Rosario Gil García is a professor at the University of Valencia (UV) in the Department of Genetics and a researcher at the Institute of Integrative Systems Biology (I2SysBio), a joint centre of the UV and the Spanish National Research Council (CSIC). In this interview she explains the development of a new biological control measure to reduce the Asian tiger mosquito population in Valencia using the bacterium Wolbachia. Rosario Gil directs the molecular research of two projects, one of the Valencia City Council and the other of the Ministry of Science and Innovation, with this objective.
How is the collaboration with the Valencia City Council on your project?
The City Council was interested in controlling the mosquito plague because it has been very annoying in recent years. Not only because they can transmit diseases, but the bite is very annoying and Valencia is a tourist city. Within the control they have of all insect pests, this is one of the most important one, that of the common mosquito and the tiger mosquito.
What diseases can Asian tiger mosquitoes transmit?
The Asian tiger mosquito, Aedes albopictus, is not the primary vector of many diseases. It is more common that they arrive with the African tiger mosquito, Aedes aegypti, which transmits many viruses such as chikungunya, Zika, dengue, Nile fever; that is, quite serious diseases. The Asian tiger can also transmit them, so it is preferable to control its population in advance to prevent the infection of the tiger mosquito population established there by any of these viruses, thus avoiding their transmission.
"Wolbachia is an insect endosymbiont, that is, bacteria that live inside insects in specialised cells"
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"Cytoplasmic incompatibility means that if a male carries a Wolbachia and mates with a female carrying another type of Wolbachia, the embryo is sterile and will not be born"
Could the ability of Asian tiger mosquitoes to transmit diseases become a public health problem on the peninsula?
At the moment we do not think so, but it cannot be ruled out because the present situation as a result of climate change has led to the invasion of the Asian tiger mosquito here. Therefore, any chance of any of these viruses becoming established could be a problem. Furthermore, there have been cases and not sporadic cases brought from outside, but some autochthonous ones. In recent years, there have been several initiatives on a local and global scale to try to use different biocontrol systems for mosquitoes, thus avoiding pesticides, given that these can affect other insects and can also be harmful to people. Therefore, if we achieve biological control, it will always be preferable.
What biocontrol systems are being carried out?
One of the biocontrol systems that has been carried out for some time against the tiger mosquito plague is the use of the bacterium Wolbachia. This bacterium has sometimes been used to prevent viruses from being transmitted and other times to prevent mosquitoes from reproducing. In Valencia, as we currently do not have virus transmission, we are trying to emulate a study carried out in the city of Rome to control the Asian tiger mosquito population by implementing the technique to achieve this purpose. However, the results will not be immediate, since some additional studies are needed. We have been characterising the mosquitoes in the city of Valencia for a few years to subsequently find the best solution against them. In short, in Valencia we are developing a system to combat the tiger mosquito plague with Wolbachia.
What is the Wolbachia bacteria?
Wolbachia is an insect endosymbiont, that is, a bacterium that lives inside insects in specialised cells and from within can do good or bad things. It’s my research line. For example, Wolbachia, depending on the insect where it is established, can be good and necessary to manufacture nutrients for the insect host that they do not have in their diet. In other cases, it is the other way around and the bacterium takes advantage of the host.
In the case of mosquitoes, Wolbachia, like most endosymbionts, is always located near the female’s ovaries to be transmitted from one generation to the next, directly through eggs or embryos. So what this bacterium does in the case of the tiger mosquito and the common mosquito, Culex pipiens, is infect the females for its own benefit. This is known as a reproductive parasite. Some of the consequences it causes are reproductive alterations that can range from eliminating the males because since it is the females that transmit it, then there would be more females; feminising the males, that is, both genetic males and females act as females that transmit the bacterium, and another of the alterations it causes is cytoplasmic incompatibility.
"We want to find a Wolbachia that is incompatible with the one carried naturally by tiger mosquitoes, produce large numbers of tiger mosquitoes infected with this new incompatible Wolbachia, and release males into the environment to mate with females"
What is cytoplasmic incompatibility?
