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Cutting-edge Approaches to Aedes aegypti Control

Mr. M. Srinivasulu

Senior Knowledge Scientist
INTRODUCTION

The aim of this paper is to review the recent trends in the control of Aedes egypti.
Aedes aegypti (Ae. aegypti) is the main vector for transmitting Flaviviruses — such as ZIKV, DENV, CHIKV and YFV — which, respectively cause Zika fever, dengue fever, chikungunya, and yellow fever.
Ae.aegypti has multiplied throughout the tropical and subtropical regions of the world and is considered to be among the most widespread mosquito species that is responsible for more than one million deaths worldwide every year.1 It is very difficult to control or eliminate the Ae.aegypti mosquito because of its quick adaptability to any and every environment.
The recent rise in the spread of Zika virus has been accompanied by an unprecedented rise in the number of children being born with unusually small heads, known as microcephaly. Microcephaly has also been linked to several other problems, including seizures, developmental delay, intellectual disability and feeding problems.2 In the absence of a vaccine, currently feasible options to control Ae. aegypti are limited to reducing the abundance of mosquito vector populations.

Mechanism of transmission of Zika virus

Zika virus is a zoonotic pathogen, naturally hosted in non-human primates. Rhesus monkeys may be the sources found in natural habitats, from which the virus could later be transmitted to susceptible human hosts. Zika virus is primarily vector-borne, but there are also secondary modes of transmission3 (mother-to-child, sexual, blood transfusion, transplantation, non-human primate bites, etc.).

Fig1: Different modes of transmission of Zika virus

Background:

In view of the recent increase in detection of microcephaly-related cases, biological approaches such as use of Bacillus thuringiensis israelensis (Bti), Gambusia fish etc., to kill mosquito larvae present in water are being considered in addition to the traditional approaches, which include use of mosquito repellents containing DEET (N, N-diethylmetatoluamide, 20 percent to 30 percent concentration), p-menthane-3, 8-diol (from lemon eucalyptus) and others.
Control of the world's most important vector-borne viral disease, Zika, is a high priority. Lack of vaccines or effective vector control methods mean that it is essential to find novel solutions to mosquito control.

Current technologies for the control of Ae.aegypti

There are multiple approaches to the control of Ae. aegypti. Use of Wolbachia bacterium, genetic modification of the mosquito species in question and use of vaccines are some recent approaches.

Bacterium-based approach for the control of Aedes aegypti

Wolbachia is a gram-negative bacteria that is an endosymbiont inside the mosquito species. However it does not cause harm to the mosquitoes. Wolbachia has been explored as a potential tool in the control of mosquito-borne diseases because of its unique ability to invade host populations rapidly. Wolbachia is found in many mosquito species in nature, but surprisingly not in Ae.aegypti.4 A transient somatic infection in male Ae. aegypti, stably transfected by Wolbachia, shows strong resistance to Flavivirus. Wolbachia bacterium inhibits viral replication in the mosquito’s midgut and disrupts the reproduction process, dramatically reducing the mosquito’s potential for viral transmission.5
Infection of the Ae.aegypti with this bacterium leads to induction of oxidative stress and an increased level of Reactive Oxygen Species (ROS) in the mosquito host. ROS elevation is linked to the activation of the Toll pathway, which is essential for mediating the expression of antioxidants to counterbalance oxidative stress. This immune pathway is also responsible for activation of antimicrobial peptides, such as defensins and cecropins.6 These antimicrobial peptides are involved in inhibition of Flavivirus proliferation in Wolbachia-infected mosquitoes. The female mosquito that acquires wolbachia by mating is sterile and they will produce wolbachia infected offsprings.7 The ability of Wolbachia to boost immunity and block Flavivirus proliferation in a newly acquired host, namely Ae. aegypti, makes it a potential “mosquito vaccine” that could be used effectively to prevent pathogen transmission. Wolbachia biocontrol represents a significant new development in the fight against a Flavivirus transmission. It could potentially be used as a multivalent strategy against all four viruses (DENV, CHIKV, YFV, and now ZIKV) transmitted by Ae. Aegypti.8

Fig2: Wolbachia-mediated resistance to flavivirus in Aedes aegypti

Sun Yat-sen University, originally known as Guangdong University, is jointly working with Michigan State University (MSU) on the Wolbachia-based project for Aedes mosquito control.
In the world’s largest mosquito farm, millions of male mosquitoes are being bred at a furious pace for release on an island in Southern China, as part of a plan to suppress a mosquito that can transmit the Zika virus. And so far, the results have been stunning.
With more testing required, the technology may still be two or three years off from being used on a wider scale.
This project9 has been funded by the Bill & Melinda Gates Foundation.
A few patents on the above have been listed below:

Publication number: US9090911B2
Assignee: Monash University
Inventors: Scott Leslie O'Neill, Conor James McMeniman, Karyn Nicole Johnson, Elizabeth Ann McGraw, Luciano A. Moreira, Peter Anthony Ryan, Brian Herbert Kay, Jeremy Colin Brownlie
Title: Modified arthropod and method of use
Abstract: A modified arthropod, an arthropod-modifying bacterium, and use thereof as an agent for control of diseases transmitted by arthropods, particularly mosquitoes, is provided. More specifically, an isolated arthropod-adapted Wolbachia bacterium capable of modifying one or more biological properties of a mosquito host is provided. The modified arthropod may be characterized as having a shortened life-span, a reduced ability to transmit disease, a reduced susceptibility to a pathogen, a reduced fecundity, and/or a reduced ability to feed from a host, when compared to a corresponding wild-type arthropod.
Summary: Arthropod-adapted Wolbachia bacterium isolated from its native host and adapted to a new host such as Aedes aegypti, this bacterium can control of diseases transmitted by mosquitoes such as Dengue, Chikungunya and Yellow fever.
Status: Alive

Publication number: US7868222B1
Assignee: University of Kentucky Research Foundation
Inventors: Stephen L. Dobson
Title: Transfected mosquito vectors
Abstract: A method is provided for producing an artificial infection in a Culicidae (mosquito) species. The mosquitoes include species within the subfamilies Culicinae and Anophelinae, and the species include Aedes albopictus, Aedes aegypti and Aedes polynesiensis infected with a Wolbachia infection. The infection may be a strain of Wolbachia which does not normally or naturally infect the selected mosquito species. The artificially infected Aedes mosquito can be introduced into a mosquito population to control the reproduction capability of the population by introducing an incompatible Wolbachia infection. The present method can be used as a novel means to limit mosquito-borne pathogens and thus control or prevent mosquito-borne diseases such as Dengue, Lymphatic Filariasis etc.
Summary: Aedes aegypti, arficially infected with wolbachia, can be introduced into a mosquito population to control the reproduction capability of the population. This method can also be used to control mosquito-borne diseases such as Dengue.
Status: Alive

Genetic engineering approach for the control of Aedes aegypti

The most recent research is a self-limiting approach to control the Ae.aegypti mosquito. A company called Oxitec has combined modern biotechnology and advanced genetics to provide effective, safe and sustainable control of Ae.aegypti mosquito population.
Oxitec insects (OX513A) are engineered to contain a self-limiting gene that causes their offspring to die, but the Oxitec insects can live and reproduce normally when they are fed a diet containing an antidote. They also contain a heritable, fluorescent marker that distinguishes them from native pest insects and helps scientists with the management of pest control programmes. Oxitec genetic control works by inserting a gene into the target organism that prevents the insect from surviving to adulthood. The pest control gene produces a protein called tTAV (tetracycline repressible activator variant), which is able to act as a switch to control the activity of other genes. It is a gene variant that has been optimised to only work in insect cells. In the engineered insects, when the tTAV gene is expressed, the nontoxic protein ties up the cell’s machinery; thus, its other genes are not expressed, and the insect dies. The proteins produced are nontoxic to the insects, so if any animals eat them, it would be the same as eating a wild insect — they will be digested in just the same way that all other insects are digested.
This method is species-specific, so the genes do not spread, and the released insects and their genes do not stay in the environment.

[Source: Oxitec]
Fig.3.a: Normal gene expression in Aedes aegypti mosquito

[Source: Oxitec]
Fig.3.b: Expression of the self-limiting gene and production of tTav protein in Aedes aegypti mosquito

There is an antidote given to the insects in the rearing facility that acts like a switch to turn off the tTAV gene preventing the tTAV protein from working. This antidote, tetracycline, an antibiotic, binds to the tTAV protein and disables it. So, in the presence of the antidote, the Oxitec insects are able to survive and reproduce in the rearing facility, but when the males are released into the wild, their offspring cannot access the antibiotic in the quantities needed to survive, so they die before reaching adulthood.10

[Source: Oxitec]
Fig.3.c: Action of tetracycline antidote in self-limiting gene

When tetracycline is added to the larval aquatic diet, it binds and inactivates tTAV, switching off the positive feedback system. The transcriptional machinery is not depleted because only a small amount of tTAV is produced, which does not affect normal cell function, and the insects survive to reproduce naturally in the production facility.

