Using Gene Drives To Change Mosquito Sex In Fight Against Zika, Dengue and Malaria – Molecular Biology is being used to perform genetic manipulation on the sex determining genes of disease carrying mosquitoes. This article explains how and why it is being done and also discusses some of the ethical implications for the human race and the future of life on this planet.

Ethiopian Child With Malaria - 438,000 were killed in 2015 by this killer disease - mainly in Africa, mostly children (W.H.O.) — Ethiopian Child With Malaria – **438,000 were killed in 2015** by this killer disease – mainly in Africa, mostly children (W.H.O.)

On Feb 17th 2016 researchers Zach Adelman and Zhijian Tu published their discovery of Nix, a sex gene in Aedes aegypti mosquitoes. These are the mosquitoes that transmit virus diseases to humans like Zika, Dengue Fever, Yellow Fever and Chikungunya. Their findings may also be applicable to the Anopheles mosquito that transmits malaria – a disease which killed 438,000 people in 2015 with 214 million new cases reported – mainly in Africa (figures from the World Health Organisation’s 2015 Malaria report).

What is the Nix Sex Gene?

As demonstrated in the research paper by Messrs Adelman and Tu it is possible to perform gender conversion for the Aedes aegypti mosquito. Their work identified the Nix ‘sex gene’ which produces a protein very early in the development of the mosquito to programme its development as a male. The mosquito develops as a female if this gene is not sufficiently expressed (in other words the sex protein that it generates is either not produced or produced incorrectly). To demonstrate the existence and influence of Nix on mosquitoes the researchers used the latest highly specific gene manipulation technology called CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats associated with the DNA cutting enzyme Cas9). This is a process that evolved in bacteria as an immune system to defend them against bacterial viruses – it can be used for very precise editing of chromosomes.

Using CRISPR-Cas9 to knock-out the Nix gene, genetically ‘male’ mosquitoes could be feminized and developed female genitals and antenna (which are not found on males). The converse occurred in genetically ‘female’ mosquitoes into which the Nix gene was ‘injected’ and made to produce it’s protein. These female mosquitoes developed both external and internal male genitalia (testes and associated male sex glands). Thus the Nix sex gene was used to successfully ‘gender reassign‘ these Aedes aegypti mosquitoes.

Why is mosquito sex important when trying to combat killer diseases?

One of the most effective ways of combating killer diseases spread by mosquitoes is to reduce the population of the insects involved (e.g. Anopheles mosquitoes for malaria and Aedes aegypti mosquitoes for Dengue, Yellow Fever, and Zika). The use of insecticides is becoming less effective due to increasing mosquito resistance. Physical approaches like eliminating the sources of standing water that provide the mosquitoes with breeding grounds need to be very thorough and strictly implemented to have a significant effect.

Spraying kerosene oil in 1912 - a less effective approach to dealing with the global malaria problem. — Spraying kerosene oil in 1912 – a less effective approach to dealing with the global malaria problem.

It is the female mosquito that feeds on human blood and is therefore responsible for transmitting the diseases mentioned above. The male of the species does not feed on human blood, preferring to derive its nutrition from nectar. Because of this scientists are particularly interested to find ways of reducing the number of female rather than male mosquitoes.

Some authorities are experimenting with releasing sterile male mosquitoes into the wild. These compete against normal ‘wild’ male mosquitoes to mate with females. Since the female only copulates once in her lifetime the number of offspring can be reduced if sufficient sterile male mosquitoes are released. To make them sterile the male mosquitoes either have their genes modified by radiation or they may contain a ‘killer’ gene which prevents them from producing fertile offspring. Some problems with this approach are:

Only males should be released (any females in the release batch would increase the number of disease carriers and therefore must be identified and destroyed)
The sterile males have to be released in large enough numbers to have an impact when competing with the fertile males that already exist in the wild
The release must be part of a continuous programme – any reduction in the numbers released would allow the remaining native fertile males to mate, multiply and produce more females.
The Aedes aegypti mosquito – vector of Zika, Dengue Fever, Yellow Fever and Chikungunya

The advantage of this sterile male approach over the use of chemical and biological insecticides (like bacteria that kill mosquitoes) is that it can get more effectively to areas with lower concentrations of mosquitoes – the released males are pretty keen on finding a partner to mate with – the fewer the number of ladies – the harder they look!

Gene manipulation approaches such as those based on the use of the Nix sex gene could be designed to address the issue of removing females from the release batch (all mosquitoes are ‘converted’ to males – thereby doubling the number of potential ‘male’ partners in the batch). The approach can be further developed to ensure that copulation with a released male will only produce more males (no female offspring). Alternatively it might be possible to ensure that any female offspring die (using a ‘lethal when female‘ gene combination). These developments involve what is termed a Gene Drive.

What is a Gene Drive?

The purpose of a Sex Gene Drive is to increase (‘drive-up’) the ratio of non-blood sucking males in the mosquito population. The term gene drive is used when a genetic attribute (like maleness) in a population is increased (or decreased) compared with the natural levels expected (e.g. for maleness the target is to have more then the usual 50% male offspring). A gene drive is inherited from generation to generation so for each mating cycle the relevant gene (e.g. Nix) continues to have its effect (increases the number of males) until eventually all offspring have the gene and there are no females left. If successful this mosquito species disappears.

