Integrated Human Practices
Entente Interdépartementale pour la démoustication du littoral méditerranéen (EID)
EID stand for “Etente Interdépartementale pour la Démoustication du littoral méditerranéen” (i.e. Interdepartmental Agreement for Mosquito control on the Mediterranean coast). They have two major objectives: controlling the nuisance caused by targeted mosquito species, originating from coastal and retro-coastal wetlands and a public health mission, justified by the progressive installation, over the last fifteen years, of the tiger mosquito, a potential vector of arboviral diseases. We, therefore, decided to contact this company to learn more about current mosquito control methods. We then spoke with Grégory L'Ambert, a medical entomologist and head of the "Methods and Laboratory Research" unit at the EID Méditerranée.
During our phone call, he told us that there were about 3500 different mosquito species. Aedes albopictus is a species coming from Asia and passed through Italy before arriving in France. It is the only species that transmits, in Europe, Dengue, Zika, or Chikungunya for example. In France and Europe there are no or few infected mosquitoes. They can only become infected by biting travelers who are themselves infected when they return from a trip.
Today, to control a virus, for which there is no vaccine, transmitted by a mosquito outside of France, the only way to cure a patient is to use a symptomatic treatment under the supervision of health professionals. EID intervenes at the level of prevention by using a treatment with insecticides. EID agents map the regions where tiger mosquitoes are, and if a traveler returns with symptoms, he must undergo blood tests. If the patient is infected, the ARS (Regional Health Agency) asks the patient for all the places he has been to in France since the onset of symptoms. EID then visits the location to search for mosquitoes. If found, a pesticide treatment (one that is authorized in France) is spread within a radius of 150m to avoid further contamination (i.e. a mosquito bite off the sick patient, which will then transmit again). The goal here is to kill the female mosquitoes that are not infected or that are already infected. This is an "archaic solution but the only way to prevent epidemics at the moment" said Gregory L'Ambert.
We also asked Gregory to answer the following question: if you were offered a solution that would not kill all mosquitoes but would still prevent the development of endemic cases, would you use this technology? His answer was mixed. It would be interesting as a professional working with health issues but it is the societal demand that "prevents it". Indeed, mosquitoes will continue to bite and as mosquito controllers, their goal is to eliminate this nuisance as well. Besides, if we wanted to put the project into practice, some critical points are put forward notably due to the creation of a GMO mosquito in the eyes of the French legislation.
We then presented to him our installation for our project to get his opinion. Our idea is to realize “sugar bait”, a sweet nectar loved by mosquitoes, containing our engineered bacteria, in specific environmental spots within a box, to feed the mosquitoes. Moreover, these boxes could release CO2 to attract them. For him, using CO2 would not be effective enough as tiger mosquitoes are usually found in places with high population concentration, meaning they would still have time to bite before eating our bait and are not highly affected by CO2 levels. Therefore the source of CO2 in the terminals to attract mosquitoes will not be enough to attract urban mosquitoes such as tiger mosquitoes. This method would be more suitable while working with mosquitoes present where humans are less concentrated such as Culex mosquitoes (common and nocturn mosquitoes, predominant in France), as they are more sensitive to CO2 because this is what allows them to find their prey (which produce while breathing and sweating).
Furthermore, we also talked about Wolbachia bacteria, not present in A. albopictus, previously used to produce sterile mosquitoes. This endosymbiotic bacterium has its advantages and disadvantages, sometimes it extends the life span or on the contrary, can reduce it. It can affect the capacity of the mosquito to be infected by diseases.
Following this meeting, we were able to change our implementation project. This allowed our project to progress in terms of perspectives of the project in the real life.
Pasteur Institute of Guyana
Mathilde Gendrin is a researcher in the "Microbiota of Insect Vectors" Unit of the Pasteur Institute based in French Guyana. She is particularly interested in the microbes naturally present in the mosquito's organs and in their influence on the mosquito physiology and behaviour. One of the main activities is the study of the implication of the mosquito microbiota in the mosquito's ability to transmit diseases.
