 3. Michigan State University, USA. 4. FAO/IAEA, Austria
SHORT SUMMARY OF PAPER
Abstract:
Effects of ionization radiation is a crucial issue in different areas of interest, from cancer
therapy and environmental safety to industrial and agricultural processes. An suitable and
stable gut microbiota influences many beneficial processes in the metazoan host. With the
increasing frequency of ionization radiation utilization, it is more easy to expose to harmful
radiation; however, little is known about how radiation influences gut bacterial community of
animal and what intestinal microflora act the role in irradiation damage progress. Here, we
analyze influence of sterilizing irradiation on the composition and structure of intestinal
microbiota community of Bactrocera dorsalis. We find that the main characteristics of
radiation-induced dysbiosis of gut community have a higher gut microbial diversity, the
major members of the gut community Enterobacteriaceae temporary down-regulated and the
minor members of the gut community Bacillaceae prolonged up-regulated relative to that
present in the gut samples. Increasing relative abundance of the minor members gut microbial
communities lead a higher nutrients-related metabolism activity that may competes with the
host for nutrient resource from food bolus in gut, resulting in a serious decline in ecological
fitness like male mating ability, flight capacity and survivorship. Reinfection of the symbiotic
bacterial species Klebsiella michiganensis BD177 to the irradiated flies can restore the
ecological fitness damaged by Co60 irradiation. Our findings raises the possibility of using
probiotics as radiation damage repair agents in sterility insect and provides insight into the
screen and administrative of beneficial bacterium and potential mechanism of probiotic
mediated radiation damage repair.
The guts of metazoans are in permanent contact with the microbial realm that includes
beneficial symbionts, nonsymbionts, food-borne microbes and life-threatening pathogens.
However, little is known concerning how host immunity affects gut bacterial community.
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Here, we analyze the role of a dual oxidase gene (BdDuox) in regulating the intestinal
bacterial community homeostasis of the oriental fruit fly B. dorsalis. The results showed that
knockdown of BdDuox led to an increased bacterial load, and to a decrease in the relative
abundance of Enterobacteriaceae and Leuconostocaceae bacterial symbionts in the gut. The
resulting dysbiosis, in turn, stimulates an immune response by activating BdDuox and
promoting reactive oxygen species (ROS) production that regulates the composition and
structure of the gut bacterial community to normal status by repressing the overgrowth of
minor pathobionts. Our results suggest that BdDuox plays a pivotal role in regulating the
homeostasis of the gut bacterial community in B. dorsalis.
Acknowledge: This work is supported by IAEA Co-ordinated Research Contract No.
18805/R0, the National Natural Science Foundation of China (No. 31572008), the earmarked
fund for the China Agricultural Research System (No. CARS-27) and Fundamental Research
Funds for the Central Universities (No.2014PY005) to Hongyu. *Corresponding author:
Hongyu Zhang. E-mail: [email protected]
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LIST OF PARTICIPANTS
Argentina Ms Silvia LANZAVECCHIA
Instituto Nacional de Tecnología Agropecuaria
Rivadavia 1439
1033 AAE BUENOS AIRES
ARGENTINA
Email: [email protected]
Australia Mr Phillip WARREN TAYLOR
Macquarie University
Balaclava Road
2109 NORTH RYDE NEW SOUTH WALES
AUSTRALIA
Email: [email protected]
Brazil Mr Jair Fernandes VIRGINIO
BioFabrica MOSCAMED
Av. C1, 992 - Quadra D 13
Lote 15,
Distrito Industrial do São Francisco
JUAZEIRO 48.908-000
BRAZIL
Email: [email protected]
China Mr Hongyu ZHANG
Huazhong Agricultural University
College of Plant Science and Technology
Shizishan Street 1
430070 WUHAN HONGSHAN
CHINA
Email: [email protected]
Germany Mr Marc SCHETELIG
Justus-Liebig-University Giessen / Fraunhofer IME
Winchesterstrasse 2
35394 GIESSEN
GERMANY
Email: [email protected]
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Germany Mr Kolja Neil ECKERMANN
Georg August University Göttingen
Institut für Zoologie, Anthropologie und Entwicklungsbiologie
Justus-von-Liebig-Weg 11
37077 GÖTTINGEN
GERMANY
Email: [email protected]
Greece Mr Kostas MATHIOPOULOS
Department of Biochemistry and Biotechnology
University of Thessaly
Ploutonos 26
LARISSA 41221
GREECE
Email: [email protected]
Guatemala 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]
Guatemala Mr Edwin Mauricio RAMÍREZ SANTOS
Medfly Program Guatemala
16 Calle 3-38 Zona 10
01010 GUATEMALA
GUATEMALA
Email: [email protected]
Italy Mr Giuseppe SACCONE
Università Degli Studi Di Napoli
Federico II
Dipartimento Di Biologia
Corso Umberto I
80138 NAPOLI
ITALY
Email: [email protected]
Mexico Mr Víctor GARCÍA MARTÍNEZ
Campana Nacional Contra Moscas de la Fruta
Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación
(SAGARPA)
Camino a Cacahoatales S/N
30860 METAPA, CHIAPAS
MEXICO
Email: [email protected]
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Panama Ms Maria Carolina CONCHA
La Comisión para la Erradicación y
Prevención del Gusano Barrenador
del Ganado (COPEG)
Screwworm Fly Production Plant
P.O.Box 0816-07636
PACORA
PANAMA
Email: [email protected]
Thailand Mr Sujinda THANAPHUM
Department of Biotechnology,
Faculty of Science, Mahidol
University
272 Rama VI Road
10400 BANGKOK,
RATCHATHEWE
THAILAND
Email: [email protected]
USA 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]
USA Mr Maxwell SCOTT
North Carolina State University
2701 Sullivan Drive
P.O.Box CB 7514
27695-7614 RALEIGH, NC
UNITED STATES
Email: [email protected]
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IAEA
(Scientific
Secretary)
Mr Konstantinos BOURTZIS
Insect Pest Control Laboratory
FAO/IAEA Agriculture &
Biotechnology Laboratories
2444 Seibersdorf
Austria
Email: [email protected]
OBSERVERS
Australia Mr Owain EDWARDS
CSIRO Land & Water Flagship
Underwood Avenue
Floreat WA 6014
Clunies Ross St
ACT 2601 CANBERRA
AUSTRALIA
Email: [email protected]
Australia Mr Heng LIN YEAP
CSIRO Black Mountain
GPO Box 1700
ACT 2601
AUSTRALIA
Email: [email protected]
Australia Ms Fleur PONTON
Macquarie University
Balaclava Road
NORTH RYDE 2109
AUSTRALIA
Email: [email protected]
Brazil 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]
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Italy Mr Giuliano GASPERI
Department of Biology and Biotechnology
University of Pavia
Via Ferrata 9
27100 PAVIA
ITALY
Email: [email protected]
Mexico Ms Martha Griselle ROBLERO ROBLERO
National Program Fruit Flies SAGARPA-
SENASICA
Camino a los Cacahotales s/n
30860 METAPA DE DOMINGUEZ,
CHIAPAS
MEXICO
Email: [email protected]