Cytoplasmic incompatibility means that if a male carries a Wolbachia and mates with a female carrying another type of Wolbachia, the embryo is sterile and will not be born. The male transmits a toxin in the sperm and the female carries the antitoxin in the cytoplasm of the egg. If the antitoxin is not the corresponding one, it does not neutralise the toxin and kills the embryo, and this is what we are trying to achieve.
The Wolbachia carried by the common mosquito is incompatible with the Wolbachia that normally infects the tiger mosquito. In our research, we want to find a Wolbachia that is incompatible with the one carried naturally by tiger mosquitoes, produce large numbers of tiger mosquitoes infected with this new incompatible Wolbachia, and release males into the environment to mate with females.
Why are only males released?
We release only males because they do not feed on blood, only females do so when they are “pregnant”, to allow their embryos to develop. When the infected males meet the female, they will mate and the embryos that emerge will die, thereby preventing them from reproducing and reducing their population. The use of Wolbachia as a cytoplasmic incompatibility tool has an advantage over another system that uses the release of sterile males that have been irradiated. Irradiation can affect their phenotype, meaning that females are not as attracted to them.
When mosquitoes carrying Wolbachia are obtained, is there any regulation for their release?
The use of Wolbachia in tiger mosquitoes does not generate transgenic mosquitoes, therefore, it is not a problem and although Wolbachia has certain host specificity, it can infect both vertically, that is, from mother to offspring, and horizontally, that is, from one another. So we are not doing anything that is alien to nature. Yes, you need a release permit, but it does not require any special permit. In addition, it has already been done in Italy and is being done in other parts of the world to prevent the transmission of viruses through mosquito bites. For the first release, a suitable space will have to be found to do a pilot, controlled trial and see if it works well.
"The use of Wolbachia in tiger mosquitoes does not generate transgenic mosquitoes"
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"Instead of bringing us an Italian Wolbachia from an Italian mosquito we could do it with a Valencian Wolbachia and a Valencian mosquito"
When did this project start?
It is a project in which the Valencia City Council, the Department of Parasitology of the University of Valencia and the company Lokímica, specialised in pest control, collaborate. I didn’t start it. My colleagues started the project and were trying to characterise the mosquitoes with the collaboration of an Italian group to try to implement the same cytoplasmic incompatibility system here as well. The idea was that instead of bringing an Italian Wolbachia from an Italian mosquito we could do it with a Valencian Wolbachia and a Valencian mosquito. Since I am dedicated to endosymbionts, they asked me if I could characterise the Wolbachia that we have naturally here and I said yes.
How is the project developing?
What they did within the scope of the project was to collect specimens of common mosquitoes throughout Valencia so that I could characterise whether or not they carried Wolbachia and what type of Wolbachia and see if they were compatible or incompatible with those of the tiger mosquito. With this we began to work and, since we did not have money to hire personnel to do the molecular analysis, what we did was design final degree projects. The students learned to do the entire process of DNA isolation, characterisation using PCR and sequencing, a round project and a very complete training. I had not thought about working with Wolbachia before until I was asked to characterise one because the research team wanted to have a native Wolbachia.
Currently, we are not only working with the Wolbachia of the common mosquito, but we are also characterising the Wolbachia of Drosophila melanogaster, which is also incompatible with that of the tiger mosquito. These fruit flies have been caught by staff from the Cavanilles Institute of the University in Requena during the harvest. We are seeing which of the two bacteria we end up with. So, on the one hand, we have people from the Department of Parasitology curing the tiger mosquito of its natural Wolbachia. And, on the other hand, we are characterising the bacteria of the common mosquito and that of Drosophila. The next step would be to inject the one we select into the tiger mosquito before releasing it.
How was the common mosquito sampling carried out?
The mosquitoes arrive dead to me. A series of traps are set in different districts of Valencia or from nearby coastal areas where there is a greater probability of finding mosquitoes: the Ayora Garden, the Monforte Gardens, Moncófar or cemeteries with vases where there are flowers in which water accumulates and the females deposit their eggs until that hatch. The eggs are normally collected, they hatch in captivity and when they have the adults, they separate the males and females to put them in small flasks that are stored at -20oC for a few days. That’s what gets to me. In each small flask there are approximately between 20 and 40 males or females. From here, we analyse them one by one and extract the DNA to check if they carry Wolbachia.
