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

Shweta PatelId No. 42537

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)

History of Sterilizatio

n 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)

METHOD:

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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• 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.

Model for sterile insect technique (SIT)

Generation

No. virginfemales inarea

No. sterilemalesreleasedper generation

Ratiosteriletofertilemales

%femalesmated tosterilemales

Pop. of fertilefemales

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

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

Some success stories…

GENETICALLY MODIFIED INSECTS

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

• 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).

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.

Why Genetically modified insects

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

useful insects.

Strategies involvingthe release of GM

insects

TRANSGENIC INSECTS

• Insects with transgene integrated into chromosome

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

• 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)

INTRODUCED TRANSGENES IN INSECTINSECTS 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

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

TYPES OF VECTOR

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.

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

PROTOCOL FOR INSECT TRANSFORMATION

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)

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.

STEPS are:

Diagrammatic Representation of Transgenesis & Paratrangenesis

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.

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

ROLE OF GMI IN ENHANCEMENT

OF PUBLIC HEALTH

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)

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)

The GM mosquito could be identified

by their green fluorescent eyes

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)

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

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.

GMI INVOLVED IN CONTROL OF

AGRICULTURAL INSECT PESTS

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

2. Med fruit fly• Males are

sterilized by irradiation prior to release (Lobo,1999)

• Sterile males mate with feral females hindering female reproduction

Medfly eggsexpressing GFP

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

• 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.

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.

Development of

transgenic Red flour

beetle

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.

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)

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

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

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.

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 .

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.

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.

• 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.

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.

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.