Of Mosquitoes and Men

In Strasbourg, the insectarium at the Moustiques laboratory is all shiny and new. It was inaugurated in 2018 and employs ten scientists. It also houses thousands of mosquitoes.

Most of the “residents” at Strasbourg’s insectarium are mosquitoes belonging to the Anopheles genus. Anopheles mosquitoes are vectors for Plasmodium, a group of parasites that cause malaria. As one of the “three great killers” alongside AIDS and tuberculosis, malaria affected no less than 219 million people in 2017, killing 435,000 people primarily in sub-Saharan Africa. The insectarium also houses colonies of Aedes mosquitoes, the vectors of the dengue, Zika, and Chikungunya viruses. These mosquitoes are not just flying syringes waiting to inject humans with parasites and viruses in exchange for a blood meal. “They also fight pathogens,” explains Stéphanie Blandin, who heads the Immune Responses in Mosquitoes team*. “Thanks to their specific genetics, some of them are able to block the development of the pathogens at the beginning of the infection. This means that they will not transmit the disease at their next meal.” These pathogen-neutralizing capacities are of interest to the specialists in Strasbourg. “The idea is to study which genes are involved in determining resistance or sensitivity to parasites in Anopheles mosquitoes, and in the antiviral response of Aedes mosquitoes. Once the genes have been identified, we develop tools that can be used to genetically modify the mosquitoes. This is known as transgenesis, and the purpose is to understand how they interact with pathogens according to their genetic baggage,” adds Blandin. The result could be the production of mosquitoes that are incapable of transmitting diseases to humans. Such mosquitoes could be released into the wild with the goal of supplanting infectious mosquitoes. This approach, which has never been tested on a real-life scale, must be proven to be not only effective, but also harmless.

  • This mosquito larva looks fluorescent when observed under a stereo microscope, proving it is a transgenic specimen. When a gene is inserted into or deleted from its genome, the larva also receives a coding gene to produce a fluorescent protein. The protein can be red, green, or blue, creating a distinction that researchers can use to categorize individuals according to which genetic modification they underwent.
    © Inserm/François Guénet
  • Éric Marois, a research officer who works with Stéphanie Blandin, head of the Immune Responses in Mosquitoes team at the Strasbourg laboratory. Since 2006, the researchers have been working on the antiparasitic response of Anopheles gambiae, one of the mosquitoes that spreads malaria.
    © Inserm/François Guénet
  • Breeding Anopheles mosquitoes is a key activity at the laboratory. Technician Nathalie Schallon is carrying one of the cages containing adult mosquitoes. The mosquitoes are bred in temperature-controlled chambers kept at 26–27°C with 75% humidity to reproduce natural conditions in the intertropical areas that form their natural habitat.
    © Inserm/François Guénet
  • “Wild type” mosquitoes with intact genetic baggage are bred in the same boxes as the transgenic mosquitoes to ensure they all develop in the same heat and humidity conditions, and they are fed the same diet. This allows the researchers to eliminate the influence of any of these factors when they study the Anopheles mosquitoes’ resistance to parasites.
    © Inserm/François Guénet
  • The researchers are involved at various stages of development in the Anopheles mosquitoes. For example, they use transgenesis on the eggs (pictured at left) by inserting or deleting certain genes. Mature mosquitoes (at right) are used to study how they react to a parasite infection or virus transmitted during a blood meal.
    © Inserm/François Guénet
  • While some mosquitoes do escape when the breeding boxes are handled in the climate-controlled chambers or on the workbenches, they never get very far. They are immediately “sucked up”! And there is zero risk of disease transmission, as these mosquitoes carry no infections. When they are infected, the process takes place in a special room equipped with glove boxes from which it is impossible for them to escape.
    © Inserm/François Guénet
  • Doctoral student Antinea Babarit is using a special brush to align mosquito eggs under the stereo microscope. This makes it possible to inject DNA into the eggs. The goal is to create a descendance of genetically modified individuals.
    © Inserm/François Guénet
  • A micro-dropper is employed for transgenesis. The tool is used under the stereo microscope to inject the transgene into the eggs of wild type mosquitoes, which will then produce transgenic offspring. Another key “tool” is CRISPR-Cas9, a complex of protein and RNA that acts like a pair scissors to cut the DNA molecule with which it is put in contact. The new gene is inserted into the “hole” left by these molecular scissors.
    © Inserm/François Guénet
  • This magnification shows what happens during transgenesis: recombinant DNA, which includes the relevant gene along with other genetic sequences, is injected into an egg to regulate the future expression of that gene or to express a fluorescent protein. The DNA inserts itself into the nucleus of a cell in the caudal region. This region contains the germ cells that will be passed on to future generations, creating transgenic lines.
    © Inserm/François Guénet
  • Since the wild type mosquitoes and their transgenic counterparts live together, the scientists must have a way to sort them before conducting experiments on their ability to resist parasites. Doctoral student Emily Green uses a flow cytometer, or a sorter, to distinguish the mosquito larvae based on the presence, colour, and intensity of their fluorescence.
    © Inserm/François Guénet
  • On the screen, Green calibrates which type of larva she needs to identify. For this experiment, she has chosen transgenic homozygotes, or larvae that have two identical genetically modified alleles on each chromosome. She will then select the “negative” larvae, which are the wild type.
    © Inserm/François Guénet
  • Genetically modified mosquitoes can also be manually identified by observing the larvae through a stereo microscope under black light as they swim in a drop of water. Black light “reveals” the larvae in which a blue fluorescent protein is expressed.
    © Inserm/François Guénet

Note :*Inserm/Université de Strasbourg/CNRS unit 1257, Immune Responses in Mosquitoes, Institute of Molecular and Cellular Biology, Strasbourg