Genetic Improvements to the Sterile Insect Technique for Agricultural & Public health Pests

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Genetic Improvements to the Sterile Insect
Technique for Agricultural Public health Pests
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Sterile insect technique

• The sterile insect technique (SIT) is an
  environmentally friendly method for the
  biological control of pests using area-wide
  inundative release of sterile insects to reduce
  reproduction in a field population of the same
  species (IPPC, 2007)

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History of Sterilization

• Irradiation of male insects
• (USDA, 1950s)
• Background
• X-rays caused sterility in male insects (1916)
• Dr. Edward Knipling (1954) in screw-worm fly
  (Cochliomyia hominivorax) - subtropical America
  livestock in Florida
• Melon fly (Bactrocera cucurbitae) from Okinawa in
  Japan(1972-1993) Koyama et al. 2004
• Tse-tse fly( Glossina austeni)from Unguja Island
  in Zanzibar,Tanzania(Vreysen et al.1996)

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METHOD
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Requirements for SIT

• Insects can be reared and sterilized in large
  quantities.
• Methods exist for distributing the sterile
  insects throughout the target area so they
  thoroughly mix with the wild population.
• The release is timed to coincide with the
  reproductive period of the target insect.
• The released, sterile insects compete
  successfully for mates in the natural
  environment.

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Continue.

• The release ratio (sterile insects to native,
  fertile insects) is large enough to overcome the
  natural rate of increase of the population, so
  that the trend in population size is downward
  after the first release.
• The target population is closed i.e., there is
  no immigration of fertile insects from outside
  the release zone.

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Model for sterile insect technique (SIT)
Generation No. virgin females in area No. sterile males released per generation Ratio sterile to fertile males females mated to sterile males Pop. of fertile females
F1 10,00000 20,00000 21 66.7 3,33,333
F2 3,33,333 20,00000 61 85.7 47,619
F3 47,619 20,00000 421 97.7 1,107
F4 1,107 20,00000 18071 99.9 Less than 1
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How SIT works

• When more sterile males are available than
  fertile males, the likelihood of mating with a
  sterile insect is high, suppressing the
  reproductive output of the fertile population.
• In generation 1, 2or3 of the males are sterile,
  so 2or3 of the matings should result in
  reproductive failure.

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• As the population of fertile males decreases,
  the ratio of sterile to fertile increases,
  depressing the population even faster.
• Once attaining a low level of fertile insects,
  it is easy to maintain the population at low
  levels with continued releases. In some cases,
  the pests are eliminated(eradicated), so no
  further releases are made

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Some success stories
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GENETICALLY MODIFIED INSECTS
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Definition-

• A genetically modified organism (GMO) is an
  organism whose genetic material has been
  altered using techniques in genetics generally
  known as recombinant DNA technology.
• Genetically modified insects are -
• Insects With newly expressed characteristics
• New characters as a result of manipulation of
  DNA in laboratory
• Changes - passed on to next generation

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• Achieved by using gamma irradiation, UV rays and
  mutagens like Ethyl methyl sulphonate
• Till now 18 different genera have been
  manipulated .
• First genetically transformed insect - reported
  when wild type eye colour gene was seen in a
  mutant strain of Drosophila.
• Next transformation was attempted in
  mediterranean fruit fly in 1995 (Loukeris).

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History of genetically modified insect

• Produced as a result of gene manipulation, a
  technique for genetic control of insects.
• In 1937,E.F.knipling-concept of genetic control
  of insect pest.
• Stated with sterilization of Screw worm flies, a
  serious pest of livestock.

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Why Genetically modified insects

• Benefit public health
• Enhance agricultural production
• Provide new forms of economically useful
  insects.

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Strategies involvingthe release of GM insects
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TRANSGENIC INSECTS

• Insects with transgene integrated into chromosome
• Transposable elements act as vectors thereby
  carrying transgenes into chromosome
  (Finnegan,1989)

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• Fusion of chromosome and transgene is promoted by
  transposable elements that cut and repair
  chromosomes
• Transgenes used for recognition of transgenic
  insects are called markers
• Promoters are used to drive the expression of
  markers (Coates,1999)

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INTRODUCED TRANSGENES IN INSECT
INSECTS GENES CHARACTER MODIFIED
Anopheles SM 1 Disease causing ability destroyed
2. Culex Defensin Disease spreading ability is lost
3. Silkworm Spider flagelliform silk Enhances quality of silk protein
4. Wolbachia Attacin and Cecopin Infective capacity is lost
5. Xylella S 1 Disease causing capacity is absent
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Requirements for gene manipulation

• 1.Gene of interest or exogenous DNA
• 2. Vector
• 3. Marker gene
• 4. Promoter

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TYPES OF VECTOR
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Transposable elements

• Transposable elements-Mobile pieces of DNA that
  do not remain fixed at one genomic location but
  move from one site on a chromosome to
  another(Liao,2000)
• Increase their copy number as they move around
  among chromosomes within individual organism.

