Teams testing mosquitoes that are being considered for field release, their advisors, policy makers and donors often talk about ‘large cage trials’, but how large must a cage be for mosquito trials to qualify for that title? Is the proper metric its volume or stimulation of behavioral characteristics for a cage that provides the data we need? Perhaps the real value of large cage testing is neither of these but its independence.
As part of the phased progression of testing transgenic mosquitoes, the concept of ‘large-cage trials’ is often an intermediate step that is recommended.
Examples of such recommendations:
Phase 1 is anticipated to begin with small-scale laboratory studies…followed by testing in larger population cages in a laboratory setting
Phase 2 initiates confined testing in a more natural setting…within a large cage that simulates the disease-endemic setting..(1)
Large cage experiments that mimic natural conditions must then be performed and models developed to extrapolate how the system will work following an open release.(2)
…laboratory studies and confined field tests (or studies that mimic confined field tests such as large cage trials and green-house studies) represent the best approaches to reduce uncertainty in an ecological risk assessment(3)
Large-cage testing is recommended for the point after which the basic phenotype of the effector of interest has been confirmed in ‘small-cage testing’ and the strain has demonstrated its potential for further development.
The broader programmatic objective is to eliminate candidates that show deficiencies (risk or performance) that warn of deficiencies upon release into the field, an effort that once committed to, is expensive, lengthy and exposes the project to risks in public perception, the scientific community and larger commentariat. Once a candidate strain is chosen to move forward past early phase testing, the costs, risk exposure and effort can be constrained by large cage testing.
Population studies that are conducted in large cages are conceptually very different from those that are typical of small cages and differ by far more than cage size, though that is usually how they are identified. Preliminary small cage studies often consist of observations of specific outcomes under controlled conditions of age, mating status, blood feeding timing etc. In contrast, population studies necessarily have a much lower level of control, thus potentially providing less certain outcomes. Successful candidate strains for development must pass more rigorous tests under a variety of conditions that result in greater confidence in their potential.
I’ve never heard concerns about e.g. Plasmodiumrefractoriness or sexual sterility being affected by different cage testing venues, but the consistency of those traits poorly predicts about the fate of a transgene in mixed populations. Less easily observed, but highly relevant biological characteristics, affecting the persistence or spread of a transgene might be affected. These are reasonable concerns because usually small-cage testing does not involve overlapping ‘stable’ populations in which the age distribution and physiological state of adults is mixed and outcomes are often constrained by the experimental design.
The range of sizes called ‘large’ that are used for mosquitoes is informative. Large outdoor cages were 6 × 6 × 2 m (72 m3) for outdoor trials of the OX3604C Aedes aegypti strain in Mexico (4). Indoor large cages for evaluating an Anopheles gambiae transgenic strain were 5.0 × 1.2 × 2.6 (18.8 m3) (5). Smaller 1.75 m2 cages in a greenhouse (probably about 3.5 m3) were used to examine competitiveness of irradiated Anopheles coluzzii males by Maiga et al. (6).
In spite of the range of sizes, all were described by the authors as ‘large’. Which is truly large? A better question is; which provides useful information that could not have been obtained in typical small laboratory cages?
To the extent that large cages elicit a wider range of behaviors and interactions of modified mosquitoes than small cages, they can reveal deficiencies that raise concerns about their behavior in nature.
Keep in mind: the objective is not to assess the phenotype of the effector or transference rate of a transgene to their progeny; it is to assess the effectiveness of the modified mosquito in a population context. These are very different. The latter necessitates all of the natural behaviors that influence fitness of the transgenic mosquito.
If we wished to define ‘large-cage’ features that require behaviors not needed for success in small cages and therefore complement those studies with useful information, what behaviors must be elicited to make the experiments relevant?
In the course of creating ‘large cages’ for testing transgenic mosquitoes, following are some behaviors that we and others have observed differ between ‘large’ and ‘small’ cages when testing An. gambiae, though many of the same issues are likely relevant to Aedes.
