Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Animal ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Protocols were approved by the Institutional Animal Care and Use Committees of the University of California (Animal Welfare Assurance Numbers A3416.01).

1. Inundative release trials on non-gene drive mosquitoes (Figure 1)

1. Cage setup and maintenance
   1. Set up three sets of triplicate 0.216 m³ cages by adding 60 second-instar wild-type (WT) larvae in each cage over three successive weeks.
      NOTE: It is not possible to determine the sex of second-instar larvae by light microscopy, so the samples added to each cage will consist of both males and females.
   2. At every week, provide adult females in each cage with anesthetized mice as bloodmeal source (Figure 2A) and an oviposition container 3 days after the bloodmeal.
      NOTE: While an alternative artificial feeding apparatus can be used, providing live anesthetized mice for bloodmeals results in better mosquito feeding performance in these large (0.216 m³) cage formats. This requires an approved animal use protocol and relevant (e.g., Institutional Animal Care and Use Committee, IACUC) approval for using mice.
      NOTE: Individual mice are anesthetized with a mixture of 4 mg/mouse ketamine HCl and 0.4 mg/mouse xylazine. Animals are injected with between 0.1 – 0.5 mL of this mixture.
   3. Hatch eggs from each cage weekly and select 60 second-instar (L2) larvae at random to be returned to their respective cages to offset mortality (weeks 4-8).
      NOTE: Steps 1.1.1 to 1.1.3 are necessary to establish a stable and distributed age-structured population in the cages – referred to as 'Initial Phase'.
   4. At week 9, assign cages assembled in step 1.1.1 randomly in triplicates for releases of the desired male release ratio.
      1. Designate one set of triplicate cages as controls to assess consistency throughout the experiment.
      2. Designate one set of triplicates for each desired release ratio (for example, 1:1 or 1:0.1 transgenic:WT males).
         NOTE: This point on is referred to as 'Experimental Phase'.
2. Replicates and release ratios
   1. Add 60 WT pupae (30 males and 30 females) to the control cages weekly.
   2. To maintain a 1:1 ratio, add weekly 30 transgenic male pupae along with 60 (30 male and 30 female) WT pupae into each respective cage.
   3. To maintain a 1:0.1 ratio, add weekly 300 transgenic male pupae along with 60 (30 male and 30 female) WT pupae into each respective cage.
      NOTE: Continued addition of wild mosquitoes to the cages maintains the cage density, which is expected to diminish weekly due to age-related adult mortality.
3. Screening of phenotypes
   1. Select a total of 300 larvae from each cage at random. With the use of a stereo microscope equipped with fluorescence filters, screen for the expression of the fluorescent dominant marker at the larval and pupal stages and score the sex of the resulting adults (Figure 3).
      NOTE: The phenotypical screening will depend on the dominant marker included in the transgene construct integrated into the mosquitoes (for example, Discosoma sp. red fluorescent protein [DsRed], cyan fluorescent protein [CFP], green fluorescent protein [GFP]), and on the promoter driving its expression (the most used in mosquito transgenesis is the 3xP3 promoter driving expression in the eyes and nerve cord).
   2. Follow this protocol for as many generations as required by the outcome parameters defined in the experimental design.
      ​NOTE: The trial is usually terminated when all mosquitoes have at least one copy of the transgene (determined by the presence of the dominant fluorescent marker) or the ratio of transgenic-to-WT mosquitoes in a cage is stabilized and does not fluctuate greatly after a few (3-5) generations.

2. Overlapping generation trials of gene-drive mosquitoes (Figure 4)

NOTE: Mosquitoes carrying gene-drive systems require written and reviewed protocols and should be approved by an Institutional Biosafety Committee (IBC) or equivalent, and others where required. Mosquito containment (ACL 2⁺ level) should follow recommended procedures^5,6,7. Specifically, the gene drive experiments should employ two stringent confinement strategies. The first is usually physical barriers (Barrier Strategy) between organisms and the environment. This requires having a secure insectary and standard operating procedures (including monitoring) for ensuring that mosquitoes cannot escape. The second confinement strategy can be Molecular, Ecological or Reproductive⁵.

