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NEA To Commence Phase Two Wolbachia-Aedes Field Study At Three Sites In April 2018

26 Feb 2018

Phase 1 has yielded valuable results, and the project is progressing to Phase 2 to further improve release methodologies for Singapore's high-density and high-rise urban landscape

Singapore, 26 February 2018 – The Phase 1 field study of Project Wolbachia – Singapore,conducted from October 2016 to December 2017,has met its objectives. The field study also surfaced challenges posed by Singapore’s high-density and high-rise urban landscape. Using valuable data collected from Phase 1, the project will progress to a Phase 2 field study, where NEA will further improve the release methodologies and tactics to mitigate these challenges. From April 2018 to January 2019, male Wolbachia-carrying Aedes aegypti (Wolbachia-Aedes)mosquitoes will be released at the same Phase 1 field study sites (at Tampines West and Nee Soon East) and their extended areas.

Project Wolbachia – Singapore

2          NEA is evaluating the use of male Wolbachia-Aedes mosquitoes to further suppress the Aedes aegypti mosquitoes in the community. When these released male Wolbachia-Aedes mosquitoes mate with urban Aedes aegypti females in the community, the resultant eggs do not hatch. The continual release of male Wolbachia-Aedes mosquitoes over time is thus expected to reduce the viability of the Aedes aegypti eggs, and bring about a gradual reduction in the urban Aedes aegyptimosquito population.

3          The Phase 1 field study – conducted at three selected study sites at Braddell Heights, Tampines West and Nee Soon East – demonstrated that the released male Wolbachia-Aedes mosquitoes had successfully competed with the urban Aedes aegypti males and mated with some of the urban Aedes aegypti females. As a result, the releases led to a 50 per cent suppression of the urban Aedes aegypti mosquito population at the study sites.

4          The Phase 1 field study also surfaced ecological challenges posed by Singapore’s high-density and high-rise housing. The impact of the releases was found to be limited by the movement of Aedes aegypti mosquitoes from the surrounding areas into the release sites; as well as by the high Aedes aegypti mosquito densities at high floors of some blocks where insufficient numbers of male Wolbachia-Aedes mosquitoes reached. The limits of the current methodology for sex-sorting of pupae also resulted in a very small percentage of fertile Wolbachia-Aedes females being released alongside the males, which may eventually limit the impact of the male releases over time. More information on the results of the Phase 1 field study can be found in Annex A.

Phase 2 Field Study

5          To mitigate high-density and high-rise challenges, the Phase 2 field study will further improve the release methodologies to distribute the male Wolbachia-Aedes mosquitoes to where they are needed. This will involve releasing male Wolbachia-Aedes mosquitoes, in the form of adults and pupae, from the ground floor as well as from high floors of apartment blocks. To mitigate any possible fertile Wolbachia-Aedes females being released alongside the males, the Phase 2 field study will also use X-ray or similar treatment of Wolbachia-Aedes mosquitoes as a further complementary step after sex-sorting of the pupae, to render infertile any Wolbachia-Aedes females that may be inadvertently released. This additional step will prevent the build-up of female Wolbachia-Aedes mosquitoes that would have resulted from the release of fertile female Wolbachia-Aedes mosquitoes over time, which may then eventually hamper the continued use of male Wolbachia-Aedes to suppress the urban Aedes aegypti mosquito population in those areas. Impact assessment has shown that X-ray or similarly treated mosquitoes pose no harm to humans or the environment. Annex B provides more details of the impact assessment. The World Health Organization’s Vector Control Advisory Group (VCAG) has assessed and concluded that the combined Wolbachia and X-ray or similar treatment approach has potential for the long-term control of Aedes aegypti and Aedes albopictus mosquito vectors. The VCAG also strongly recommended further entomological and epidemiological field trials to validate the use of this intervention.

6          Male Wolbachia-Aedes mosquitoes will be released at the same Phase 1 field study sites (at Tampines West and Nee Soon East) and their extended areas. About one to six male mosquitoes per person will be released per week, during any release periods.  Mosquitoes will be released around the blocks and along common corridors, and will not be released directly into homes. Please see Annex C for the location maps and boundaries of the three study sites. NEA researchers will be setting up mosquito traps and pupal containers at various locations. We seek the co-operation of members of the public not to remove or tamper with the mosquito traps and pupal containers. Please see Annex D for images of the mosquito traps.

7          Data collected from the Phase 2 field study will strengthen our planning for a subsequent suppression trial. NEA’s careful and thorough studies, to be conducted over a timeframe of several years, is necessary to ensure the proper evaluation and application of the Wolbachia technology in our urban landscape. NEA hopes to use the Wolbachia technology to complement its existing vector control efforts, to reduce the risk of Aedes-borne diseases, such as dengue and Zika.

