FIELD EVALUATION ON THE IMPACT OF WOLBACHIA INFECTED

AEDES AEGYPTI AND AEDES ALBOPICTUS ON DENGUE &

CHIKUNGUNYA TRANSMISSION

PREPARED BY:
MEDICAL ENTOMOLOGY UNIT, INSTITUTE FOR MEDICAL RESEARCH (IMR)
VERIFIED BY:
DR LEE HAN LIM, UNIT ENTOMOLOGI PERUBATAN, INSTITUT PENYELIDIKAN PERUBATAN
(IMR)

1.0 Dengue the challenges

Dengue is a disease transmitted by mosquitoes, which causes sudden high fever, severe
headache, pain behind the eyes, muscle and joint pain, rashes and bleeding in some cases.
Dengue virus (DENV) is transmitted through the bite of an infected female Aedes mosquito.
There are two main Aedes species that can transmit DENV: Aedes aegypti and Aedes albopictus.
Dengue is found especially in tropical and subtropical regions of the world. Dengue fever is
caused by four closely related virus types, which are called serotypes. The four DENV serotypes
are DENV-1, DENV-2, DENV-3 and DENV-4 (WHO, 2014).
In Malaysia, recent statistics obtained from Crisis Preparedness and Response Centre (CPRC),
Ministry of Health (MOH) showed that 83,594 of dengue cases were recorded in Malaysia from
3 January 2016 to 4 October 2016 and among these cases, 186 deaths were recorded (MOH,
2016).

Figure 1. Worldwide distribution of dengue (WHO, 2013)

Ae. aegypti is the main vector for dengue (Rudnick, 1986). Ae. aegypti is a small mosquito that
has white banded legs a white lyre-shaped marking on its head. It usually bites indoor during day
time and prefers to lay its eggs in man-made containers with clean or clear water. These
containers may include water storage containers, flower pots, discarded tires, plates under potted
plants, cemetery vases, flower pots, buckets, tin cans, clogged rain gutters, ornamental fountains,
drums, water bowls for pets, birdbaths and other suitable places. The eggs of Aedes mosquitoes
are able to withstand desiccation (dryness), allowing them to survive long periods and increasing
its efficacy to spread (Figure 2a).

a) Ae. aegypti

b) Ae. albopictus

Figure 2. Taxonomy of a) Ae. aegypti and b) Ae. albopictus

Meanwhile, Ae. albopictus (secondary vector) is a small dark mosquito. It is also known as the
Asian tiger mosquito. It has a distinct median line of white scales (Figure 2b). They are mostly
found outdoors and prefer to lay their eggs in natural containers like tree holes and plants. They
are also day biters.

Dengue virus is spread through a human-to-mosquito-to-human cycle of transmission (Figure 3).

Female Aedes becomes dengue vector after feeding on the blood of a person infected with the
dengue virus. Female mosquitoes are capable of spreading the disease within 10-12 days after
biting an infected person. The virus will multiply in the salivary glands of the mosquito and will
be transferred into a healthy human body during mosquito bites.

Figure 3. The dengue virus is spread through a human-to-mosquito-to-human cycle of

transmission (source: www.eliminatedengue.com)

It is possible to get dengue fever multiple times since there are 4 types (DENV-1, DENV-2,
DENV-3 and DENV-4) of the dengue virus that cause dengue fever. Individuals infected by a
particular dengue virus serotype will become immune to that type of dengue virus; however they
can still get fever from the other types of dengue. Being infected subsequently by a different type
of dengue virus will lead to increased risk of developing severe dengue. Severe dengue causes
bleeding and shock, and can be life threatening.
The transmission of the virus to mosquitoes must be interrupted to prevent the illness. The
prevention of dengue requires control or eradication of the mosquitoes that are able to carry the
virus. These include source reduction, bite protection by using mosquito repellents when being
outdoors during daytime, wearing light-colour clothing and long sleeves and long pants when out
of the house, installing insect screens on doors and windows, the proper use of insecticides and
the use of mosquito nets during day time.

