Dengue, Mosquitoes & GenesAn information pack toaccompany the Oxitec film

CONTENTS

Introduction

Dengue Fever a growing problem

Who is at risk?

Why are they at risk?

How can we treat it?

Introducing Haedes and Aegypta: all about the Aedes aegypti mosquito

Dining out with Aegypta: how and why do mosquitoes suck blood?

How does the Aegypti mosquito transmit Dengue fever?

Getting on top of the problem: how we can make life uncomfortable for Aegypta and friends

Using genes to control insects: the Oxitec solution

What exactly is a gene, and what is meant by genetics?

Applying genetic modification to insect control: The Oxitec solution

Using sterile insects for population control

The Oxitec approach: genetic engineering of sterile insects

How can we breed genetically sterile mosquitoes?

Why genetic modification works

More on the science: How does Oxitec make genetically modified insects?

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IntroductionWe hope you enjoy this short film about Dengue Fever and the Aedes aegypti mosquito.

At Oxitec, were concerned about Dengue Fever because

of the devastating impact it has on so many peoples

lives, and because of the huge costs which Governments

and communities must meet in order to try to control this

dangerous disease. Thats why were using advanced

science, and a process called genetic modification, to

develop a new solution to the Dengue threat by

targeting the mosquitoes which spread it.

As you watch the video, we hope youll learn more about

the Dengue Virus, about the mosquitoes which spread

the disease, and about how Oxitecs cutting-edge

solution is providing a new approach to controlling them.

Weve designed this information pack to be a

supplement to the video, and provide a helpful resource

for anyone wanting to learn more about Dengue Fever,

mosquitoes, and the Oxitec approach.

Dengue Fever -a growing problem

Dengue Fever is a dangerous and debilitating disease,

and its a growing threat to global health. Like Malaria,

Dengue is spread by a bite from an infected mosquito

(we call diseases of this type mosquito-borne).

Although it is not usually fatal, Dengue Fever is an

extremely serious disease. Dengue symptoms range from

mild and flu-like to high fever, rash, severe headache,

pain behind the eyes, muscle and joint pain. The joint

pain can be so severe that Dengue has been given the

name breakbone fever. Nausea, vomiting, and loss of

appetite are also common. These symptoms can last for

weeks!

In the Film, youll hear from Hannah Strange and

Professor Paul Reiter about the ordeal that they suffered

when they contracted Dengue Fever. The experiences

they describe are not uncommon.

Unfortunately, for some patients, Dengue Fever can be

even more dangerous. In the more severe form, known as

Dengue Hemorrhagic Fever (DHF), blood vessels start to

leak and the blood fails to clot, causing bleeding from

the nose, mouth, and gums. Without prompt treatment,

the blood vessels can collapse, causing a critical

condition called Dengue Shock Syndrome. Ultimately,

this can lead to fatality: about 25,000 people die from

Dengue Fever every year.

Hannah StrangeDengue Fever Sufferer

As well as pain and suffering for those people unfortunate enough to catch Dengue, the disease is also a serious financial

burden for the Governments and communities which are struggling to cope with it. Governments spend a lot of money on

efforts to control the spread of the Dengue mosquito, but these efforts are not very effective, as well hear about later. At

the same time, the cost of looking after and treating people affected by Dengue can be huge both for Governments and

the individuals concerned. Here are a few facts about the costs of dealing with Dengue:

2.5bn people are now estimated to be at risk from

Dengue

Between 50-100 million people are infected each

year, with around 25,000 deaths

In the Americas, the average economic cost of

Dengue fever is estimated at $2.1bn

In the Americas and Asia, a study found that the

average cost of each hospitalised case was $1,394

The economic costs of Dengue can be of the same

order of magnitude as those associated with

Tubercolosis, sexually transmitted diseases

(excluding HIV/AIDS), Chagas Disease and

Leishmiasis.

$1,3942,500,000,000

TB+25,000

$2,100,000,000

After Malaria, Dengue Fever is the second most

widespread mosquito-borne disease in the world. The

World Health Organisations has estimated that between

50 and 100 million people suffer from Dengue Fever

each year: thats more than the population of the UK

every year!

Dengue is also the fastest-growing mosquito-borne

disease. Since the 1970s, the number of countries

experiencing Dengue outbreaks has grown from 9 to

more than 100.

Today, up to 40% of the worlds population, or 2.5 billion

people, is thought to be at risk[1]. Dengue fever occurs in

most tropical areas of the world. It is common in Asia,

the Pacific, Australia, Latin America and the Caribbean

and is continuing to spread. It has now reached North

America. A recent Natural Defence Resource Council

report shows that 28 US states are now at risk[2].

