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)
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 procedures5,6,7. 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.
3. Non-overlapping generation trials of gene-drive mosquitoes (Figure 5).
Transgenic anopheline mosquitoes generated to bear non-gene drive or autonomous gene-drive modifications are set up for cage trials as described in the Protocols section. The representative results shown here depict the phenotype dynamics of the best-performing replicates of each of the cage trials experiments performed by Pham et al. (2019)2 for Anopheles stephensi mosquitoes. The three trials (1 – 3, respectively: inundative non-gene drive, overlapping gene-drive and non-overlapping gene-drive) varied in different parameters, such as the size of the cage (0.216 m3 vs 0.005 m3), whether or not the target population was age-structured, source of blood meal (mice or artificial feeder) and release ratios. As a means of representation, Figure 6 displays the observed data selected from the same release ratio (1:1) for all three protocols used, on the course of seven generations.
The 1:1 non-drive release reaches >80% transgene introduction within 6-7 generations. For gene-drive transgenic cage trials, the 1:1 releases in both the non-overlapping and overlapping protocols reach this level within 3-4 generations, thus, validating the expectation that a single release of a gene drive system can be more efficient than non-drive inundative releases for transgene introduction. The faster trajectory can also be confirmed by the slope of the trendlines. Both gene-drive protocols, despite different set ups, present similar angles and slope trends. At the end of observation, non-drive cages achieve ~80% of individuals bearing the transgene, while cages with gene-drive individuals reach complete (or near complete) introduction. Complete data and processing details on individual experiment results using the protocols described here can be found in Figures 1-3 of Pham et al. (2019)2, Figures 2-3 of Carballar-Lejarazú et al. (2020)3 and Figure 3 of Adolfi et al. (2020)4.
Figure 1. Non-drive inundative release trial schematic. Nine 0.216 m3 cages are set up with 60 wild-type second-instar (mixed-sexes) larvae added to each. Beginning week 3, females are provided a bloodmeal weekly and eggs are collected and hatched. Until week 8, 60 larvae are randomly selected and returned to their respective cages weekly to create an age-structured population in the cages (initial phase). Beginning week 9, the nine cages are randomly assigned in triplicate according to their transgenic:wild-type male release ratios (experimental phase). Cages A (Control) have no transgenic pupae added. Females are provided a bloodmeal weekly and eggs are collected, hatched, and reared to pupae. 30 male and 30 female wild- type pupae are added back to their cages. Cages 1:1 have an additional 30 transgenic male pupae added. Cages 1:0.1 have an additional 300 transgenic male pupae added. 300 larvae from each of the 9 cages are selected randomly and screened for the fluorescent marker. This procedure was repeated weekly until transgene fixation (stabilized ratio of transgenic-wildtype mosquitoes after a few generations). Adapted from Pham et al. (2019)2. Please click here to view a larger version of this figure.
Figure 2. Blood feeding of cage populations. (A) Anesthetized mice or (B) Hemotek blood feeders are offered for blood feeding female mosquitoes on the 0.216 m3 cages or the small 0.005 m3 cages, respectively. Please click here to view a larger version of this figure.
Figure 3. Screening phenotypes for non-drive, overlapping gene-drive and non-overlapping gene-drive cage trials. Fluorescent images of a larva, pupa and adult of transgenic or wild-type phenotypes. In this example, An. stephensi individuals were screened for the DsRed marker driven by the 3xP3 promoter in the eyes (DsRed+ or DsRed-), visible in all three stages, and adults were screened for sex (♀ or ♂). Note the background fluorescence in wild-type larvae associated with the food bolus in the midgut. Please click here to view a larger version of this figure.
Figure 4. Overlapping gene-drive cage trial schematic. Six 0.216 m3 cages are set up in triplicate according to their gene-drive:wild-type male release ratios. 120 wild-type males and 120 wild-type females were added to each cage. Cages with a 1:1 gene-drive male release ratio had an additional 120 transgenic males added. Cages with a 1:10 male release ratio had an additional 12 transgenic males added. Until full introduction of the transgene, every 3 weeks, adult females are provided with bloodmeals and eggs are collected and hatched. A total of 240 larvae were selected randomly and returned to their respective cages. Three-hundred (300) larvae are selected randomly and screened for the dominant marker. They are later screened as pupae and adults for eye-color and sex. No additional transgenic males are added to the original cages. Adapted from Pham et al. (2019)2. Please click here to view a larger version of this figure.