[Source: Oxitec]
Fig.4: Positive and negative feedback system of self-limiting gene

A few salient features of this technology in which GM mosquitoes are produced are:

  1. Amore effective approach as compared to the existing technologies
  2. Safe
  3. Sustainable
  4. Non-persisting in the environment

Oxitec was originally incorporated in 2002 by Oxford University's ISIS Innovation technology transfer company. In August 2015, Oxitec was purchased by U.S.-based Intrexon Corp in a deal valued at $160 million.
Oxitec has developed an innovative new solution, through world class science, to control harmful pests. Oxitec’s solution can help governments and communities around the world keep people healthy in a way that is sustainable, environmentally friendly and cost effective.
The following are some interesting patents related to this technology:

Publication number: US9121036B2
Assignee: Oxitec Limited
Inventors: Luke Alphey
Title: Expression system for insect pest control
Abstract: Promoters active in insects can be enhanced by positive feedback mechanisms and associated with repressible lethal effects
Summary: Positive feedback system produces large amounts of tTAV, which binds to more and more transcriptional machinery, eventually making the transcriptional machinery unavailable for other essential gene expression.
Status: Alive

Publication number: US9125388B2
Assignee: ISIS Innovation Ltd
Inventors: Luke Alphey
Title:Biological control
Abstract: The invention relates to a non-human multicellular organism carrying a dominant lethal genetic system, the lethal effect of which is conditional, wherein the lethal effect of the lethal system occurs in the natural environment of the organism.
Summary: A non-human multicellular organism carrying a recombinant dominant sex-specific lethal genetic system, the expression of a dominant lethal genetic system capable of sex specific lethality, in order to eliminate one sexual entity.
Status: Alive

Vaccination approach to control ZIKV

Bharat Biotech, a company based out of India, started working on a Zika vaccine in November 2014 even before the Zika outbreak in Brazil in April 2015. Bharat Biotech was the first company to file a patent on a vaccine to combat Zika.11
Bharat Biotech has submitted necessary information to Indian Council of Medical Research (ICMR). The company is now looking to seek government approval to expedite regulatory clearances12.
The status of vaccine development for ZIKV is given below:


Outlook on recent technologies used in the control of Ae.aegypti

The US Food and Drug Administration’s Center for Veterinary Medicine (FDA-CVM) has affirmed that genetically engineered mosquitoes (OX513A) have no significant impact on the environment.
Efficacy trials in Brazil, Panama and the Cayman Islands have tested self-limiting OX513A Aedes aegypti mosquito to reduce the wild mosquito population. In each of these trials, the population of Aedes aegypti was reduced by more than 90 percent, which is an exceptional level of control compared to conventional methods such as insecticides.
The U.S. Food and Drug Administration (FDA) gave its support for the biotech company Oxitec to release genetically modified mosquitoes into the Florida islands in an effort to stop the spread of Zika13.
In India, GM mosquitoes were launched on January 23, 2017, in a few districts of Maharashtra for outdoor caged trials. Based on the results, the efficiency of genetically modified mosquitoes to suppress wild female Aedes aegypti mosquito populations that transmit dengue, chikungunya and Zika was found to be 99 percent. Male mosquitoes imported from the U.K. were able to mate with locally available wild female mosquitoes, and the longevity of the imported mosquitoes was the same as that of the wild ones. Gangabishan Bhikulal Investment and Trading Limited (GBIT) and Oxitec plan to conduct open field trials in the country.14
India is also looking at another alternative, such as use of Wolbachia-infected Ae. aegypti mosquitoes to control the spread of Zika, dengue and chikungunya. For this, India has signed a memorandum of understanding with Monash University14.

Conclusion:

In the absence of an effective vaccine that protects humans from Zika fever, Wolbachia-infected and genetically modified mosquitos may prove promising alternatives in the control of mosquito populations and prevent Zika infections.

References:
  1. https://en.wikipedia.org/wiki/Aedes_aegypti
  2. http://www.cdc.gov/ncbddd/birthdefects/microcephaly.html
  3. http://link.springer.com/article/10.1186/s12941-016-0128-2/fulltext.html
  4. http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1000833
  5. http://www.pnas.org/content/109/1/E23/F6.expansion.html
  6. http://uk.reuters.com/article/us-china-health-mosquitoes-idUKKCN10D0MV
  7. http://www.nature.com/articles/srep28792
  8. http://www.aiche.org/chenected/2016/02/gates-foundation-backs-tech-stop-spread-zika-and-dengue
  9. http://www.oxitec.com/our-solution/technology/the-science/
  10. http://qz.com/609291/this-indian-biotech-firm-is-the-worlds-first-to-ready-a-zika-vaccine-for-testing/
  11. http://www.thehindu.com/sci-tech/health/bharat-biotech-seeks-govt-approval-of-two-zika-vaccine-candidates/article8188330.ece
  12. http://www.popsci.com/fda-okays-oxitec-proposal-for-gmo-mosquito-release-in-florida
  13. http://www.thehindu.com/sci-tech/health/GM-mosquito-trials-to-control-dengue-chikungunya-launched/article17093840.ece