8          Dr Raman Velayudhan, Coordinator, Department of Control of Neglected Tropical Diseases, World Health Organization, Geneva said, “The positive results and further data from larger scale studies would certainly strengthen the evidence base for this new Wolbachia-Aedes vector control tool. Aedes mosquito control will remain a challenge until we have effective sustainable methods to suppress the vector population over a longer period of time. Climate change could favour the multiplication of mosquitoes and cause increased dengue burden, and vector population suppression methods have good potential to control disease transmission.”

9          NEA is very encouraged by the support of the public, as well as the continual interest in Project Wolbachia – Singapore. Prior to and during the Phase 2 field study, NEA will be providing more information to residents and local stakeholders at the study sites.

10        The co-operation and support from residents and local stakeholders at the selected sites will be crucial in ensuring the success of the Phase 2 field study. Residents do not have to do anything differently, but continue to carry out mosquito control procedures and practise the 5-Step Mozzie Wipeout as normal.

11        Members of the public can find out more about Wolbachia technology by visiting www.nea.gov.sg, or contacting NEA at 1800-CALL-NEA (1800-2255 632) if they have any enquiries. We are thankful for the support from residents, various stakeholders and volunteers.

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For more information, please contact us at 1800-CALL NEA (1800-2255 632) or submit your enquiries electronically via the Online Feedback Form or myENV mobile application.

 

ANNEX A

KEY FINDINGS FROM PHASE 1 FIELD STUDY

1)    Wolbachia technology has the potential to suppress the Aedes aegypti mosquito population in the community. 

At sites where the male Wolbachia-Aedes mosquitoes were released, much fewer Aedes aegypti adult mosquitoes were found.
Fig 4 - Location - Tampines St 81

Fig. 1. Impact of male Wolbachia-Aedes mosquitoes on the urban Aedes aegypti population at the study sites

 

At the release sites, half of the collected Aedes mosquito eggs did not hatch, which provided strong indication that the released Wolbachia-Aedes males had successfully competed with the urban Aedes males and mated with some of the urban Aedes aegypti females.

A larger reduction in egg hatch and adult population will be necessary to achieve suppression of the urban Aedes mosquito population. If we have lower levels of urban Aedes to start with, it will be easier to achieve this goal. Continued community effort in keeping the mosquito population low is thus important to enable effective suppression through the release of male Wolbachia-Aedes mosquitoes.

2)    Singapore’s high-rise and densely built urban environment poses unique challenges.

i.  Aedes aegypti mosquitoes moved easily from surrounding areas into the release sites. This reduced the suppression effect of Wolbachia-Aedes at the release sites.

ii.  Wolbachia-Aedes released at ground level achieved less suppression for high-rise blocks that had more Aedes aegyptimosquitoes at higher floors. 
Fig 2 - Movement and migration

3)    More frequent releases can help keep the number of Wolbachia-Aedes males in the field more constant, and thus achieve better suppression effect.
Fig 3 - Lifespan

4)    Long term small releases of female Wolbachia-Aedes over time may eventually result in Wolbachia-Aedes taking over as the dominant mosquito strain, and hamper the continued use of Wolbachia-Aedes to suppress the Aedes population in those areas. 

i.  Although only a very small number of female Wolbachia-Aedes mosquitoes may slip through the sorting process during production, the cumulative effect of successive releases over time may eventually result in Wolbachia-Aedes taking over as the dominant mosquito strain, and hamper the continued use of male Wolbachia-Aedes mosquitoes to suppress the Aedes population in those areas.

ii.  Existing manual methods for removing female Wolbachia-Aedes mosquitoes, when production numbers are small, cannot be scaled up cost-effectively to cater for higher production numbers. X-ray or similar treatment will be used to complement existing sorting process to render any remaining small number of females infertile.

ANNEX B

IMPACT ASSESSMENT ON THE RELEASE OF X-RAY OR SIMILAR TREATED WOLBACHIA-AEDES MOSQUITOES FOR SUPPRESSION OF THE URBAN AEDES AEGYPTI MOSQUITO POPULATION

Summary

The impact assessment process involved critical reviews of existing knowledge and research, consultations with domain experts from the International Atomic Energy Agency (IAEA), and laboratory experiments carried out at the Environmental Health Institute (EHI), of the National Environment Agency (NEA). No ecological risk has been identified. A potential risk identified is the reduction in competitiveness of the X-ray or similarly treated male Wolbachia-Aedes mosquitoes, leading to ineffective suppression of the urban Aedes aegypti mosquito population at the study sites, thus necessitating the need to release larger numbers of such male mosquitoes.