Figure 5. Non-overlapping gene-drive cage trial schematic. Nine small 0.005 m3 cages are set up in triplicate according to their gene-drive:wild-type male release ratios. Cages with a 1:1 male release ratio have 100 wild-type females, 50 wild-type males, and 50 gene-drive males added. Cages with a 1:3 male release ratio have 100 wild-type females, 75 wild-type males, and 25 gene-drive males added. Cages 1:10 male release ratio have 100 wild-type females, 90 wild-type males, and 9 gene-drive males added. Females are provided a blood meal and eggs collected and hatched. For 1:1 and 1:3 cages, 200 larvae are selected randomly and used to populate new cages, separate from that of their parents, for the next generation. An additional 500 larvae are selected randomly and reared to pupae, when they are screened for the dominant marker gene. The 500 pupae are then reared to adults and scored by sex. All remaining larvae are screened for the marker. For the 1:10 cages, all larvae are scored in generations 1-12 and 200 larvae reflecting the existing transgene frequency are used to populate new cages. Beginning at generation 13, these cages are set up identically to the 1:1 and 1:3 cages. Adapted from Pham et al. (2019)2 and Carballar-Lejarazú et al. (2020)3. Please click here to view a larger version of this figure.
Figure 6. Predicted transgene fixation dynamics for the different population replacement cage trials. Representation of the expected phenotype dynamics of the best-performing replicates for each of the cage trials experiments performed by Pham et al. (2019)2, monitored over 7 generations. Experiments set ups are described in the Protocols. The predictions are based on data from all 9 experiments on the 1:1 release models (triplicate replicates for each of the three different cage trial protocols). The X-axis is the generation number after initial introduction and the Y-axis is the proportion of larvae showing the DsRed marker phenotype (DsRed+) over time. Dashed lines represent linear trendlines of the data. The DsRed+ phenotype results from having at least one copy of the modified allele. Hence the results reflect the spread of the transgene, expedited in the gene drive system, reaching (near) full introduction at the end of the observation. For the variability between replicates and full detailed data on the experiments, please refer to Pham et al. (2019)2, Carballar-Lejarazú R et al. (2020)3 and Adolfi A et al. (2020)4. Images adapted from Pham TB et al. (2019) Experimental population modification of the malaria vector mosquito, Anopheles stephensi. PLOS Genet 15(12): e1008440. doi: 10.1371/journal.pgen.1008440, Adolfi A et al. (2020) Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi. Nat Commun 11(1): 5553. doi: 10.1038/s41467-020-19426-0 and Carballar-Lejarazú R et al. (2020) Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae. Proc Natl Acad Sci USA 117(37):22805-22814. doi: 10.1073/pnas.2010214117. Please click here to view a larger version of this figure.
Supplemental File: The construction of the 0.005 m3 colony cage. Please click here to download this File.
Artificial feeders | Hemotek | SP6W1-3 | Starter pack – 6 feeders with 3ml reservoirs |
Cage, commercial | BioQuip | 1450D | Collapsible Cage, 24 X 24 X 24" – 0.216 m3 (60 cm3) |
Cage tub (popcorn) | Amazon.com | VP170-0006 | 0.005 m3 (170 fl oz) |
Dissecting microscope with fluorescence light and filters | Leica | M165FC | |
Glue sticks | Michaels | 88646598807 | Gluesticks 40 pk, 0.4X4” |
Hot glue gun | Woodwards Ace | 2382513 | Stanley, 40 watt, GR20 |
Nylon screen (netting) | Joann.com | 1102912 | Tulle 108" Wide x 50 Yds – ~35.6 cm2 (14 in2) |
Oviposition cups | Fisher | 259126 | Beaker PP grad 50 mL |
Razor cutting tool | Office Depot | 487899 | Box cutters |
Scissors | Office Depot | 978561 | Scotch Precision Ultra Edge Titanium Non-Stick Scissors, 8" |
Stapler | Office Depot | 908194 | Swingline Commercial Desk Stapler |
Surgical sleeve (stockinette) | VWR | 56612-664 | ~48 cm (19”) cut from bolt ~15 cm (6”) X ~23 m (25y) |
Zip ties | Home Depot | 295715 | Pk of 100, 14” cable ties – 35.6 cm (14 in) |
Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.
Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.
Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.