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Use of viral vectors

• Viral systems offer promising techniques for
  expression of foreign genes (Hahn,1992)
• Viral transducing systems allow long term and
  stable cytoplasmic expression of foreign DNA
• Viruses engineered with antisense RNA are found
  complimentary to yellow fever viral sequences

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PROTOCOL FOR INSECT TRANSFORMATION
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Sperm mediated transformation

• Factors like low reproductive rates and egg
  properties prevent DNA introduction
• So, virgin queens are inseminated with a mixture
  of linearized DNA and semen (Robinson,2000)

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PARATRANSGENIC INSECTS

• Paratransgenesis was first conceived by Frank
  Richards (1996)
• Paratransgenesis is a technique that attempts to
  eliminate a pathogen from vector populations
  through transgenesis of a symbiont of the vector.
  The goal of this technique is to control
  vector-borne diseases.

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STEPS are
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Diagrammatic Representation of Transgenesis
Paratrangenesis
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Chagas disease

• Is caued by parasite Trypanosoma cruzi spread by
  kissing bug (Rhodnius prolixus ) which is
  associated with the symbiont Rhodococcus
  rhodnii.. The strategy was to engineer R. rhodnii
  to express proteins such as Cecropin A that are
  toxic to T. cruzi or that block the transmission
  of T. cruzi.

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Requirements for Paratransgenesis

• The Symbiotic bacteria can be grown in vitro
  easily
• They can be genetically modified, such as through
  transformation with a plasmid containing the
  desired gene
• The engineered symbiont is stable and safe
• The association between vector and symbiont
  cannot be attenuated
• Field delivery is easily handled

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ROLE OF GMI IN ENHANCEMENT OF PUBLIC HEALTH
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1.Genetically modified malaria causing
mosquitoes

• Mosquitoes spread malaria and kill 2.7 million
  people per year world wide (Rasgon,2007)
• Mosquitoes are engineered to produce protein that
  disrupt malarial parasite life cycle within
  insect .
• Gene (SM 1) prevents malarial parasite from
  penetrating into mosquito mid gut and reaching
  salivary glands (Braig and Yan, 2002)

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• Green fluorescent protein (GFP) inserted into
  transgenic mosquitoes make their eyes glow green
  under UV light
• Transgenic mosquitoes - With high survival rate
  and lay more eggs Anopheles stephensi is one of
  the genetically engineered common mosquito
  species to resist malaria (Catteruccia, 2003)

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The GM mosquito could be identified by their
green fluorescent eyes
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2.Genetically modified Yellow fever causing
mosquitoes

• Mosquito like Aedes aegypti spread yellow fever
• Ken Olson, a virologist created GM mosquito to
  replace these breeds.
• Produce antibacterial protein, limiting its
  ability to transmit disease (Adelman, 2002)

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3. Sleeping Sickness

• This disease is also referred to as African
  Sleeping sickness(Askoy,2003)
• It affect more than fifty thousand people per
  year
• It is caused by Tsetse fly and kissing bug
• Controlled by paratransgenesis

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4.Genetically modified Dengue Fever causing
mosquitoes

• Dengue Fever is caused by viruses transmitted by
  mosquitoes Aedes aegypti
• It infects 50-100 million people annually with
  2.5 billion worldwide at risk
• 6,000 of such GM mosquitoes have already been
  released in the Malaysian forests in January of
  this year.
• Oxitec scientists has led to such GM mosquitoes
  also released in the wild in the forests of the
  Cayman Islands.

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GMI INVOLVED IN CONTROL OF AGRICULTURAL INSECT
PESTS
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1.Pink boll worm

• Sterile insect technique programme (SIT)
• Protects more than 900,000 acres of cotton
• Million of male pink boll worm moth were
  sterilized by irradiation(Pelloquin,1999)
• Moths are engineered to contain gene from jelly
  fish(GFP)
• A lethal gene (t Ta) is introduced from
  bacteria(Briggs,2001)
• It alters the metabolism of the moth larvae

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2. Med fruit fly

• Males are sterilized by irradiation prior to
  release (Lobo,1999)
• Sterile males mate with feral females hindering
  female reproduction

Medfly eggs expressing GFP
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3. Pierces disease

• It is the lethal infection of grape vines xylem
  by bacteria Xyllela Species(Bextine,2004)
• This bacteria is carried by the vector Glass
  Winged Sharp Shooter
• There is no control measure for this disease
• Controlled by paratransgenesis

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• Anti Xyllela effector proteins (S 1)were isolated
  and modified to carry anti bacterial toxins
  against Xyllela(Miller,2007)
• Others insects like Codling Moth, Cabbage looper,
  Onion fly and parasitoids like Trybliographa
  species are controlled under SIRM programme.