Background

Project Wolbachia – Singapore takes advantage of the cytoplasmic incompatibility (CI) attribute of the naturally occurring Wolbachia bacterium, to help suppress the urban Aedes aegypti mosquito population in Singapore, for further reduction of the dengue risk. Impact assessment and laboratory studies conducted since 2012 at the Environmental Health Institute (EHI), of the National Environment Agency (NEA), have shown that the Wolbachia technology is safe and poses no harm to humans or the environment. From 2016 to 2017, NEA carried out the Project Wolbachia Phase 1 field study, to gain an understanding of the behaviour of male Wolbachia-Aedes mosquitoes, by releasing these mosquitoes in our urban environment. The Phase 1 field study – carried out at three study sites at Braddell Heights, Tampines West and Nee Soon East – yielded valuable data on the bionomics of the released male Wolbachia-Aedes mosquitoes, such as their longevity in the field, and their vertical and horizontal dispersal ranges. The data also indicated the ability of male Wolbachia-Aedesmosquitoes to compete with urban Aedes aegypti males to mate with urban Aedes aegypti females, leading to a 50 per cent suppression of the urban Aedes aegypti mosquito population at the study sites (www.nea.gov.sg).

The Phase 1 field study also surfaced ecological challenges posed by Singapore’s high-density and high-rise housing. The impact of the releases was found to be limited by the movement of Aedes aegypti mosquitoes from the surrounding areas into the release sites; as well as the high Aedes aegypti mosquito densities at higher floors of some blocks, where insufficient numbers of male Wolbachia-Aedes mosquitoes reached. In addition, a very small percentage of fertile Wolbachia-Aedesfemales being released alongside the males could also have limited the impact of the releases.

As highlighted in our earlier impact assessment on the Wolbachia-Aedes technology, a very small number of female Wolbachia-Aedes mosquitoes may be released along with the male Wolbachia-Aedes mosquitoes, due to imperfect sex-sorting. The current sex-sorting method uses a mechanical size-separation device to separate the smaller male pupae from the larger female pupae. Whilst the device has consistently achieved a high accuracy of separating male from female pupae (over 99.7% accuracy), a very small number of the smaller-sized females may be segregated with the males. Though the small number of females will have negligible potential for disease transmission, as viral replication is blocked by the Wolbachia present, long-term releases of female Wolbachia-Aedes mosquitoes may potentially hamper the continued use of Wolbachia-Aedes technology to suppress the urban Aedes aegypti mosquito population in those areas. This is due to the biological mating compatibility between female Wolbachia-Aedes mosquitoes and males with or without Wolbachia. These matings could lead to the propagation of offspring with Wolbachia, and as these compatible matings increase over time and produce more progeny with WolbachiaWolbachia Aedes aegypti population may dominate in the field. The female Wolbachia Aedes will be biologically compatible with the released Wolbachia-Aedes males and will thus hamper the continued use of the same strain of male Wolbachia-Aedes to suppress the mosquito population.

Use of a combined SIT/IIT approach for the suppression of Aedes mosquitoes

One approach to address the potential establishment of Wolbachia-Aedes mosquitoes in the community is to subject the sex-sorted pupae to X-ray or similar treatment, to sterilise any female Wolbachia-Aedes mosquitoes that may have slipped through the sorting process. X-ray or similar treatment affects the fertility of female mosquitoes more than the virility of male mosquitoes, and laboratory studies have shown that a low dosage X-ray or similar treatment can sterilise the females without diminishing the virility of the males.

The potential of using radiation for the control of mosquito populations was first demonstrated in the late 1950s, when eggs from matings between female Anopheles quadrimaculatus and treated males failed to hatch. However, earlier field studies had shown that male mosquitoes exposed to the treatment dose required for sterility (over 100Gy) had rendered them unfit and uncompetitive due to somatic cell damage. Further scientific research later showed that lower dosage treatment could render the females infertile without diminishing the virility of the males.

Coupling the Wolbachia-based Insect Incompatibility Technique (IIT) with the classical irradiation-based Sterile Insect Technique (SIT) thus has the potential to produce competitive male Wolbachia-Aedes mosquitoes, whilst rendering the small number of female Wolbachia-Aedes mosquitoes infertile. Laboratory studies have shown the potential of this approach for Aedes albopictusAedes polynesiensis and Culex pipiens mosquito species, with promising results. Encouraging findings from an ongoing field study in Guangzhou, China, have also shown the feasibility of this approach for the Aedes albopictusmosquito (Prof. Xi Zhiyong, personal communication). In 2017, the World Health Organization’s Vector Control Advisory Group (VCAG) assessed and concluded that the combined SIT/IIT technology has the potential for long-term control of Aedes aegypti and Aedes albopictus mosquito vectors. VCAG strongly recommended further entomological and epidemiological field trials to validate the use of this intervention. 

Impact assessment

Impact of X-ray or similarly treated Wolbachia-Aedes mosquitoes on the environment

Does treatment of Wolbachia-Aedes mosquitoes render them radioactive or cause them to emit radiation?