- Sugar location. Sugar water is easily located in small cages. However, in large cages, one must add an attractant such as honey or verbenone (7). Why? Sucrose has no scent, the cue that adults use to locate it. However, when a mosquito is bumping into sugar water sources regularly in a tiny cage, smelling and searching is not necessary.
- Swarm mating. Mating is the primary interface between released mosquitoes and the wild population. Mating is an essential component of any genetic control method. This is particularly important for Anophelesmosquitoes that mate in swarms. (For Ae aegyptistudies, is it relevant whether mating is occurring around a host?)
- Mating competitiveness. We have observed that cage size has a strong effect on mating competitiveness (8). While initial testing in small cages showed no effect of a transgene on competitiveness (9), testing in large cages later demonstrated strong inferiority.
- Assortative mating. I’ve received a report that this has been observed in large cages(10)but in small laboratory cages, members of the gambiae complex readily mate with one another, an event that rarely occurs in nature. Some cage size/design is sufficient to accomplish this and further efforts to understand what is necessary to elicit this behavior would be useful.
- Oviposition site location. We observed in cage experiments in Italy that females could not find the same oviposition dishes we used in small cages; they required special illumination or eggs were laid on the floor. While special illumination is not ‘natural’, it indicates that the ease of finding oviposition sites in small cages does not translate to larger cages and requires capabilities that are unnecessary in small cages.
- Resting site seeking and use. Small cages almost never require an adult to locate a suitable site for resting. Adults are forced to accept whatever is provided, and often there is absolutely no consideration for providing anything. Finding suitable resting sites is essential for wild mosquitoes. It requires them to sense humidity, light and temperature and to move there. In the absence of that, they are prone to predation and desiccation.
- Stresses of variable temperature and humidity.Even indoors, few studies attempt to vary the temperature and humidity even though this is easily done in most modern programmable control systems. The stresses of flying and need for sugar water and resting sites differ depending on temperature and humidity.
- Locating blood sources.Host seeking is stimulated by a combination of heat, odors and CO2. A female’s ability to locate blood, even using only one of these, is often adequate in a small cage. Large cages require longer-distance sensing than small ones and the addition of scent and CO2is helpful to attain high blood feeding rates in large cages.
- Spatial complexity. An uninterrupted interior with no internal structure is typical for insectary cages. Spatial complexity – objects, plants, partitions – increase the need for adults to navigate around obstacles, follow less obvious cues to locate blood, sugar and oviposition sites, provides alternative resting places and can stimulate swarms. Complexity presents a more difficult environment that necessitates greater sensitivity and more complex behaviors.
- Overlapping generations and stable age distributions. Large cage trials usually don’t permit anything but ‘stable’ populations consisting of all ages of adults, mating status and physiological status. Small cage trials often consist of same-aged adults introduced into cages in a scramble for matings. That’s anything but similar to what mosquitoes will encounter when released into the environment.
- Flight distance. In spite of the direct relationship of ‘large’ to this parameter, it is perhaps the most difficult to objectively parameterize. The typical energy reserve replenishment of wild mosquitoes may not be tested in cages that are too small. However, it was observed that OX3604C transgenic males had inferior flight performances compared to their wild-type counterparts (11) and failed to eradicate populations of Ae. aegypti in large cages in the field (12) due to their poor mating capabilities. This was possibly due to their reduced flight capacity that resulted in poor mating performance in large cages. However, I have no estimate of what dimension challenges mosquito flight. Even relatively small ‘large cages’ (9.1 m3) seem to have different demands on energetics (7), and the authors of that study question the relevance of life history observations in small cages.
This is my proposal for a short list of merits of large cage testing that are not possible in small cages; the same issues have been articulated previously (13). There are certainly more subtle ones but even these strongly justify considering their use.
