2. GeneticGenetic
Improvements toImprovements to
the Sterile Insectthe Sterile Insect
Technique forTechnique for
Agricultural &Agricultural &
Public health PestsPublic health Pests Shweta
Patel
Id No.
42537

3. 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)

4. 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)

5. METHOD:

6. 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.

7. 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.

8. Model for sterile insect
technique (SIT)
Generati
on
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 2:1 66.7 3,33,333
F2 3,33,333 20,00000 6:1 85.7 47,619
F3 47,619 20,00000 42:1 97.7 1,107
F4 1,107 20,00000 1807:1 99.9 Less than 1

9. 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.

10. Continue…
• 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

11. Some success stories…

12. GENETICALLY
MODIFIED INSECTS

13. 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

14. • 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).

15. 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.

16. Why Genetically modified
insects
• Benefit public health
• Enhance agricultural production
• Provide new forms of economically
useful insects.

17. Strategies involving
the release of GM
insects

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

19. • 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)

20. INTRODUCED TRANSGENES IN INSECT
INSECTS GENES CHARACTER
MODIFIED
1. 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

21. Requirements for gene
manipulation
1.Gene of interest or exogenous DNA
2. Vector
3. Marker gene
4. Promoter

22. TYPES OF VECTOR

23. 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.

24. 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

25. PROTOCOL FOR INSECT
TRANSFORMATION

26. 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)

27. 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.

28. STEPS are:

29. Diagrammatic Representation of
Transgenesis & Paratrangenesis

30. 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.

31. 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

32. ROLE OF GMI
IN
ENHANCEMENT
OF PUBLIC
HEALTH

33. 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)

34. 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)

35. The GM mosquito could
be identified by their
green fluorescent eyes

36. 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)

37. 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

38. 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.

39. GMI INVOLVED IN
CONTROL OF
AGRICULTURAL INSECT
PESTS

40. 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

41. 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

42. 3. Pierce’s 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

43. • 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.

44. 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.

45. Developmen
t of
transgenic
Red flour
beetle

46. 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.

47. 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)

48. 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

49. 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

50. 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.

51. 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.

52. Continue…
• 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 .

53. 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.

54. 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.

55. • 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.

56. 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.

57. 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.