[1] World Health Organisation Dengue Factsheet: http://www.who.int/mediacentre/factsheets/fs117/en/index.html

[2] Natural Resources Defence Council (2009) Fever Pitch: Mosquito-borne Dengue Fever Threat Spreading in the Americas http://www.nrdc.org/health/dengue/files/dengue.pdf

The rapid spread of Dengue Fever is due to the global

migration of the mosquitoes which spread the disease.

The main culprit is Aedes aegypti, and youll hear more

from these in the video! The mosquitoes eggs are

extremely hardy: they can survive for months without

water, allowing them to be transported all over the

world, for example in used tyres and plant containers,

before hatching and infesting a new area.

In the video, youll hear from the scientist Professor Paul

Reiter who explains how the Aedes aegypti mosquito has

spread so rapidly around the world. The life-cycle of a

mosquito is about 3 weeks from hatching, to adult, to

reproduction. If each female can lay up to 500 eggs, as

Professor Reiter explains, it is easy to see how quickly a

new area can become infested!

Who is at risk? Why are they at risk?

How can we treat it?

There is currently no treatment for Dengue fever. One of

the difficulties in developing an effective drug or vaccine

to combat the virus is that Dengue Fever is actually

caused by four different, but closely related, types of

virus.

Scientists call these related viruses serotypes: the four

Dengue serotypes are named DENV1, DENV2, DENV3

and DENV4. When a person is infected by one of these

serotypes, they develop life-long immunity to that type,

but not to the other three. In fact, a person who has been

exposed to one type may be at risk of developing a more

serious illness if they are infected by another type later

in life. Scientists dont completely understand the reason

for this, but it is likely to be a result of the way our

immune systems respond to the different viruses.

Because of the risk of these complications, a new vaccine

has to be effective against all four types of Dengue virus.

A vaccine of this type is in development, but it could be

many years before it can enter production. Even then, a

vaccine of this type could still risk actually increasing

peoples sensitivity to one or more Dengue types. Its

important to remember as well that there are an

estimated 2.5 billion people currently thought to be at

risk from Dengue. Vaccines are a safe and effective way

to control disease, but they are expensive. With so many

people at risk from Dengue, some countries may struggle

to fund a vaccination programme. If a vaccine

programme was implemented, it could be combined

with mosquito control strategies to provide even better

protection against Dengue.

With no drug, and a potential vaccine a long way off, our only way of controlling Dengue Fever is to target the mosquitoes which carry it.

Bednets are often used in hospitals within tropical regions, but they are of limited use in preventing Dengue transmission because the Aedes aegypti mosquito bites during the day

Introducing Haedes & Aegypta: all about the Aedes aegypti mosquitoSo youve met Haedes and Aegypta the animated stars

of our film! As you may have noticed, Haedes and

Aegypta get their names from Aedes aegypti, which is

the Latin name for the mosquito which carries Dengue

Fever.

Aedes aegypti is a prolific pest. Originating in Africa, it

has spread around the world, hitching rides in shipping

containers, used tyres, and other transported goods

which can provide ideal vehicles for the mosquitoes

eggs. As Aegypta proudly points out, Aedes aegypti are

highly adaptive: once transported to a new habitat, they

breed quickly and rapidly become established in an area.

As the expert earlier in the video tells us, female Dengue

mosquitoes can produce up to 500 eggs, so Aegyptas

300 children arent unusual!

If you have a quick think about how

often you might find some of these

containers lying around in your

own home, you can start to see

how difficult it might be to try to

get rid of Aedes aegypti once it has

become established in an area.

They included:

The Dengue mosquito is primarily an urban pest. That means that it prefers to live in and around human habitation.

It may seem a bit far-fetched to find Haedes and Aegypta setting up home in a fruit bowl, but in fact, thats exactly the

kind of environment which Aedes aegypti mosquitoes prefer. For example, scientists studying the preferred breeding sites

of Aedes aegypti mosquitoes in Singapore City listed a number of common household containers which were frequently

found to contain Aedes aegytpti larvae, or young.

If youve ever been bitten by a mosquito, youll probably

be aware that they feed on blood! What you may not

have been aware of is that it is only female mosquitoes

which actually do this males dont bite. In the video,

youll hear from Haedes and Aegypta about their

different dietary habits: as Hades explains, hes a

vegetarian: what he means by this is that, like all male

mosquitoes, he gets his meals from feeding on nectar,

fruit and other sugar sources.

