CONTENTS

 Sterile insect technique

 Genetic sexing mechanism (GSM)
 RIDL
 Comparison of SIT and RIDL
 Development of RIDL strain for insect control
 RIDL positive feedback system
 Markers and monitoring
 Benefits of RIDL
 Case studies
 Current status of OXITEC RIDL Strains
 Conclusion
 Future perspective
 Application of genetic engineering for insect control was first
discussed during a meeting sponsored by Rockefeller
foundation in 1979 (Levin, 1980).
To address important concerns:
1. Socio-economic challenges

2. Public health challenges

The first application of the new transgenic insect technology is

likely to be improvements in the sterile insect technique (SIT).
 Sterile Insect Technique
 Mass rearing and release of sterile insects

 Controlling insects by sterilisation was first conceived by

Dr. Edward F. Knipling in 1937.

 In 1954, Successful area-wide SIT programme- New World

Screw Worm in northern and central USA.

 Other notable Success

 Drawbacks of SIT

 Sex-separation
Released sterile females- detrimental to control programme

 Negative effect of sterilization

 Difficulties in discrimination

 Highly expensive method

 Genetic sexing mechanism (GSM)
 Sex sorting genetic system

 Principle: Autosomal translocation (Robinson et al., 1989)

 The first medfly GSS (T(Y:5)101) with white pupae mutation

(Robinson and van Hemert 1982)

 Franz et al. (1996)- isolated tsl marker from medfly, in which

homozygous females can be eliminated during embryogenesis by
incubating eggs in a 34°C water bath
Mutations as visible markers
Brown pupal phenotype - males
Mutant white pupal phenotype - females

Franz et al., 1996

Mediterranean fruit fly genetic sexing: large scale
field comparison
 Strains
Male-only strain: Vienna-4/Tol-94(tsl gene) @ 2000/ha/week
Standard bisexual : Petapa @ 4000/ha/week
Field evaluations over a 2-yr period (2001-02) in
southwestern Guatemala

 Estimated number of wild flies population

 Induced egg sterility(%)

Rendon et al., 2005

Fig.1: Plan of layout of each plot for release
Each sub plot- 1 square km
Total size of plot: 36 square km

Rendon et al., 2005

 2001 2002

Weeks of the year

Fig.2: Estimated number of wild flies population(FTD) in areas of petapa and VIENNA4/Tol-94 strains
released

Rendon et al., 2005

 2001 2002

Weeks of the year

Fig.3: Comparison of induced egg sterility between Petapa and VIENNA 4/Tol-94 strains

Rendon et al., 2005

 RIDL
 Major modification of SIT

 Insects are not sterile

 Principle: Tet-on and Tet-off system

 Tet-on : suppression of DL gene

 Tet-off : female specific lethality

Types
Bisex RIDL
Female-specific RIDL
 SIT and RIDL

no. of generations

Fig.6: Comparison of the effectiveness of SIT and RIDL

Fig.5: Outline of SIT and RIDL
Andreasen et al., 2007
 Development of RIDL strain for insect control

Develop RIDL strain Mass rearing Removal of females Male-only release

DL gene

RIDL male Wild female

Fig.7:Population reduction using RIDL based system

Wilke et al., 2009

 RIDL positive feedback system

Fig.8: Tetracycline-repressible lethal systems

tTa :Tetracycline-repressible trans-activator fusion protein.
tetO: Female Specific DNA.

Thomas et al., 2000; Horn and Wimmer, 2003

Markers
 Fluorescent dusts or food dye

 The fluorescent proteins are derived from marine corals or

jellyfish

 RIDL strains have a heritable, fluorescent marker

 The markers can also be read with PCR techniques in the

laboratory to provide a further tool for identifying the captured
insects
Fig.9: Fluorescent protein markers in RIDL strains of Mediterranean fruit fly
Benefits of RIDL

 Species-specific, cost effective and chemical-free

 Works well, even at very low pest population
 Better-quality insects than SIT
 Biosecure, as self-limiting in the wild
 Genetic marker for monitoring
 Easily produced using existing mass-rearing protocols
 Effective barrier to re-invasion

Asokan, 2007
 In 2000, Thomas et al., developed RIDL system in Drosophila
melanogaster using Drosophila transcriptional control elements