What is the objective of this measure?
Try to reduce the tiger mosquito population with controlled releases. If everything goes well, we believe that next year we can do the first controlled release in September and see that it works. Then, in the next campaign, what would be done is releasing males in different doses. However, we experimentally do not have the capacity to produce large quantities of mosquitoes because we have small facilities. The City Council intended, and I hope it will continue to do so, to launch a larger facility. There is a site with possible facilities that could be prepared for breeding mosquitoes, both those cured and those infected with incompatible Wolbachia.
"We believe that next year we can do the first controlled release in September and see that it works"
What objectives have already been achieved?
At present, we have characterised the Valencia tiger mosquitoes, some Culex to be able to extract Wolbachia from them and the Requena Drosophila to be able to use them as well. All this in terms of molecular characterisation to detect what we want to introduce. Furthermore, in the laboratory we have tiger mosquitoes that carry the native Wolbachia strain and we have mosquitoes cured of it. Therefore, we are at the point where we would have to introduce the new Wolbachia into the eggs. Now we need a micromanipulator to introduce the bacteria into the eggs and hire the person who has to do it.
What stage(s) in the project rehearsal do you consider critical?
The truth is that we are solving all the problems that arise, but there is still a lot to do. As soon as we have the micromanipulator we would have to do thousands of injections to get mosquitoes with the new Wolbachia. Additionally, we would need the females they mate with to be naturally infected (which they seem to be) to achieve incompatibility and we would also need to see if it works in nature. Another limitation is that we cannot do it at any time of the year, but it has to be done at the end of summer with sufficient numbers of mosquitoes.
At this moment, where is the agreement with the Valencia City Council at?
We have just signed a new agreement with the city council. It's a small agreement, but everything related to the facilities is further behind. Everything that is characterisation will continue. The idea is to keep the agreement alive because we need to hire personnel who are maintaining the mosquito populations and we also need to move forward with larger facilities.
"In the laboratory we have tiger mosquitoes that carry the native Wolbachia strain and we have mosquitoes cured of it. Therefore, we are at the point where we would have to introduce the new Wolbachia into the eggs"
Are there any aspects that could be improved or negative in your research?
Everything is going well at the moment, but we are always fighting for money, material and personnel. It would be easier if we had a large center dedicated to this and we are a small group with a very limited number of personnel. Therefore, we go step by step.
What is your contribution within the research team?
To carry out the characterisation of the bacteria and if at any time it is necessary to also to characterise the insect with molecular markers. The idea would be that when we select a Wolbachia and since I also work in genomics, we would do the genomic study. With this we could have the genome of a Valencian Wolbachia, which would have a Valencian name and we could publish it and deposit it in the databases. We are also studying communication between the host and the symbiont. What we want to study is whether there is trafficking of molecules that allow communication between Wolbachia and the insect.
"We could have the genome of a Valencian Wolbachia, which would have a Valencian name and we could publish it and deposit it in the databases"
What does the contribution of collaboration with Italian researchers mean to your research team?
At the moment there is a person from the parasitology team who is doing his international thesis, co-directed with a member of the Italian group that carried out the study in Rome. He has spent some time in Italy learning all the techniques and now what we are going to do is apply them here.
What long-term goals do you have in research with Aedes albopictus? What do you think will be the distribution of this new mosquito in the country in the near future?
We are going to have Asian tiger mosquitoes throughout Spain, especially in areas where there is more humidity. This year the tiger mosquito population has been delayed because we have had a very dry spring. However, in the months of June and July with rainy days, the reproduction of this pest has increased. I am working on other model systems and the techniques that are used to try to understand how the insect and the host communicate with each other can also be applied in this research and I love this. From here you can do molecular studies, microscopic studies, studies that allow you to better characterise the system and this may also help fight the pest in a different way.