2.0 Wolbachia, the fascinating endosymbiont bacteria

Recently, another increasingly popular method to overcome dengue issue is using Wolbachia
approach. Wolbachia is a naturally occurring bacterium that is present in up to 60% of all
different types of insects including mosquitoes, ticks, flies and nematodes populations. Some of
the major disease vectors are not naturally infected, including Ae. aegypti and some anopheline
mosquitoes. Wolbachia live inside the cells of various organs, but most frequently appear in
ovaries and testes (Werren, 1995).
Interestingly, some Wolbachia strains are able to cause pathogen interference (inhibition of
replication or growth of pathogens), including viruses in their newly introduced invertebrate
hosts. This means that Wolbachia needs to be transferred from other insects into mosquito, by
injecting the mosquito eggs through a technique called microinjection. For example, the
introduction of Wolbachia strain B isolated from Ae. albopictus (wAlbB) and fruitfly,
Drosophila melanogaster (wMel) into a new host, such as mosquito, has shown to inhibit
replication of the virus in the Aedes mosquitoes (Joubert et. al., 2016). Besides, wMel has shown
inhibition towards chikungunya and ZIKA (Frentiu et al, 2014, Dutra et al., 2016).

Figure 4. Wolbachia infected mosquitoes proven to block ZIKA transmission

3.0 Wolbachia-based vector control approach

Wolbachia-based vector control strategy can be utilized using two approaches; i) Population
Replacement and (ii) Population Suppression.
Population Replacement
The goal of population replacement is to replace the wild mosquito populations with Wolbachiainfected mosquitoes. The replacement strategy involves the release of MALES AND
FEMALES infected with Wolbachia over a number of weeks and these Wolbachia infected
mosquitoes then mate with the wild mosquito population. Wolbachia is maternally transmitted
and passed on from generation to generation. Over time, the percentage of mosquitoes carrying
Wolbachia grows until it remains high without any further releases. Wolbachia-infected
mosquitoes are unable to transmit viruses to people and therefore, reducing the risk of outbreaks
in the released areas. Studies have shown that Wolbachia can sustain itself in mosquito
populations without continual reapplication, which makes this method sustainable and cost
effective in the long term (Eliminate Dengue, 2016).

Figure 5. Population replacement approach

Population suppression
The second approach - population suppression goal is to suppress the wild mosquito population
to below threshold where disease transmission can be interrupted. Suppression approach will
involve the release of ONLY MALE Wolbachia-infected mosquitoes. The Wolbachia infected
males will mate with the wild female mosquitoes. The eggs produce by the wild female will not
hatch and subsequently the population will decrease over time, with sustained released of the
Wolbachia infected males. This technique requires the release of a large number of Wolbachiainfected males. Nonetheless, this approach needs subsequent & sustained release as the
population of wild mosquitoes gradually returns over time.

Figure 6. Population suppression approach

4.0 Wolbachia- from lab to open field

In Malaysia, the Minister of Health has instructed the research arm in the MOH to conduct
Wolbachia-based control strategy utilizing the population replacement approach to stop the
escalating number of dengue cases every year. This study is led by the Institute for Medical
Research (IMR) in collaboration with Institute for Health Behavioural Research (IHBR). This is
a collaborative effort with University of Lancaster, UK and University of Melbourne, Australia.
At present, two sites have been identified to be suitable for population replacement trial run. The
sites are 1) Keramat and 2) Shah Alam. There are some other selection criteria to be included in
selecting trial sites such as; i) presence of high population of both Ae. aegypti and Ae. albopictus,
ii) the size of the release sites should be at least 0.6 km2, and iii) logistically convenient from the
mosquito breeding facility in IMR.
To date, eight countries had field relesaed Wolbachia infected mosquito. Australia, Vietnam,
Indonesia, Brazil and Colombia are working on population replacement approach. Meanwhile,
three other countries which are China, Thailand and Tahiti are using the population suppression
approach.

Table 1. Countries involved in Wolbachia mosquito field release

Country

Vector Infected

Idea under evaluation

Australia

wMel infected
Ae. aegypti

Replaced wild
population by wMel
infected Ae. aegypti

Goal
Population
replacement

Details
Released in Cairns (9
months)
Medium (50 - 80%) to
high (more than 80%)
level of wMel infection

Vietnam

Indonesia

wMelpop
infected Ae.
aegypti

Half the life of adult

females

wMel infected
Ae. aegypti

Replaced wild
population by wMel
infected Ae. aegypti

wMel infected
Ae. aegypti

Replaced wild
population by wMel
infected Ae. aegypti

Population
replacement

No locally acquired
dengue cases reported
wMelpop- failed due to
high fitness cost (death
of infected adults)
wMel- released in Tri
Nguyen Island (17
weeks)