The discussion on Zoom started with an introduction on disease transmission by vectors. Most of the vectors discussed were mosquitoes, especially A.aegypti and A.albopictus, which carry Dengue, Chikungunya, Zika. She taught us that the only mosquito acting as a vector is the one able to feed itself with human blood, i.e. female mosquitoes. Indeed, male mosquitoes do not feed on blood but only on nectar.
She explained to us the different stages of the infection of a mosquito. The first step is the entry of the virus through the midgut of the mosquito during a blood meal from an infected person. The virus then multiplies in the epithelial cells. After this first phase of replication, the virus passes into the hemolymph (equivalent to blood in humans) and then into various tissues, to finally reach the salivary glands where the virus multiplies again in the epithelial cells.
We also talked about the definition of the R0 which allows us to determine the efficiency of transmission of the virus by the vector, the mosquito and which we heard a lot about during the COVID-19 pandemic. If the R0 coefficient is greater than 1, the virus will succeed in infecting more than one host causing an epidemic. This coefficient depends on three factors:
the duration of contagiousness after infection
the likelihood of infection after contact between an infected person and a susceptible person
the frequency of human contact
The higher of these three factors are, the higher the R0 will be. If the R0 remains below 1, the virus will infect less than one person per case.
Then, we moved to the microbiota of the mosquito which is the host of a large community of microbes. These microbes can be bacteria, viruses, fungi, protozoa, nematodes, and mites... Numerous studies have focused on bacteria but viruses are also very present in these species. The microbiota of the mosquito evolves according to the stages of growth of the insect: the eggs develop in water, the latter can itself be a source of microbes, colonization can also occur during the laying of eggs, by a transmission from the female. The larva eliminates a large part of its intestinal microbiota just before its transformation into a pupa and its metamorphosis, leading to adults with very few microbes in the gut. Dr. Gendrin, also told us the microbiota of the mosquito could have an impact on the vectorial competition of these mosquitoes, namely it can protect the host via induction of the immune system (1). Conversely, some bacteria such as Serratia marcescens, derived from the intestines of A.aegypti, allow a better vectorial competence of viruses. This bacterium secretes a protein, named SmEnhancin, capable of destroying the mucus layer that normally protects the intestine. By affecting it, the capacity of the virus to infect the epithelium is greater (2).
Finally, we discussed current strategies using Wolbachia (endosymbiont). In the case of A.aegypti in particular, females usually mate only once. If they mate with a male carrying Wolbachia, they are sterilized for life. This strategy allows to "suppress the population”, thus limiting the risk of infection by viruses while reducing the nuisance linked to mosquitoes. This strategy can also be called "population replacement". In this case, males and females carrying Wolbachia are released in very large quantities, so that the bacterium colonizes the mosquito population. This population replacement strategy is currently used, also on A.aegypti, in 12 countries including Australia, Brazil, and New Caledonia.
This exchange with Dr. Gendrin, allowed us to have a better understanding and interpretation of epidemiological data, especially by explaining the R0 coefficient. But above all, during this intervention, we learn more about the microbiota of the mosquito and its implication in the vector transmission of viruses. For the continuation of our project, we decided to use a commensal bacterium of the mosquito. Following the exchange, we are particularly interested in the Asaia genus, naturally present in the adult mosquitoes' gut and in the salivary glands and reproductive organs. The advantage of this bacterium presented by Dr. Gendrin is also its capacity for vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for our design (3).
Heu, K. and al.; Le microbiote de moustique et son influence sur la transmission vectorielle ; Biologie Aujourd’hui ; 119-136 (2018)
Han G. and al. ; Mosquito Microbiota and Implications for Disease Control ; Trends in Parasitology ; 98-111 (2020)
Abbas, R. and al. ; Isolation and identification of Asaia sp. in Anopheles spp. mosquitoes collected from Iranian malaria settings: steps toward applying paratransgenic tools against malaria ; Parasites & Vectors, volume 11; article number: 367 (2018)
Dorothée Murat & Anne Walburger
National Research Center (CNRS)
Anne Walburger and Dorothée Murat are two researchers at the Bacterial Chemistry Laboratory. Anne Walburger works on metalloprotein biogenesis and anaerobic respiration in microorganisms, and Dorothée Murat works on the cell biology of bacterial motility. They also teach biocontainment in Master's degree at the Faculty of Sciences of Aix-Marseille University.
