The protocol described here are appropriate to evaluate transgenic mosquito performance and comparisons with wild type counterparts in a small laboratory cage trials. The small-cage trials are important to provide empirical values that will aid to fill implementation of new technologies for malaria elimination. The cage trials can be adapted to other experimental designs and are suitable to assess the dynamics of a threat of a desire trans-gene.
Following the introduction transgenic individuals. Begin the initial phase of the experiment by setting up a three 0.216 cubic meter cages in triplicates. And adding 60 second-instar wild type larvae to each cage over three successive weeks.
Every week, provide adult females in each cage with anesthetized mice as a blood meal source. And an oviposition container three days after the blood meal. Hatch eggs from each cage weekly.
From week four to eight, select 60 second-instar L2 larvae at random to return to their respective cages. At week nine, randomly assign one set of triplicate cages as controls and one set for each desired release ratio. Add 60 wild type pupae with 30 males and 30 females to the control cages weekly.
Maintain a one to one ratio in each respective cage by weekly adding 30 transgenic male pupae along with 60 wild type pupae. For a one to 0.1 ratio, add 300 transgenic male pupae weekly into each respective cage along with 60 wild type pupae. Repeat adding mosquito pupae in the cage, providing blood meal and hatching eggs every week.
Select a total of 300 larvae from each cage at random for screening. Screen the larvae under a stereo microscope with fluorescence filters for the expression of the fluorescent dominant marker at larval and pupal stages. Score the sex of the resulting pupae.
In the gene drive experiment, employ a barrier strategy by keeping mosquitoes in a secure insectary and following standard operating procedures with regular monitoring. Prepare a cage set up with two sets of triplicate 0.216 cubic meter cages for each desired transgenic to wild type male release ratio. To achieve a male release ratio of one to one, add 120 transgenic males, 120 wild type males and females at the pupa stage to each replicate cage.
Provide a blood meal using anesthetized mice to four to seven day old females in each cage. After hatching eggs in a larval tray, select approximately 240 first instar L1 larvae at random from each cage and rear them to pupae before returning to their respective cages. Select and screen larvae for eye marker and sex phenotype as described before.
Assemble a cage setup of triplicate 0.005 cubic meter cages with an equal number of males and females for each specific release ratio of transgenics to wild type males. Using an artificial feeding apparatus, provide blood meals to the four to seven day old females in each cage on two consecutive days. Three days after the second blood meal, add an oviposition container and remove the containers after three days.
After scoring all the dead and the live adults remaining in the cage by sex, store them at minus 80 degrees Celsius for molecular analysis. Hatch eggs and run select 200 L1 larvae and set the larvae aside to populate new cages for the next generation. Then randomly select another 500 larvae from each cage set aside for screening.
Rear the selected 200 larvae to pupa and 500 larvae to L4 instar stage. Return pupa from 200 trays to populate new cage. Screen randomly selected larvae for eye marker and sex phenotype As before.
In the representative analysis, the expected phenotype dynamics of the best performing replicates are shown for the different population replacement cage trials protocols. The results showed the linear increasing proportion of larvae with DsRed marker phenotype over seven generations. The one to one non-drive release reached little more than 80%transgene introduction within seven generations.
The gene drive protocols achieved this within three to four generations. Thus validating that a single release of a gene drive system can be more efficient for transgene introduction. At the end of the observation, the spread of the transgene had reached almost complete introduction in gene drive systems.
Important parameters, such as release ratio population age-structure, cage size, and others will vary depending on the purpose of the experiment and specific aims. Experiments to assess fitness parameters such as fertility and fecundity can be done. Further more empirical data from the cage trials can be used to parameterize mathematical models of population dynamics.