Female mosquitoes like Aegypta also feed on nectar and

other sugar sources as their primary source of energy.

However, females need to feed on blood in order for

them to produce eggs.

What makes Aegypta and the rest of her species

especially troublesome to man, and makes her such a

good carrier of dengue and other diseases, is that she

prefers human blood. Scientists say she is

anthroprophilic, which means human loving.

The images below show how the female mosquito has

become adapted to take blood meals from people and

animals. In these highly magnified images, it is easy to

see the females long feeding mouth parts, or proboscis.

In comparison, the males proboscis is much smaller, and

doesnt have some of the specialised mouthparts found

in females. When a female mosquito bites a human or

animal, she will insert this proboscis into a blood vessel,

using a back-and-forth motion of her head to create a

sawing motion that drives the piercing parts of the

proboscis into the skin. Special chemicals in her saliva

prevent the blood from clotting, so she can easily suck

the blood into her stomach. It is the bodys reaction to

this saliva which causes the area around the mosquito

bite to swell up and itch as most of us will have

experienced! When a female feeds, her stomach can

expand to many times its original volume, allowing her

to consume more than her body weight in blood (she can

struggle to fly afterwards!).

Dining out with Aegypta: how and why do mosquitoes suck blood?

Most of us would agree that mosquitoes blood feeding habits can be extremely annoying! But in the case of Aedes

aegypti, its their ability to transmit Dengue Fever that changes them from a mere annoyance into a potentially deadly

threat.

In the video, youll have heard the reporter asking Aegypta and Haedes about Dengue Fever. As Aegypta explains, the

Dengue virus is picked up by the mosquito when they blood feed on an infected person. After infecting the mosquito, the

virus takes about 5-7 days to replicate itself and pass through the mosquitos body eventually reaching its salivary

glands. Once there, it can enter the mosquitos saliva, so when the mosquito bites another person, the virus can pass into

their bloodstream.

Once the mosquito has become

infected with Dengue, it remains

that way for the rest of its life. So

every time she bites a human, she

can pass on the deadly virus.

How does the Aedes aegypti mosquito transmit Dengue fever?

Female mosquitoes mouth parts using a scanning electron microscope

Diagram showing the life-cycle of Dengue Virus

A = Labium tip (the labellum); this

guides the mouthparts into the

skin.

B = Labrum; this is the tube through

which the blood is sucked up.

C = Maxilla; these sharp serrated

edges aid penetration of the skin.

D = Hypopharynx; saliva is

delivered through this tube.

Because of the threat posed by the Dengue mosquito,

people have been trying to reduce or eradicate

populations of the pest for many years. Unfortunately,

despite considerable time and money expended by

governments and communities, these efforts have until

now not been very successful.

In the video, Haedes explains part of the problem. Most

of the control methods which have been used before

now have relied on adding chemicals to potential

breeding sites to kill mosquito larvae, or involve spraying

or fogging with chemical pesticides which are designed

to kill off the adult mosquitoes. The image below shows

fogging in progress.

Because Aedes aegypti live inside peoples homes

remember all those empty bottles, tin cans, vases and

bowls they breed in chemical fogging like this actually

requires the pesticide to penetrate every room of a

house. You can probably imagine just from looking at the

picture below that this is not a very pleasant process, so

people are often understandably reluctant to open their

doors and windows to let the chemicals into their house.

As Haedes suggests, they may not always realise just how

close the mosquitoes are living.

As well as being unpleasant for people, chemical

pesticides can also be damaging to the environment and

disrupt natural habitats. One of the biggest problems

with these chemicals is that they dont just harm

mosquitoes: other insects will also be killed (because of

this, we sometimes say that pesticides are

indiscriminate).

As a result, pesticide use can damage the ecology of an

area, reducing populations of insects which may be

important food sources for birds and fish, or which are

pollinators for local plants and flowers.

Haedes also mentions another problem with this

approach: resistance. Pesticides rarely, if ever, kill all the

mosquitoes in an area there will be some which are

naturally resistant to the chemicals used. Because these

mosquitoes survive they will be able to pass on their

resistance genes to their children, so in the next

generation more mosquitoes will be resistant. Over time,

the proportion of resistant mosquitoes will increase in

this way, until eventually all or most insects in a

population may be resistant. As Haedes explains, many

Aedes aegypti populations are now resistant to the

majority of commonly-used chemical pesticides.

Getting on top of the problem: how we can makelife uncomfortable for Aegypta and friends

Because of all these problems, a new approach to controlling Aedes aegypti is needed.