What is the potential for success if we apply this new tool?
In places where this tool has been used, the growth of mosquito populations has been significantly limited.
If mosquitoes with Wolbachia were stopped, would the Aedes albopictus population without this bacterium slowly return to the levels at which they were established?
We want releases to continue to be made and, if possible, alternative releases to be made so that there is never a problem of sudden appearances of a population with the new Wolbachia. So we must continue to make controlled releases regularly, but it is not expected that the less frequent one will stabilise.
"In places where this tool has been used, the growth of mosquito populations has been significantly limited"
In addition to reducing the population of tiger mosquitoes, can the biological control measure with Wolbachia also reduce the capacity for disease transmission when mosquitoes carry this bacterium?
Yes, we have seen that mosquitoes infected with Wolbachia have less capacity to transmit viruses and there is probably an incompatibility between them. In the case of dengue in the African tiger mosquito, unlike the Asian tiger mosquito, it rarely has Wolbachia. And by introducing the bacteria in your system you can stop dengue; The number of mosquitoes is not reduced, but dengue cannot be transmitted.
“All areas of science are equally important”
What advice would you give to future researchers in the area of invasive arthropods?
All areas of science are equally important. You can never know what is going to be really important because when you discover some natural phenomenon or some explanation or relationship between living beings or even physical aspects of nature, you never know what the application is going to be. For example, Einstein was awarded the Nobel Prize for the photovoltaic effect and not for the theory of relativity. I believe that he had not thought at that time that it would be used to make solar panels to obtain energy. Open curiosity is very good and if you also have a problem that you want to solve and you are looking for a way to solve it, it also helps you, but it is still curiosity to solve the problem. That is, understand it, define it and then solve it.
In addition to being part of the Asian tiger mosquito research team, are you also carrying out other research projects?
At this moment, I am the main researcher of a Ministry project called “Host-symbiont communication and its usefulness in the biological control of pests and pathogens (SYMB-CONTROL)”. The main study model that we have established in the laboratory is the German cockroach, Blattella germanica. We are using this model system because it has an endosymbiont but it also has an intestinal microbiota and, since it eats almost the same thing as us, its intestinal microbiota is quite similar to ours.
We are working to understand how this intestinal microbiota is established and how we can manipulate it because it can allow us to understand aspects of the human microbiota. We want to study if the microbiota of the cockroach Blattella germanica contains antibiotic resistance genes and if it could be a reservoir for this type of genes that are, of course, bad for us. Other research that is being carried out is to see if they can also be vectors of bacteria that may be pathogenic. Therefore, they are all aspects that are related to human health.
"We want to study if the microbiota of the Blattella germanica cockroach contains antibiotic resistance genes and if it could be a reservoir for this type of genes"
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"We are studying antimicrobial peptides that could be an alternative to the use of antibiotics that can keep some bacteria at bay"
And what status is this project in?
We have obtained microbiota-free cockroaches, so we can see what happens when they do not have intestinal bacteria. We also want to see how the insect maintains control of the different bacteria that live in the intestine and those that live in specialised cells without them mixing with each other. In addition, we want to see if they communicate with each other or if they provide any benefit to the host. To do this, we are studying cockroach transcriptomes, which can be from the entire individual or just from the intestine. In another parallel project, we are also looking at whether different stress conditions related to temperature can affect them, to see how the microbiota is modified in response to stress.
On the other hand, we are studying antimicrobial peptides that could be an alternative to the use of antibiotics that can keep some bacteria under control. There is increasing resistance to antibiotics and small peptides generated by the body itself have been detected, which are part of the innate immunity of insects. We are trying to detect what the antimicrobial peptides are in our system. In addition, we want to see how the host sends signals to the microbiota and vice versa, using extracellular vesicles as a connection system. It is a system in which even RNA can be transmitted. We have isolated extracellular vesicles from cockroach hemolymph and are analyzing their cargo, which is what the vesicles carry inside.
You are also coordinator of the doctoral program in Biodiversity and Evolutionary Biology at the University of Valencia. What role does she play as a doctoral coordinator?