1. Cage setup
   1. Set up two sets of triplicate 0.216 m³ cages for each desired transgenic:WT male release ratio.
      1. To achieve a male release ratio of 1:1, add 120 transgenic males, 120 WT males and 120 WT females at the pupa stage to each replicate cage.
      2. To achieve a release ratio of 1:10, add 12 transgenic males, 120 WT males and 120 WT females at the pupa stage to each replicate cage.
         ​NOTE: Different release ratios can be tested (1:1, 1:3, 1:10, etc.) and the number of mosquitoes used to initiate the experiments varies accordingly. However, it is important to consider the effects of low numbers on the statistical evaluation of the data.
2. Population maintenance and screening
   1. Provide 4-7 days old females in each cage with a blood meal using anesthetized mice (Figure 2A).
   2. Three days after the blood meal, insert an oviposition container in each cage.
   3. Hatch eggs in a larval tray, select ~240 first instar (L1) larvae at random from each cage, rear them to adulthood, and return them to their respective cages.
   4. Provide additional (2-3) blood meals every 3-4 days for the newly emerged adults as described in step 2.2.1.
      NOTE: No additional transgenic males are added during any of the subsequent generations.
   5. Select a total of 300 larvae from each cage at random and screen them for the presence of the dominant marker phenotype at the larval and pupal stages using a fluorescence stereo microscope and score emerging adults for sex (Figure 3).
      NOTE: As before, the phenotypical screening will depend on the dominant marker and promoter included in the gene-drive system and integrated into the transgenic mosquitoes (e.g., DsRed, CFP or GFP). If homozygous or heteroallelic disruptions of the targeted genes result in a visible phenotype (for example, genes related to eye pigmentation), screening of this trait will depend on which stage it is easiest to visualize the altered phenotype.
   6. Follow this protocol for as many generations as required by the outcome parameters defined in the experimental design.
      ​NOTE: Each generation (delimited by the blood meal) takes ~three weeks. The trial is usually terminated when all mosquitoes are deemed homozygous for the gene-drive construct or the populations stabilize at a maximum percentage of mosquitoes carrying at least one copy of the gene-drive construct.

3. Non-overlapping generation trials of gene-drive mosquitoes (Figure 5).

1. Cage setup
   1. Set up triplicate 0.005 m³ cage populations for each specific release ratio of transgenics to WT males to be investigated (for example, three sets of triplicate cages each set up with 1:1, 1:3, 1:10 release ratios). Set up all cages with an equal total number of males and females.
      NOTE: The Supplementary File is a video demonstrating the construction of the 0.005 m³ colony cage.
      1. Add 50 transgenic males, 50 WT males, and 100 WT females to each of three replicate cages to achieve a 1:1 male release ratio.
      2. Add 25 transgenic males, 75 WT males, and 100 WT females to each of three replicate cages to achieve a 1:3 male release ratio.
      3. Add 9 transgenic males, 90 WT males, and 100 WT females to each of three replicate cages to achieve a 1:10 male release ratio.
         NOTE: Different release ratios can be tested and the number of mosquitoes used to initiate the experiments can vary accordingly. However, it is important to consider the impact of low numbers of mosquitoes on the statistical analyses. These are single releases; no additional transgenic males are added at any subsequent generation.
2. Population maintenance and screening
   1. Provide the 4-7 days old females in each cage with blood meals using an artificial feeding apparatus (Figure 2B) on two consecutive days.
      NOTE: Routine blood meals for females consist of a commercially available source of blood (e.g., calf's blood) provided from a feeding apparatus. Live anesthetized mice are used only to provide bloodmeals in larger (0.216 m³) cage formats for better feeding performance.
   2. Add an oviposition container 3 days after the second bloodmeal. After three days, remove the oviposition containers.
      NOTE: At this step, 5-10 females can be selected at random from each cage and placed individually in vials to assess additional fitness parameters, such as fertility and fecundity, if needed.
   3. Score by sex all adults (dead and alive) remaining in the cage and store them at -80°C for molecular analysis.
   4. Hatch eggs and randomly select 200 L1 larvae from the 1:1 and 1:3 ratio cages to populate new cages for the next generation.
      NOTE: Due to the low frequency of starting transgenic individual in the 1:10 ratio cages, random sampling may lead to excessive loss of transgenic progeny in the next generation to carry on the population.
   5. To ensure an accurate sampling for the 1:10 cages and sufficient numbers of transgenic mosquitoes, screen all larvae for the dominant marker and select 200 larvae reflecting the observed transgene frequency to populate the new cages.
      NOTE: The 1:10 cages can be maintained identically to 1:1 and 1:3 cages when they reach a transgene frequency of ≥80%.
   6. Select 500 larvae from each cage at random for an in-depth analysis. Screen under a fluorescence stereo microscope for the expected marker phenotypes at the larval and pupal stages and score sex of adults (Figure 3).
      NOTE: 'Exceptional' phenotypes can be selected to be further crossed and analyzed molecularly to monitor resistant allele formation.
   7. This protocol can be followed for as many generations as required by the outcome parameters defined in the experimental design.
      NOTE: Each generation is delimited by the bloodmeal and takes ~three weeks. The trial is usually terminated when all mosquitoes are deemed homozygous for the gene-drive construct or populations stabilize at a maximum prevalence of transgenic mosquitoes. And as before, screening for phenotypes will depend on the dominant markers and promoter integrated in the transgenic mosquitoes (for example, DsRed, CFP, GFP) or in the targeted genes if they present a visible phenotype (for example, genes related to eye pigmentation).