The problem raised during this exchange is the possible leakage of our system in Nature in case of failure of the latter. Some solutions were offered to us: use of auxotrophic genes, systems based on temperature or pH, etc. The researchers at the CNRS - Centre National de Recherche Scientifique - told us pH and temperature methods are not discriminating enough. Together we, therefore, thought about developing a lock system to contain the expression of the toxins.
The "timer lysis currency" system naturally contains a strong lock system, the expression of the lysis genes is strongly regulated and thus allows to minimize a possible resistance to the lysis protein. Therefore, all regulatory systems of this lysis protein should be kept. To lock the bacterial death system as much as possible, it was proposed to use nucleic acid toxins. They could be produced at the same time as the lysis protein, allowing the degradation of the genetic material of our bacteria and thus limiting its dispersion.
Concerning the locking of toxin expression, it would be possible to develop a double recognition system: association of two ligands on a receptor involving the production of the toxin. For this, it would be necessary to work on two virus detection systems allowing in fine the induction of the promoter of the operon by two proteins interacting together.
This exchange took place in the form of a brainstorming session allowing us to think together about the different possible systems that could be used to secure our project. Following this meeting, we were able to apply the advice of the two professionals in the laboratory. This allowed our project to progress in terms of construction and perspectives.
Vet and director of the Diversity Research Foundation
During the festival Lumexplore, we participated in the conference of Hélène Soubelet, a vet and director of the Diversity Research Foundation. She works with researchers on societal issues in order to popularize the results of science. So the conference was about: how a pandemic, such as the Covid-19, a coronavirus, emerged.
In fact, for several years, many researchers have tried to warn us of this risk of pandemics but in vain. The main causes of death in the world are mainly due to our way of life: sedentary lifestyle, alimentation with too much fat / too much sugar, lack of sport, etc… On the contrary, in poorer countries, people rather die of infectious diseases. In 2019 a report was published on “Global Assessment on Biodiversity and Ecosystem Services” which says that each year five new diseases appear, with three which are related to animals, called zoonotic diseases. During the seminar, she highlighted the fact that disease like tuberculosis, Ebola which develops in poor countries, are studied by pharmaceutical industries slower than diseases that first developed in Western countries. So the first reason why these infectious diseases appear more and more is the biodiversity changement due to agriculture, deforestation, etc... (30% of new emergence in the world), and the second one is wild animal trade. Indeed, all around the world, all exploitable soils are exploited, however, these soils contain 25% of the biodiversity. The next question of the seminar was: why in degraded environments do infectious diseases increase? There is a lot of mechanisms to explain that but one of the most obvious is the notion of balance between “dilution of the risk of emergence” (epidemiologic impasse) and the “amplification of the risk of emergence”. And to illustrate this she used the example of Pallusdism which is a vector-borne disease. This parasite is carried by mosquitoes, which are very adapted to anthropized environments. In fact, mosquitoes need water holes for reproduction. But by deforesting humans are creating water holes, these are too small to contain predators, so you will find mosquitoes’ larvae. And also, by cutting trees, they reduce the number of birds that are also predators for mosquitoes. And the last thing is that by cutting trees they increase the temperature of the air so mosquitoes’ larvae could be bigger and more resistant.
So this seminar permits us to understand the role of environmental degradation and the emergence of new viruses in the world. But above all, during this presentation, we learn more about the importance of biodiversity and the mechanism of vector-borne disease spreading.
Professor Bruno Coutard
UMR190 Emerging Viral Diseases, Faculty of Medicine Aix-Marseille
Bruno Coutard, researcher at the Faculty of Medicine at Aix-Marseille University, is working on emerging viral diseases. He also teaches virology and bacterial genetics at Polytech Marseille Engineering School.