Using genes to control insects: the Oxitec solutionWeve heard how traditional methods to control Aedes aegypti can be harmful to people and the environment, and arent

very effective in any case. In the film, the reporter then asks Haedes and Aegypta about the Oxitec approach. This is a

new technology based on advanced genetic science, which uses the natural instincts of the mosquitoes themselves to

track down other mosquitoes and stop them reproducing.

Before taking a look at the Oxitec solution in more detail, you can read a bit more about genes and genetic modification;

an incredibly powerful science which has already been central to recent advances in medicine, agriculture and

understanding diseases.

Most people have heard of genes even if its not always quite clear what

they are, and what they do! A gene is the name given to a section of DNA.

In both people and mosquitoes, genes produce proteins that influence

everything from height to eye color. Offspring inherit these genes from

their parents

Genetics is the name given to the science of studying and manipulating

genes, and understanding how they are passed on from parent to offspring.

Over the last 50 years, since the discovery of the structure of DNA, genetics has

contributed to many important scientific developments, such as understanding

and treating inherited diseases like cystic fibrosis; developing more effective

targeted cancer medicines, like Herceptin; or enabling us to manufacture

large amounts of human insulin to treat diabetics.

Using modern genetics, scientists now have the ability to manipulate and

combine genes in many different ways. This allows them to study how genes

work, as well as combining different genes in ways that can be enormously

useful in medicine, agriculture and other applications.

What exactly is a gene, andwhat is meant by genetics?

Genetic modification (GM), also known as genetic

engineering, is a biotechnology technique which can be

used to add to or alter the genes within an organism. A

genetically modified organism, or GMO, is an organism

that has had its existing genes altered, or new genes

added, through a process of genetic modification.

Genetically modified organisms were first developed in

the late 1970s and since then their use in a range of

industries has become widespread. Perhaps without

appreciating it many people today are reliant on this

biotechnology for medicines and foods.

One of the first products of genetic modification was

insulin produced by genetically modified bacteria.

Through insertion of the human gene for insulin into the

bacterias DNA, the bacteria effectively acts as

As weve seen earlier in the video, the challenge we face in trying to control Aedes aegypti is to find ways of targeting the

mosquitoes where they live; in peoples houses and gardens. Whats more, we need to have ways of doing this which dont

harm people or other animals and plants in the environment, and which avoid the use of chemicals which mosquitoes are

often resistant to. One potential approach which meets some of these goals is known as the Sterile Insect Technique or

SIT.

a chemical factory, synthesizing the human protein

exactly. This has provided a quick and easy way to

produce pure insulin for diabetics.

Since the 1980s, there have been a number of other

human proteins produced through genetic engineering,

such as growth hormone or blood clotting factors.

Genetic modification has also been used in a wide

variety of other medical applications: the Hepatitis B and

HPV vaccines have been developed using genetic

modification, and there is potential for scientists to

develop an entirely new field of cancer vaccines based

on this biotechnology.

Oxitec has applied the science of genetic modification to

the problem of controlling populations of Aedes aegypti.

Applying genetic modification to insect control: The Oxitec solution

Injecting DNA: Genetic modification allows scientists to add to or alter

genes within an organism

SIT uses the natural instincts of the released male

mosquitoes to seek out females, so it is much more

effective than traditional means at targeting

difficult-to-reach pest populations, like Aedes aegypti. It

is also species-specific: it affects only the target pest, and

doesnt harm other insects.

Unfortunately, using radiation to produce sterile insects

in this way can cause problems. Not surprisingly, being

hit by a large amount of radiation isnt very good for the

male insects! Often, irradiated males are very sickly, so

wild females prefer not to mate with them. If that

happens, they wont be very effective at controlling the

population. While SIT has been used successfully against

some insect pests, mosquitoes are easily damaged by the

process of irradiation, and to date there have not been

any successful programmes of mosquito population

control using radiation-based SIT.

The concept of SIT was first developed in the 1950s. The

basic technique is to dose male insects with radiation,

which makes them sterile. By sterile, we mean that

although the males do produce sperm and can fertilise

the females eggs, their offspring are inviable meaning

that they die at a very early stage of development.

The sterile males are then released into the

environment, where they mate with wild females.

Females usually only mate once, so a female which

mates with a sterile male doesnt produce any offspring.

As a result, the population as a whole is reduced.

Eventually, with enough sterile releases, the population

of the target insect in an area can be dramatically

reduced or even eliminated.