 Tetracycline-repressible transactivator fusion protein (tTa)

 Tetracycline responsive element (tRe)
 Female specific fat body enhancer (Yp3 )

 Sex specific expression system (Yp3tTa)

 Non- Sex specific expression system(Hsp26tTa)

x specific expression system (Yp3tTa)

Fig.10: tTa Expression in Yp3 using tRe-lacZ reporter

A: wild type female on normal media
B: Yp3-tTa, tRe-lacZ male on normal media
C: Yp3-tTa, tRe-lacZ female on normal media
D: Yp3-tTa, tRe-lacZ female on media with tetracycline (0.1 micro gram/ml)

Thomas et al., 2000

Non- Sex specific expression system(Hsp26tTa)

Table1: Tetracycline-repressible lethality with Yp3-tTa

Yp3-tTa: female-specific tTa

Hsp26-tTa: non-specific tTa

Thomas et al., 2000

 Repressible female-specific lethal genetic
system through terminator gene (hid)

 In 2004, Henrich and Scot (2004)- developed tetracycline-

repressible female-specific lethal genetic system in the
Drosophila melanogaster by expressing ‘‘terminator’’ gene
under the control of yp1-tTa.
 hid- head involution defective, proapoptotic gene

 Expression of hid under yp1-tTa

 Tetracycline-regulated female-killing system

Fig.11: Expression of hid under the control of yp1-tTA

Henrich and Scot , 2004

 Tetracycline-regulated female-killing system

Table3: Viability of males and females carrying one copy each of the tetO-hid
and yp1-tTA constructs

Henrich and Scot, 2004

 Dominant Lethal Genetic System for Control of
Mediterranean fruit fly

• RIDL strains, LA656, LA928f1 and LA928m1 through

insertion of piggyBac-based transposans

• Medfly transformation with transposans was done by using

standard microinjection method

 Repressible dominant lethality in transgenic medfly

 Effect of tTA on adult male longevity
 Effect of tTA on adult male mating competitiveness

Gong et al., 2005

 a, b: LA656
c, d: LA928f1
e, f-: LA928m1
C1: no. of wild progenies pupating
C2: no. of transgenic progenies pupating
C3: no. of wild progenies eclosing
C4: no. of transgenic progenies eclosing

Fig. 12: Repressible dominant lethality in transgenic Ceratitis capitata

Gong et al., 2005
Fig.13: Effect of tTA on adult male longevity

Gong et al., 2005

 Five wild adult females --- mating area (5 transgenic
males & 5 wild males)
Mating period- 6hrs
Genotype of mating partner was inferred by analysis of
progeny
• Fluorescent progeny- female mated with transgenic
male
• Non fluorescent progeny- female mated with wild-type
male

Fig.14: Effect of tTA on adult male mating competitiveness

Gong et al., 2005

LATE-ACTING DOMINANT LETHAL GENETIC SYSTEM
AND MOSQUITO CONTROL

 Developed piggyBac-based transposon, LA513A (OX513A)

using tTAV and DsRed2.

 Lethal phase in LA513A using tetracycline-repressible lethal

genetic system

Fig.16 : The structure and function of transposon LA513A

Phuc et al., 2008
Fig.15: Schematic representation of RIDL positive feedback system in mosquito

Gong et al., 2005

Table 5: Tetracycline-repressible lethality in LA513A

Phuc et al., 2008

Female-specific flightless phenotype for mosquito control

 Constructed transgenic Aedes aegypti OX3604C, which is

highly penetrant, dominant, late-acting, female-specific
lethality

 Promoter: Aedes aegypti Actin-4 (AeAct-4)

 OX3604C crossed with wild type and their progenies reared in

the absence of tetracycline

Fu et al., 2010
 OX3604C with tRe- DsRed2
• tTAV under the control of a promoter fragment from AeAct4.
• translation initiation codon (ATG) 5’
• VP16 under control of tRe
• fluorescent marker (DsRed2) under control of the Hr5-IE1 promoter

Fu et al., 2010
A

A : late fourth-instar female larvae.