The advantage of using gene drive approaches is that a smaller number of mosquitoes need to be released because the new attribute (e.g. to be male) is self sustaining and increases in the population from generation to generation. As well as trying to influence the proportion of males in the mosquito population (as with ‘driving up’ the Nix gene) other genes could be used in gene drives such as those which make the mosquitoes resistant to being infected by the specific parasite or virus that causes harm to humans (e.g. the malaria parasite or Zika virus). Researchers are also trying to develop gene drives which reduce the ability of female mosquitoes to have offspring.

A gene drive to create males of the Anopheles gambiae mosquito (which transmits malaria in Africa) is close to being field tested by Austin Burt and colleagues in Africa. These mosquitoes are like humans in that their sex is determined by the possession of X and Y chromosomes (XX = female, XY = male). Normally half the mosquito sperm would have a Y chromosome and half would contain an X. All the eggs produced by the female have a X chromosome so upon mating you would then have a population of half XX (female) and half XY (male). The altered mosquitoes with the gene drive only produce sperm containing a Y sex chromosome.

Using a meganuclease based gene drive system Burt and his team have produced Malaria transmitting mosquitoes in which all the males only produce sperm with an Y chromosome and any females mating with these mosquitoes will only produce males. This trait is then ‘driven’ into the next generation so that any wild females mating with these ‘altered’ male offspring also only produce males. Eventually this would destroy the population due to lack of any remaining females (this project is funded by the Gates Foundation).

Among the gene drives being developed using the CRISPR/Cas9 techology one also reduces female mosquito fertility and in another case the mosquito is given a gene to make it resistant to the parasite that causes malaria (protozoa belonging to the Plasmodium family). This is in addition to a potential drive incorporating the Nix male sex determining gene to drive an increase in populations of wild Aedes aegypti (Dengue, Zika, Chikungunya, Yellow Fever) and Anopheles gambiae (Malaria) mosquitoes. CRISPR Associated Protein see Sex Gene Drives in Mosquitoes by Chris Duggleby

Ethical Considerations

Gene manipulation of species released into the wild is a very controversial subject. The debate about genetically modified crops has received considerable press and political attention. Crops can’t fly on their own! The idea of releasing genetically modified mosquitoes into the wild will certainly need to be thoroughly researched and given very careful consideration. The scientific community is clearly not ready for such a step today but it will be within the next 5-10 years. The speed of developing and testing gene drives is dependant on the life cycle of the species involved – the mosquito life cycle is very short so a system can be tested in a couple of years (a human gene drive would require decades or centuries to fully field test because we are much older before we reproduce).

One of the particularly worrying things about using gene drive technology is that it does not just change the current generation but the technology alters all the offspring for generations to come. Once released into the wild it will be impossible to stop working in the offspring that inherit the new genes. We would be altering the make up of the natural gene pool over time. Some processes are being designed which could neutralise the impact for example by using another gene drive to ‘re-inject’ and drive back up the levels of a ‘corrected’ gene in the population. However, these systems would not remove any of the changes to organisms in the wild that have already been created by an earlier ‘rogue’ gene drive. The neutralizing systems would be aimed at diluting the impact of these changes on the ecosystem by adding large numbers of ‘corrected’ organisms.

Those in favour of using such new technologies will want to take advantage of their potential to save lives (like the nearly half a million lives lost to malaria each year – mainly young children). Human activities have helped these mosquitoes to enter into new regions of the World and some will argue that eliminating the insects from these new areas will simply be reinstating the natural state. One of the reason Zika has been so successful in spreading throughout the American continent is that it is a new disease here and the local population has not previously been exposed to the virus. There is therefore no natural immunity (much like the small pox, influenza and measles that Europeans introduced into the Americas, Australia and Africa killing huge numbers of the indigenous populations on those continents).

Fortunately the responsible members of the scientific community are very wary of proceding with field trials of these new techniques until the ethical and regulatory issues have been thoroughly discussed and a consensus with both authorities and the general public is reached on the most appropriate way forward. However these tools are so powerful that it may only take an accidental release of an ‘engineered’ organism into the wild to have a potentially devastating impact on the genetic composition of the native species.

Of particular concern is the potential use of this technology by a rogue state or terrorist organisation for the purposes of biological warfare or to simply terrorise those of opposing beliefs. One of the characteristics of the CRISPR/Cas9 system is that is is relatively easy to design and use compared with previous genetic engineering systems. Laboratories with basic skills in modern molecular biology and genetic engineering could (and are) developing these organisms.

My personal fear is that we could be releasing into nature a powerful self replicating system – if we can add passenger genes to gene drive systems surely it is conceivable that other genes could try and hitch a lift. For example cancer genes – those involved with cancer biology will be well aware of how some viruses have managed to insert human type oncogenes (cancer causing genes) into their genome (For techies: I am thinking here of the postulation that a non-carcinogenic evolutionary predecessor of the Rous Sarcoma Virus incorporated the src oncogene into its genome – converting it into the cancer causing agent of today). It would be a cruel outcome if a genetic device that we introduce into nature to save millions of lives is able, through mutation or genetic recombination, to itself become a powerful pathogen. Clearly we need to ensure any safeguards are foolproof and novel systems are thoroughly tested.

We have reached a very serious junction in which humanity could either wisely develop systems to eradicate painful and deadly diseases or destroy the balanced ecosystem upon which we depend. Watch this space!

Chris Duggleby