The Sterile Insect Technique was successfully used to

eradicate screw-worm (a pest of cattle) in North

America. It has also been successful in reducing

populations of other pests, such as eradicating the Tsetse

fly, which causes sleeping sickness, in Zanzibar.

Using sterile insects for population control

Oxitec has developed a new way to control mosquitoes

using genetic modification. Our approach is similar to

the sterile insect technique, but because we use genetics

to stop our insects from reproducing, we eliminate the

need for damaging irradiation.

Scientists at Oxitec have developed a way to modify

mosquitoes by adding a gene which produces a protein

that stops their cells from functioning normally.

The gene produces a protein called tTA, which is a

special kind of protein able to act as a switch that

controls the activity of other genes.

Our modified mosquitoes produce high levels of this

protein because it actually activates its own gene,

producing lots more of itself. Although its not toxic itself,

it ties up some of the cells essential machinery. It can

interact with other proteins which are needed for

controlling genes in the cell, and in this way it stops the

cell from turning on other genes which are essential for

it to survive.

All this means that the modified mosquitoes become

very sick, and die before reaching adulthood.

The Oxitec approach: genetic engineering of sterile insects

If the gene in the modified mosquitoes kills them, how does that make them

sterile? That depends on another special property of the gene, and the tTA

protein it makes: when the mosquitoes are reared in the presence of

tetracycline, it stops the tTA from working - in effect, it acts like an antidote.

So when we feed the modified mosquitoes with this supplement in the lab,

they stay perfectly healthy. But when the male mosquitoes mate with females

in the wild, their children inherit the lethal gene. Tetracycline is not present in

the environment in sufficient quantities to allow survival, so without the

antidote in their diet, the children of the modified mosquitoes die.

Because of this, the Oxitec genetically modified mosquitoes are effectively

sterile. When radiation is used to sterilise insects, as we saw earlier, their

offspring die at a very early stage of development before hatching. With the

Oxitec technique, the insects offspring die later in life, but the effect is the

same: when a genetically modified male mates with a wild female, her

children will die before reaching adulthood, so the population is reduced.

How can we breed genetically sterile mosquitoes?

So why use genetics? As we saw earlier, a major problem

with using irradiation is that the released males are

often sick, so females may not choose to mate with them.

In the video, Aegypta at first seems convinced that shell

be able to recognise Oxitecs males and avoid them. With

irradiated males, she might - but thats the clever part of

using genetic modification; because Oxitecs insects

dont have to be irradiated, they are fit and healthy. Like

all male mosquitoes, they will naturally seek out females

and mate with them. This means that Oxitecs approach

will be much more effective than other treatments, like

pesticides, at targeting mosquitoes in difficult-to-reach

places peoples homes and gardens. And unfortunately

for Aegypta and her friends, they wont be able to tell the

difference until its too late

Why genetic modification works

No wonder Aegypta looks worried!

More on the science: How does Oxitec make genetically modified insects?

To make a genetically modified mosquito, Oxitecs

scientists have to find a way of incorporating the new

gene into the mosquitos own DNA, from where it will be

copied into every cell of the mosquitos body.

The process begins with mosquito eggs. These are tiny,

cigar-shaped objects about 1mm long. Using special

glass needles, so sharp that the point can only be seen

clearly under a high-powered microscope, Oxitecs

scientists can inject very small amounts of DNA into the

end of a mosquito egg. The amount of DNA injected into

each egg is miniscule thousands of times smaller than

a typical raindrop!

Many of the eggs injected in this way wont survive. In

others, the DNA which is injected wont be incorporated

into the mosquitos cells. But in a very few eggs, the new

DNA will be taken up by the mosquitos cells and will be

cut and pasted into the mosquitos own genome. If this

happens in the sperm cells of a male mosquito, or the

egg-producing cells of a female, the new DNA can be

passed on to their offspring.

After being injected, the eggs are hatched, and the

resulting mosquitoes carefully looked after until they

reach adulthood. Then they are bred with other

mosquitoes, and if the injected DNA has entered sperm

or egg cells, then it will be passed on to their offspring.

The DNA which was injected contains the lethal gene,

but it also contains a fluorescent gene which allows the

genetically modified mosquitoes to be identified using a

special microscope. So Oxitecs scientists can look at the

offspring of the mosquitoes which were injected to

identify those which contain the new DNA.

The scientists may have to inject thousands of mosquito

eggs to obtain just one individual which has the new

DNA incorporated into their genome. But from this single

insect, a new strain of genetically modified mosquitoes

can be made.