No. Treated mosquitoes are not radioactive, as the mosquitoes do not come into physical contact with the radioactive source. X-ray or similar treatment (less than 30 Gy) applied to the Wolbachia-Aedes mosquitoes does not render them radioactive – just like X-ray screening of baggage at airport and seaport checkpoints, does not cause the baggage to become radioactive or emit radiation. The use of such treatment for commercial sterilisation of medical devices is also widely implemented throughout the world. The reference dose for sterilisation of medical devices is 2500 Gy.

Could there be an adverse effect when predators eat, or when other animals come into contact with, the treated Wolbachia-Aedes mosquitoes? 

For the same reason as above, predators that eat, or other animals that come into contact with, the treated mosquitoes will not be affected by the treatment. X-ray or similar treatment is commonly applied to our food to improve food safety, quality and prolong shelf-life; and to agricultural products as part of phytosanitation measures for removing and reducing pests that may be harmful or destructive to these products. The doses applied under these circumstances are more than 100 times higher than that required to sterilise insects to control pest populations.

Impact of X-ray or similarly treated Wolbachia-Aedes mosquitoes on public health

Will treatment increase the biting frequency of female Wolbachia-Aedes mosquitoes?

No. Studies carried out at EHI, have shown that treated female Wolbachia-Aedes mosquitoes have similar biting behaviour when compared to non-treated female mosquitoes.

Will treatment cause female Wolbachia-Aedes mosquitoes to lay more eggs?

No. Studies carried out at EHI have shown that treated female Wolbachia-Aedes mosquitoes have reduced fecundity (over 99.9% reduced) when compared to non-treated female Wolbachia-Aedes mosquitoes. 

Will treatment affect the level of cytoplasmic incompatibility (CI) induced by treated male Wolbachia-Aedes mosquitoes, such that mating with urban Aedes aegypti females will result in viable offspring?

No. Studies conducted at EHI have shown that treatment has no impact on cytoplasmic incompatibility (CI) between the male Wolbachia-Aedes mosquitoes and urban female Aedes mosquitoes. All eggs laid by urban Aedes aegypti females that had mated with treated Wolbachia-Aedes males did not hatch.

Will treatment lower the ability of male Wolbachia-Aedes mosquitoes to compete with urban Aedes aegypti males to mate with urban Aedes aegypti females?

Studies conducted at EHI and at other international research laboratories have shown that treatment reduced the mating competitiveness of male mosquitoes. To compensate for this reduction in mating competitiveness, a larger number of male Wolbachia-Aedes mosquitoes may need to be released. The Project Wolbachia Phase 2 field study will aim to understand the impact of X-ray or similar treatment on the fitness of the male Wolbachia-Aedes mosquitoes in the urban setting.

Despite the reduced competitiveness, mass release of irradiated insects to control or eradicate various agricultural pest species has been successful and well documented since the 1950s, The New World screw worm fly (Cochliomyia hominivorax) – an important veterinary parasite affecting livestock – was eradicated from the US and all Central American countries in the 1950s. Since then, SIT has also successfully eradicated or suppressed: the Mediterranean fruit fly (Ceratitis capitate) in many parts of Central and South America, South Africa and Australia; the melon fly (Bactrocera cucurbitae) in Okinawa and all of Japan’s south-eastern islands; the codling moth (Cydia pomonella) in British Columbia, Canada; and a tsetse fly species that transmits African animal trypanosomiasis in Zanzibar, Tanzania. 

Will the increase in the number of treated male Wolbachia-Aedes mosquitoes to be released lead to an increase in the number of Wolbachia-Aedes females being unintentionally released?

The number of Wolbachia-Aedes females unintentionally released will increase proportionally with the increase in the number of Wolbachia-Aedes males released. However, the X-ray or similar treatment will render any released females infertile.  The Wolbachia in the females will also help block the transmission of dengue virus. So even though this small number of released Wolbachia-Aedes female mosquitoes may still bite, they would neither be able to transmit disease nor reproduce. Furthermore, the number released would be only be a negligible fraction of the population of urban Aedes aegypti females at the release sites. It would therefore not add significantly to any mosquito bite encounters that is already present at these release sites due to the presence of the urban Aedes mosquitoes. 

ANNEX C

LOCATION MAPS OF THREE STUDY SITES

Fig 4 - Location - Tampines St 81

Fig. 1. Location map of Tampines West study site: Tampines Street 81
Fig 5 - Location - Tampines Ave 4
Fig. 2. Tampines West study site: Tampines Avenue 4
Fig 6 - Location - Nee Soon

Fig. 3. Nee Soon East study site

ANNEX D

IMAGES OF MOSQUITO TRAPS 

Mosquito Traps