Population
replacement

62% level of wMel

infection
Released in Yogyakarta
(6 months)
>80% level of wMel
infection
Not available

Brazil

wMel infected
Ae. aegypti

Replaced wild
population by wMel
infected Ae. aegypti

Population
replacement

Colombia

wMel infected
Ae. aegypti

Replaced wild
population by wMel
infected Ae. aegypti

Population
replacement

Not available

China

wPip infected
Ae. albopictus

Suppressed the wild

population due to
cytoplasmic
incompatibility

Population
suppression

Released ratio 5:1

USA

wPip infected
Ae. albopictus &
Ae. aegypti

Suppressed the wild

population due to
cytoplasmic
incompatibility

Population
suppression

Tahiti

Ae.
polynesiensis

Suppressed the wild

population due to
cytoplasmic
incompatibility

Population
suppression

Ae. aegypti

100% no egg hatching

(after 5 months of
release)
Pending

Population suppressed
for 30 weeks

Relevant references
1. World Health Organisation. Dengue Guidelines for diagnosis, treatment, prevention and
control. Geneva: World Health Organization, 2009.
2. Ministry of Health, Malaysia, 2016. http://idengue.remotesensing.gov.my/idengue/index.php
3. Rudnick, A. (1986). Dengue fever studies in Malaysia. In T. W. Lim & J. L. Ireland (Eds.),
Bulletin no. 23 from the Institute for Medical Research, Malaysia (pp. 155167). Book
Section, Kuala Lumpur, Malaysia: Institute for Medical Research.
4. Werren, J. H., Guo, L., & Windsor, D. (1995). Distribution of Wolbachia in neotropical
arthropods. Proceedings of the Royal Society of London B, 262, 197204.
5. Joubert, D. A., Walker, T., Carrington, L. B., De Bruyne, J. T., Kien, D. H. T., Hoang, N. L.
T., ONeill, S. L. (2016). Establishment of a Wolbachia Superinfection in Aedes aegypti
mosquitoes as a potential approach for future resistance management. PLoS Pathog, 12(2).
6. Frentiu, F. D., Zakir, T., Walker, T., Popovici, J., Pyke, A. T., van den Hurk, A., ONeill,
S. L. (2014). Limited dengue virus replication in field-collected Aedes aegypti mosquitoes
infected with Wolbachia. PLoS Negl Trop Dis, 8(2).
7. Dutra, H. L. C., Rocha, M. N., Dias, F. B. S., Mansur, S. B., Caragata, E. P., & Moreira, L.
A. (2016). Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes
aegypti mosquitoes. Cell Host & Microbe.
8. Eliminate Dengue, 2016. http://www.eliminatedengue.com/our-research/Wolbachia
9. Brelsfoard, C. L., & Dobson, S. L. (2011). Short Note: An update on the utility of Wolbachia
for controlling insect vectors and disease transmission. Asia Pacific Journal of Molecular
Biology and Biotechnology, 19(3), 8592.
10. Bian, G., Xu, Y., Lu, P., Xie, Y., & Xi, Z. (2010). The endosymbiotic bacterium Wolbachia
induces resistance to dengue virus in Aedes aegypti. PLoS Pathogens, 6(4).
11. Hoffmann, A. A., Montgomery, B. L., Popovici, J., Iturbe-Ormaetxe, I., Johnson, P. H.,
Muzzi, F., ONeill, S. L. (2011). Successful establishment of Wolbachia in Aedes
populations to suppress dengue transmission. Nature, 476(7361), 454457.
12. van den Hurk, A. F., Hall-Mendelin, S., Pyke, A. T., Frentiu, F. D., McElroy, K., Day, A.,
ONeill, S. L. (2012). Impact of Wolbachia on infection with chikungunya and yellow fever
viruses in the mosquito vector Aedes aegypti. PLoS Neglected Tropical Diseases, 6(11),
1892.