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4. Transgenic Red flour beetle

• It is a worldwide pest of stored products
• Genes responsible for regulating pheromone
  secretion are mutated (Dabron, 2002)
• Specific gene expression is knocked out by RNA
  interference.

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• Development of transgenic Red flour beetle

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RELEASED COMMERCIALLY

• Predatory mites-In 1997 in US.
• Pink bollworm-in 2001 in Mexico.
• Anapheles mosquito-In 2002 in New Delhi and UP.
• Screw worm fly-Exported from Libya to Kenya and
  Central America.

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Hybrid Sterility

• Males Females of different strains can produce
  non-viable offspring
• Incompatible strains can be generated through
  several ways
• Direct genetic manipulation
• Microbially-mediated (Cytoplasmic
  Incompatibility)
• This phenomenon has been clearly demonstrated in
  crosses between Heliothis virescens males and
  Heliothis subflexa females (Laster et al. 1996)

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Wolbachia and Reproduction

• Vertical transmission cytoplasmic inheritance
  Causes male killing and sterility
  in males
• Induces parthenogenesis
• Cytoplasmic incompatability (conflict between
  cytoplasmic and nuclear components)

Insect egg containing Wolbachia
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Cytoplasmic Incompatability and vertical
transmission

• If both male and female insects are infected
  with Wolbachia the progeny will be infected
• If the female is infected and the male is not
  infected, the progeny will all be infected.
• If the female is not infected and the male is
  infected there will not be any progeny

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RIDL

• RIDL (release of insects carrying a dominant
  lethal)insects contain a genetic modification
  that causes their offspring to die, but the RIDL
  insects can live and reproduce normally when they
  are fed a diet containing a supplement.
• RIDL males are released to mate with wild female
  pest insects their progeny inherit the RIDL gene
  and do not survive to adulthood.

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Inherited sterility in insects

• The inherited sterility in insects is induced by
  substerilizing doses of ionizing radiation. When
  partially sterile males mate with wild females,
  the radiation-induced deleterious effects are
  inherited by the F1 generation. As a result, egg
  hatch is reduced and the resulting offspring are
  both highly sterile and predominately male.

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• The silk worm Bombyx mori was the first insect
  in which inherited sterility was reported.
• Then inherited sterility was reported in the
  greater wax moth Galleria mellonella , codling
  moth Cydia pomonella .

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LIMITATIONS

• Instability of the introduced genes
• Transgenes were reported to get rapidly lost
  under field conditions.
• Experimental release of transgenic predatory
  mites showed that very few individual contained
  the transgene only after three generations while
  in laboratory strains, it was persistent for over
  one fifty generations.

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What are the limitations of SIT?

• Geography. The eradication zone must have either
  natural barriers to prevent the immigration of
  the target pest from outside.
• Economics. Cost of rearing, sterilizing, and
  releasing a large numbers of insects can be very
  high.
• Desirability of sterile males. The lab-reared and
  sterilized males must be equally or more
  competitive than the native males in mating with
  the native females. They may become less
  desirable after many generations and need renewal.

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• Knowledge about the pest. reproductive behavior,
  population dynamics, dispersal, and ecology of
  the insect.
• Accurate estimation of the native population
  density
• Timing. The development of the lab-reared colony
  must be synchronous with that of the wild
  population.
• Resistance. Native females may be able to
  recognize and refuse to mate with sterile males.

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FUTURE PROSPECTS

• Transgenic insect approach will help to control
  harmful insects and create beneficial insects.
• Creation of transgenic insects with increase
  fitness.
• Biosafety research on transgenic insect has to
  gain important in international symposia.
• Risk assessment guidelines require more
  clarification.

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Conclusion

• SIT has been, and continues to be, a hotbed of
  genetic innovation. transgenic technology offers
  a much wider spectrum of advances in genetic
  tools for SIT, from heritable marking to
  alternative methods for sterilisation. it is,
  increase the range of pest species that can be
  targeted by this environmentally friendly,
  species-specific method of control.

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