While one can debate whether large cage testing provides anything ‘more natural’, that question can be a red herring. It provides something which should be standard in transgenic mosquito development; independent trials under radically different conditions. Independent trials build our confidence that the salient behaviors are robustly reproducible and will be observed in a wide variety of conditions. When independent trials are performed under similar controlled conditions their value is questionable. It makes sense to perform them in greatly different conditions that are within the realm of ‘realistic’. This is what their performance upon release will require.
I hope this blog has helped readers consider the benefits – indeed I believe the necessity – of large cage trials for modified mosquitoes. The name ‘large cage’ belies their true function. Large cage studies’ are really
population studies that elicit a wide range of behaviors that cannot be observed in small cages.
I’d love to hear comments, corrections, advice and questions.
1. WHO T, FNIH. Guidance Framework for Testing of Genetically Modified Mosquitoes. Geneva: WHO; 2014 Oct p. 159.
2. James AA. Gene drive systems in mosquitoes: rules of the road. Trends Parasitol. 2005 Feb;21(2):64–7.
3. Committee on Gene Drive Research in Non-Human Organisms: Recommendations for Responsible Conduct, Board on Life Sciences, Division on Earth and Life Studies, National Academies of Sciences, Engineering, and Medicine. Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington (DC): National Academies Press (US); 2016 Jul 28.
4. Facchinelli L, Valerio L, Ramsey JM, Gould F, Walsh RK, Bond G, et al. Field cage studies and progressive evaluation of genetically-engineered mosquitoes. Barrera R, editor. PLoS Neglected Tropical Diseases. Public Library of Science; 2013;7(1):e2001.
5. Facchinelli L, North AR, Collins CM, Menichelli M, Persampieri T, Bucci A, et al. Large-cage assessment of a transgenic sex-ratio distortion strain on populations of an African malaria vector. Parasites & Vectors. BioMed Central; 2019 Feb 6;12(1):137.
6. Maïga H, Damiens DD, Niang A, Sawadogo SP, Fatherhaman O, Lees RS, et al. Mating competitiveness of sterile male Anopheles coluzzii in large cages. Malaria Journal. 2014;13(1):460.
7. Stone CM, Hamilton IM, Foster WA. A survival and reproduction trade-off is resolved in accordance with resource availability by virgin female mosquitoes. Animal Behaviour. 2011 Apr;81(4):765–74.
8. Facchinelli L, Valerio L, Lees RS, Oliva CF, Persampieri T, Collins CM, et al. Stimulating Anopheles gambiae swarms in the laboratory: application for behavioural and fitness studies. Malaria Journal. BioMed Central; 2015 Jul 15;14(1):271.
9. Windbichler N, Papathanos PA, Crisanti A. Targeting the X chromosome during spermatogenesis induces Y chromosome transmission ratio distortion and early dominant embryo lethality in Anopheles gambiae. Stern DL, editor. PLoS Genet. Public Library of Science; 2008 Dec;4(12):e1000291.
10. Nignan C, Sawadogo SP, Niang A, Maïga H, Diabate A. Demonstration of assortative mating among members of the gambiae complex in large cages, personal communication.
11. Bargielowski I, Kaufmann C, Alphey L, Reiter P, Koella J. Flight performance and teneral energy reserves of two genetically-modified and one wild-type strain of the yellow fever mosquito Aedes aegypti. Vector-Borne and Zoonotic Diseases. 2012 Dec;12(12):1053–8.
12. Facchinelli L, Valerio L, Ramsey JM, Gould F, Walsh RK, Bond G, et al. Field Cage Studies and Progressive Evaluation of Genetically-Engineered Mosquitoes. Barrera R, editor. PLoS Neglected Tropical Diseases. 2013 Jan 17;7(1):e2001.
13. Stone CM, Taylor RM, Foster WA. An effective indoor mesocosm for studying populations of Anopheles gambiae in temperate climates. Journal of the American Mosquito Control Association. 2009 Dec;25(4):514–6.