During our meeting, Pr. Coutard gave us an overview of arboviruses and possible solutions to fight against them.
We learnt that arboviruses come from Africa and are today present on the entire planet. In France, Aedes aegypti and Aedes albopictus are vectors of arboviral diseases, yet A. albopictus is the most present in the country and the only mosquito described as tiger mosquito, that is why we chose to work on it.
Nowadays, there is no vaccine against Chikungunya and Zika viruses, yet one is commercialized for Dengue: DENGVAXIA. However, it is very controversial (risk of making people sicker, no protection, children deaths after vaccination…). Overall, vaccine production against arboviruses is very complicated.
After we exposed our project, we talked for a long time about our experimental strategy. ConA protein has a great affinity for glycosylation on the viral membrane. Pr. Coutard was really enthusiastic about our idea and genetically engineered bacteria
After the wetlab, we, unfortunately, faced an issue with our system: the specificity of the recognition of the virus and of the signalization pathway. For the detection, ConA is not specific enough and can recognize various viruses. As mosquitoes are often infected by insect viruses, this can be highly inconvenient. Thus, our system would not work because all the mosquitoes would die because engineered bacteria would always recognize a virus. In the signalization pathway, TonB is a protein that can react via quorum sensing mechanisms, which can be a problem when the bacterium is in the mosquito midgut, surrounded by plenty of bacteria using the same communication system. We exposed those issues to the professor, to find together a more specific recognition system to avoid an uncontrolled activation of our signalization. Pr. Coutard suggested us to use nanobodies, also called vHH. They are small antibody fragments from the variable part, with high specificity to a chosen target. Some epitopes are conserved within the Flavivirus genus, and could allow us to target a wide range of arboviruses.
In our design, we could use two different vHH:
One could be located on the bacterial membrane, replacing ConA. This vHH could be produced to recognize specifically a viral peptide from the targeted virus (epitope from a flavivirus for instance). More in detail, the recognition site will be hidden thanks to a linear viral epitope. The latter must have a lower affinity than the viral peptide of interest. In this condition, when a virus has infected a mosquito, it will induce the detachment of the linear viral epitope and will be able to bind with a strong affinity to membrane vHH. The purpose is to increase viral recognition specificity and avoid unintended system triggering.
The second vHH could be used for signalization. The initial idea is previously explained. From this, TonB will be engineered, as well as the designed peptide. The latter will keep the interaction loop domain and the ConA interaction domain, this time specifically for a vHH. The middle domain will be changed into an epitope-specific for the second vHH. The latter will replace the external part from TonB (green). In summary, after the first step of the viral recognition, the interaction loop domain will be detached from vHH interaction site, the specific epitope will bind specifically to the second engineered vHH to induce a phosphorylation cascade thanks to TonB internal part. As long as the recognition is specific, phosphorylation is no longer necessary to be specific. Consequently, our system will be more specific to trigger bacterial lysis and further mosquito’s death.
Together, with Pr. Coutard, we were able to design a novel system, more specific, easier to produce, and with the highest security.
To end the interview we wanted to have his opinion on our system in the distant future. According to him, there are different subjects: ecology, genetics, health… For the ecologic and genetic aspects, we must be careful because we aim at introducing engineered bacteria in the mosquitoes’ gut, meaning we introduce new genes in the environment. Some cases of recombination in natural host bacteria could happen. As mosquitoes are part of the food chain, their major predators, bats, could ingest the recombinant strain (transgene ingestion) containing our engineered bacterial DNA. The latter will be disseminated uncontrollably in the environment and could impact more than the targeted mosquitoes.
Moreover, when we think about numbers of autochtone cases in France, there are still few of them detected per year. Consequently, our technology might not be interesting in our country considering its cost. However, the ARBO-BLOCK system might have a higher interest for countries overrun by arboviral-vectors and the disease they caused. For the human health aspect, there are still few cases of autochtone infections and infected patients still, mainly from abroad. So, our technology could be hard to sell in France and seems more adapted to another market. Our project is, therefore “interesting and relevant with this approach” but could face issues regarding legislation.