Published: May 1st, 2021
The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.
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.
Strategies based on genetically-engineered mosquitoes are being pursued to control transmission of vector borne pathogens such as those that cause malaria1. These include technologies 1) aimed at decreasing the numbers and densities of Anopheles mosquitoes (population suppression), or 2) aimed at impairing the ability of vectors to transmit parasites responsible for human disease (population modification, replacement, or alteration) wherein strains of vectors are engineered to express effector genes that prevent pathogen transmission. These genetic approaches have been bolstered by the advent of CRISPR/Cas9-based gene drives, with proofs-of-concept in parasite-transmitting mosquitoes of effective spread of payload traits as well as anti-parasitic effector molecules in caged populations.
Small laboratory cage trials represent a first step for evaluating the characteristic of transgenic strains as part of a phased approach to their further development towards field applications2. Specific outcome considerations include heritability of the introduced DNA in a competitive environment, penetrance and expressivity of the phenotype, and stability. Relevant experimental design features include the size of the cages, mosquito densities, number of replicates, overlapping or non-overlapping generations, age-structured target populations, single or multiple releases of engineered strains, male-only, female-only or mixed-sex releases, release ratios, blood meal sources (artificial or live animal), and screening procedures.
We describe here protocols used to evaluate strains of anopheline mosquitoes for inundative releases (no gene-drive system) and those that carry autonomous gene-drive systems mediated by Cas9 endonucleases and guide RNAs (gRNA). Applications of these protocols appear in Pham et al. (2019)2, Carballar-Lejarazú et al. (2020)3, and Adolfi et al. (2020)4.
Inundative release trials evaluate the spreading rate of a designed transgene under Mendelian inheritance following multiple releases of a large number of transgenic mosquitoes into a wild population. Without the attachment of the transgene to a drive system, data from inundative release trials provide information regarding the fitness and dynamic of the transgene of interest in a stabilized population.
When mosquito populations contain an autonomous gene-drive system, small cage trials are designed to assess the dynamics of the spread of the desired transgene by determining the rate of dominant marker increase following a single introduction of transgenic males. Autonomous gene-drive elements carry the genes encoding the Cas9 nuclease, gRNA and dominant marker linked in such a fashion as to be active in subsequent generations.
'Overlapping' generations refer to the simultaneous presence of multiple generations in the same cage to create an age-structured continuous population, while 'non-overlapping' refers to single discrete generations in each consecutive caged population2. Gene-drive cage experiments can be terminated once the initial dynamics of the drive (conversion) rate can be determined (8-10 generations depending on the construct), and while they provide information on the short-term stability of the transgene within the mosquito population, they may not reveal what happens when and if the dominant marker frequencies reach or are close to full introduction (every mosquito carrying at least one copy of the gene-drive system).
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.
Genetically-engineered mosquitoes that have pathogen blocking ability or bear sterility genes constitute new tools to control vector-borne diseases. Given the multiplicity of parameters that comprise these alternative approaches, a critical step in their research consists of laboratory-confined experimental evaluations that allow a fast and safe prediction of the potential outcomes of a synthetic transgene release for control purposes1.
Because the monitoring of the transgene dynamics in caged populations can extend for several months, one of the central aspects of the protocols is the consistency in experimental design between replicates (including mosquito rearing, cage size, age-structured populations, fixed release ratios, stable blood meal sources and minimally invasive screening procedures).
Male-only releases are considered ideal because male mosquitoes neither transmit pathogens nor feed on humans, therefore they can safely introduce heritable characteristics into wild populations. In laboratory cage experiments, it is possible to detect transgenic strains with reduced male mating competitiveness and other fitness loads associated with transgene integration. However, direct and specific experiments, such as those conducted in large cages10, can be conducted to properly analyze male competitiveness, as well as female fecundity in more natural mosquito densities2. Furthermore, empirical data from the cage trials can be used to parameterize models of cage population dynamics, including resistant allele formation, and provide useful information on effectiveness and possible adjustments in the proposed technology.
The protocols described here can be easily adapted to other experimental designs as required, with minimal requirements regarding regular insectary infrastructure and conditions. In addition, except for the commercial cages and microscopes, most of the materials are inexpensive and allow low-cost multiple replicates and iterations of the trials. Notably, this also allows multiple transgenic strains to be pre-screened in small cage trials in order to prioritize best-performing candidates to be moved forward in the phased testing pathway and to suspend testing on those showing sub-optimal performances.
Finally, concern regarding the use of genetically modified organisms motivates the elaboration of frameworks for the development, evaluation, and application of genetic strategies for prevention of mosquito-borne diseases5,8,9. The relevance and execution of the protocols defined here are consistent with these guidelines.
The authors have no disclosures.
We are grateful to Drusilla Stillinger, Kiona Parker, Parrish Powell and Madeline Nottoli for mosquito husbandry. Funding was provided by the University of California Irvine Malaria Initiative. AAJ is a Donald Bren Professor at the University of California, Irvine.
|Starter pack – 6 feeders with 3ml reservoirs
|Collapsible Cage, 24 X 24 X 24" - 0.216 m3 (60 cm3)
|Cage tub (popcorn)
|0.005 m3 (170 fl oz)
|Dissecting microscope with fluorescence light and filters
|Gluesticks 40 pk, 0.4X4”
|Hot glue gun
|Stanley, 40 watt, GR20
|Nylon screen (netting)
|Tulle 108" Wide x 50 Yds - ~35.6 cm2 (14 in2)
|Beaker PP grad 50 mL
|Razor cutting tool
|Scotch Precision Ultra Edge Titanium Non-Stick Scissors, 8"
|Swingline Commercial Desk Stapler
|Surgical sleeve (stockinette)
|~48 cm (19”) cut from bolt ~15 cm (6”) X ~23 m (25y)
|Pk of 100, 14” cable ties - 35.6 cm (14 in)
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