Mosquito transgenes and courtship songs

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A recent study shows how disruption of the mosquito double sex gene affects the promotional songs produced by women.

Anopheles gambiae during mating: Source: Sam-Cotton, www.scienceupdate.com

Mosquito advertising and mating is an aerial matter. Male mosquitoes gather in large swarms, swirl around, and attract women by the pheromones they emit. But smell isn’t the only sense associated with advertising; sound is also. When a woman flies into the flock, she is recognized by the sound of her wing flapping, and men identify women using a phonotactic response to the specific frequency of sound produced by their wing flapping. A man and a woman then coordinate with each other by changing the frequency of their wingbeats in a form of mutual coordination that is very specific.

The head and antennae of a male Anopheles gambiae. Credit Alexos Simoni, Imperial College London. source

Mosquito ears

Wingbeats create vibrations in the air that a mosquito can hear because they cause vibrations in the antennas. These movements are picked up by flagellar ears, which are located near the base of the antennae. Mosquito antennae are made up of segments, with the third being divided into sections known as flagellomeres.

Double sex disturbed

In the anopheline mosquito Anopheles gambiae, sexual differentiation is controlled by the gene doublesex (Agdsx).

CRISPR / Cas9 mutants were produced in which the female-specific isoform of the doublesex (dsxF) gene was disrupted. Mutant women who were homozygous for the destroyed allele had an intersex phenotype and were sterile. When these mutants were introduced into cage populations of wild-type mosquitoes, the populations eventually collapsed. This finding bode well for their usefulness in a mosquito control program in which dsxF mutants could be genetically engineered. However, in order to assess the possible spread of the dsxF allele, further experiments were required to test their fitness, especially with regard to advertising and mating with wild types.

This is where promotional songs come into this story.

Members of the Ear Institute, University College London, tested flight tones generated by flapping wings and reacted to these tones (phonotaxis) in dsxF + / +, dsxF +/- and dsxF – / – mutant mosquitoes raised in the Department of Life were sciences, Imperial College. During the experiments the temperature was controlled and the wing length was used as a proxy for the size.

Their experiments answered the following questions:

  • Was there a difference between flight tones in the different genotypes of male and female mosquitoes?
  • Did men and women of all three genotypes react differently to acoustic stimulation?
  • Did dsxF + / + males react differently to the flight tones of the three female genotypes?

Flight sound

Individual mosquitoes were tied to tungsten wire and mounted in a custom-made cage that held a particle velocity microphone (experimental details can be seen here). Mosquitoes were encouraged to fly and the microphone values ​​were recorded. The wing beat frequencies were determined for different subsections of a flight period and the median was calculated and used to assign a flight tone to each mosquito.

The flight tones of all men were larger than those of women, but there was no significant difference between the male genotypes. However, the flight tones of the female groups differed in a dose-response manner, with the dsxF – / – female flight tone being significantly higher than the other female groups but still lower than the males. The flight tone of dsxF + / + mutants was the lowest.

Phonotaxis assays

Groups of mosquitoes were housed together in same-sex cages and were each exposed to pure tones at frequencies of 100, 400 or 700 Hz for one minute. Mosquitoes approaching the sound source were counted to determine what frequency they responded to.

Female mosquitoes did not respond to any of these frequencies, while all male genotypes responded most to tones of 400 Hz, which are most similar to wild-type women. In addition, there was no evidence that male flight behavior was influenced by the dsxF – / – allele.

More specific tones

Finally, male dsxF + / + mosquitoes were exposed to the mean sound frequencies of the wing beats of dsxF + / +, dsxF +/- and dsxF – / – women to see how wild-type males responded to these female types. A dose response was noted, with most responding to the flight tone produced by wild-type women and the least responding to that of the dsxF – / – group. Male dsxF – / – mosquitoes also responded more strongly to wild-type female wing frequencies. Therefore, wild-type and mutant men preferred the song made by wild-type women and may be less likely to mate with mutant women.

The future

The release of male transgenic mosquitoes as part of a population control strategy is a goal pursued by several research groups. One of many considerations that must be evaluated before implementing this strategy is the likelihood that freed males will be able to compete successfully with those in the natural population, that is, with their reproductive capacity.

Since the acoustic fitness of these male transgenic lines does not seem to be influenced, male hearing behavior could lead to successful mating by selecting wild-type females in mixed swarms of wild-type and transgenes. Since the homozygous mutant women are sterile, attracting dsxF + / + women would be very beneficial and could allow the mutation to spread slowly, which could lead to population breakdown. However, the conditions in flocks in the wild would be much more complex than the environment created by these experiments, and other factors will also play a role in mate selection, such as pheromone production. This is clearly a promising start and a thorough study of acoustic advertising and other aspects of the suitability of these transgenic lines in laboratory and half-field conditions is required.

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