(Developing IFMs are the bright pair of spots in
each thorax)
B: female and male pupae
C: Male and female adults
B

Fu et al., 2010
Table 6: Flight ability of females

n: Total no. of female adults analyzed

% of females that could not fly Fu et al., 2010
 Field performance of a genetically sterile strain of
pink bollworm

• OX1138B: Genetically sterile strain(DsRed2)

• APHIS: Standard SIT strain
• Sterilized by irradiation(200 Gy)
• Both strains were mixed in equal proportion & released in 3
cotton fields

 Accumulation of sexual active sterile males in the release area

(pheromone traps)
 Persistence and Dispersal of released moths beyond the
release area

Simmons et al., 2010

OX1138B construct

nls: nuclear localization signal

Piggy Bac: transposan
DsRed2: fluorescent protein
Opie2: promoter from baculovirus
(orgyia pseudostugata).

Simmons et al., 2008

 Filled bars = OX1138B
white bars = APHIS

Fig.18: In-field recapture data during release period

Simmons et al., 2008

 Filled bars = OX1138B
white bars = APHIS

Fig.20: persistence and dispersal of moths beyond release areas

Simmons et al., 2008

• Biotech company from Oxford University at Milton Park in
Oxfordshire, UK.
• Set up in 2002 with protected IP for commercializing
environment-friendly techniques to control insect pests.

Pipeline of products designed to address insect pests of Public Health and Agriculture
 OX513A
 OX513A is a female specific Aedes aegypti RIDL strain
 Late acting lethality
 DsRed marker

Current Status
 It has regulatory approvals for import and contained testing in
Brazil, Cayman Islands, France, India, Malaysia, USA and
Vietnam

 Open field trials have taken place in Grand Cayman and

Malaysia, and are currently also underway in Brazil
Fu et al., 2010
 OX3604C
 OX3604C is a female-flightless Aedes aegypti RIDL strain

 They are likely to be rapidly eaten by predators

 Late lethality

Current Status
 Available in a Mexican genetic background
 Consortium member has obtained a permit for import into
Mexico and will evaluate the strain in large scale outdoor
cages in Chiapas in December, 2011.
Fu et al., 2010
 OX1138
 Genetically Engineered Pink bollworm (Pectinophora
gossypiella)
 OX1138 contains a fluorescent marker

Current Status
 The strain has been evaluated in three years of open release
trials by the USDA-APHIS Plant Protection and Quarantine
and CPHST.

 The USDA has conducted an Environmental Impact

Assessment (EIS).

Simmons et al., 2010

 OX3647
 OX3647 is a female-specific Mediterranean fruit fly (Ceratitis
capitata) RIDL strain

 It allows 100% male release by removal of the supplement

from the diet of the final release generation

Current Status
 It has been tested at Oxitec for conditional lethality, marker
performance and mating competitiveness

Fu et al. 2010
 OX3402
 Pink bollworm bisex RIDL strain with fluorescent marker

 Improved homozygous strain

 It offers a replacement to radiation sterilsation

Current Status
 CPHST- to be test under field conditions in Dec, 2011 in
south-western USA.

Simmons et al., 2010

 OX3713A
 OX3713A is a female-specific Olive fly (Bactrocera oleae)
RIDL strain

 In addition to genetic sexing, the strain may also be used

without irradiation for population suppression

Current Status
 OX3713A has now been optimized by Oxitec. It is now
available for evaluation for open field trials

Gong et al. 2008

CONCLUSION

 GSM is less effective for SIT

 Release of sterilized male insects induce several times more

sterility into wild female population

 Use of DsRed2 facilitates the discrimination of transgenic

strains from wild ones
 Yp3, Yp1 and hid can be used as female specific promoters for
RIDL in insects of economic importance

 Construction and use of strains harboring tTA could induce

lethality in early developmental stages of heterozygous
progenies

 LA513A has a highly penetrant, fully repressible late acting

dominant lethal genetic system
FUTURE PERSPECTIVE

 Factors that influence the transposable element behavior in

insects to be measured

 Identification of male specific promoter for development of

bisexual RIDL strains

 Incorporation of genetic sexing system into RIDL programme

could potentially provide a very significant improvement in
cost effectiveness

 Combination of RIDL with other control methods could be

used as an effective component of IVM