It is a programme with ten lines of research in which anything related to biodiversity and evolution is studied, both at the molecular level and at the functional or ecological level. I coordinate the academic committee that evaluates people who want to access the programme and checks that they meet the requirements to be admitted up to the limit that the program allows us. We evaluate research plans and progress reports and ensure they meet appropriate standards. So, when the time comes to deposit the thesis, we are the first filter that allows us to see if the thesis is ready to be deposited. We review that the committee of experts that has been proposed is adequate to evaluate the thesis.
"The first value, and the most important in research, is curiosity"
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"Other values in research are vocation and a lot of patience"
What personal values do you consider important in your work?
The first value, and the most important in research, is curiosity. If you are curious, you try to find the answer to things, you look for information, the bibliography and then you seek to answer those questions that are not yet described in the bibliography. For that you need to be curious. Of course, you have to have a vocation because this requires a lot of effort and many hours of work. Without a vocation it is very difficult to concentrate on something in which you will not always see the result easily. Therefore, other values in research are vocation and a lot of patience, which is also one of the things that are important.
What are you most passionate about in your area of research?
What I like is research, in general, because I am curious. Anything that means learning. And all biological phenomena seem fascinating to me. I always make the joke of saying that I am a microbiologist by de-formation because in reality I believe that it is very difficult to define a science by the size of the object of study. From the beginning, I started doing things with microorganisms, first with yeast, and then I've been working with bacteria, but I’ve done very different things with all of them and I’ve always liked everything I've been doing. At first I studied the yeast wall to find a target with which we could attack fungal infections, which is what was done at that time in the Department of Microbiology of the Faculty of Pharmacy. Afterwards, I went to the United States to do my postdoctoral stay and entered a human genetics laboratory using yeast as my model system to study tumor suppressor genes in humans.
"I entered a human genetics laboratory using yeast as my model system to study tumor suppressor genes in humans"
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"In 2001 I started working in the evolutionary genetics laboratory and since then I have continued with this line, which is currently my passion"
And when you return to Spain?
When I returned to Spain I did not have the opportunity to work in a human genetics laboratory, but yeasts did give me scope and I started working first with brewing yeasts and then with wine yeasts. However, the race was interrupted by difficult moments to stabilise researchers. That was hard. I applied for a position to work at the Cavanilles Biodiversity Institute of Evolutionary Biology in an evolutionary genetics group. I am a pharmacist and had never studied genetics or evolution, and I got it. I completely changed what I was working on, I stopped focusing on yeasts to start working on insect endosymbiont bacteria, which at that time was something I didn’t even know what it was. It was also the moment when genomics began to emerge to study entire genomes instead of studying individual genes and I got into this line. In 2001 I started working in the evolutionary genetics laboratory and since then I have continued with this line, which is currently my passion.
Any memorable anecdotes about your work that you would like to share?
Every day there is some anecdote. Right now, I remember when my reinstatement contract had expired, I went home and told my husband, “Well, I will study for public examinations to be a high school teacher”. He told me “You? “You will die, you need to do science like breathing.” I understood that my husband had realised that I needed it, which is very good because my husband came with me to the United States, but he is not a scientist. Another anecdote is that I had a programmed daughter – like almost all female scientists who need to have programmed children– before reading my thesis, because what I didn’t want was to be breastfeeding her at the time I went abroad to have a postdoctoral stay. My husband was a civil servant, so he could ask for a leave of absence to be able to accompany me and take care of the baby. We were very lucky because he was able to ask for “maternal leave”, and I say maternal and it is not a mistake because it was the first year that a man could ask for it. The form said: “the civil servant mother” and then my husband’s name “has requested permission to care for a child under three years of age”. Subsequently, he requested a leave of absence for two more years. We spent three years abroad and he was the housekeeper. The state where we were was quite sexist and when he said that his profession was a housekeeper, people would stare at him and he would explain that his wife was the one who worked. They called my husband Mr. Gil because since I am Rosario Gil, obviously he had to be Gil and I was the one who had changed my name.