Sample Questions and Answers

Q Is Wolbachia a GMO?
A No, Wolbachia has not been genetically modified, and is classed as a biological control agent.
It already exists in the environment naturally (within invertebrate hosts). It is also not being used
as a biopesticide because we are not using it to suppress populations.
Q Do the Wolbachia strains that will be released occur naturally in Malaysia?
A Yes, in the common Drosophila fruit flies and in other mosquito species.
Q If Wolbachia occur naturally in Aedes albopictus, why dont they block dengue transmission?
A Natural Wolbachia strains occur at lower density and dont have a big impact on virus
transmission, while the introduced Wolbachia will be at much higher density within the
mosquito cells and will block dengue.
Q Could Wolbachia increase transmission of some viruses?
A Increased transmission efficiency of human viruses has never been observed for a stable
mosquito Wolbachia infection; the viral inhibition seems to be a general effect for RNA viruses
and occurs for both flaviviruses (including dengue) and alphaviruses (chikungunya)
Q Could the virus mutate to be transmitted efficiently by mosquitoes carrying the released
Wolbachia?
A This is unlikely as there are multiple mechanisms of viral blocking by Wolbachia within the
mosquito cell, making the system naturally resistant to viral escape mutations. There is no
evidence to date of viruses having evolved in this way.
Q Could Wolbachia be transmitted to humans?
A No, Wolbachia or Wolbachia proteins are not transmitted in mosquito saliva. These Wolbachia
can only grow in insect cells; no Wolbachia can survive / grow in vertebrate cells. Humans have
been exposed to Wolbachia in countless ways for millions of years and there is no evidence that
there has ever been transmission to humans.
Q Could Wolbachia cause ecological damage in the environment?
A No, Wolbachia can only survive and replicate inside host invertebrate cells, they do not escape
and cause damage.
Q Could Wolbachia infect / damage other insect species?
A Wolbachia is only transmitted from mother to offspring (like mitochondria), not as an
infectious agent, so it is extremely difficult for it to move into other insect species; this is
estimated to occur only once every million years. In any case the Wolbachia strains to be used
are already common in flies / other mosquitoes, and do not cause ecological damage to the host
insect.

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Q Why do female mosquitoes have to be released?

A Wolbachia population spread is dependent on female release due to its mode of maternal
transmission, it is not transmitted by males. So only by releasing females will the Wolbachia be
introduced into a population.
Q Why not just release males to suppress the mosquito population?
A This is a much less efficient strategy that requires ongoing intervention a much larger scale
of release would be needed and it would have to be conducted continuously to have any effect on
dengue transmission, so would be prohibitively expensive. As soon as male releases are stopped,
the mosquito population bounces back. In contrast, the replacement strategy requires just a short
period of release, then Wolbachia spreads by itself and maintains itself stably at a high frequency
from then on, blocking virus transmission.
Q I heard that Singapore is trialling Wolbachia male releases? Why cant we do the same?
A Singapore is starting with some experimental releases of very large numbers of males to see if
they can suppress the mosquito population. This might have an effect when mosquito densities
are very low as they are in Singapore, but is not feasible for a large area, and requires a very
large number of mosquitoes to be produced.
Q Would people notice an increase in mosquito biting?
A This is extremely unlikely - releases are conducted during drier times of lower mosquito
population density, for a short period only, and the released males would be sterilizing wild
females so any increase (if detected at all) would only be minor. Wild mosquito populations
can also be suppressed with insecticides prior to starting releases. Aedes (the mosquito being
released) are not the main mosquito biting nuisance in cities, Culex are much more common.
Q Will the public support releases?
A Careful community engagement and education will be conducted at each site prior to any
releases. There has been considerable enthusiasm for Ae. aegypti Wolbachia releases in previous
trials in Australia, Indonesia, Brazil and Vietnam
Q What happens if individuals / families are opposed to releases?
A They can be given a veto and releases would not be conducted near their homes.
Q When can we expect to see dengue suppression occurring?
A Once the Wolbachia has established, the mosquitoes carrying the Wolbachia can be screened
to test if they are less likely to carry and transmit viruses. But showing that Wolbachia decreases
dengue in humans will only become possible once Wolbachia has been established in several
areas which will take some time.
Q How long will the Wolbachia effects last?
A Their effects are permanent. Mosquitoes will stay infected with Wolbachia, it is a selfmaintaining control method.

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Q Can we still undertake our usual mosquito suppression measures while releases are
occurring?
A Yes completely. All normal methods of mosquito control like suppressing breeding sites
continue while releases are undertaken.
Q What happens when there is a dengue outbreak in the area where releases are being
undertaken?
A Because the Wolbachia infected mosquitoes block dengue transmission, releases could
continue, but this will be up to the community. They can also be stopped in a local area for a
period.
Q Why dont you start releases first in field cages? Wolbachia have already been shown to
invade field cages successfully many times, so these experiments do not provide any new
information. Moreover, Wolbachia have now been released successfully in many countries
without any issues and they are already present naturally in the environment in many insects.

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