Published: March 12th, 2021
The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.
The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.
Transmission of mosquito-borne pathogens to humans causes millions of cases of disease and deaths each year worldwide. In the absence of effective, approved vaccines for mosquito-borne diseases, such as Zika or dengue fever, one of the most effective ways to reduce transmission is to reduce disease-vector mosquito populations. Vexingly, an increasing number of mosquito species, traditionally targeted by pesticides, are displaying increasing levels of pesticide resistance1. Simultaneously, government agencies have aggressively deregistered or banned previously approved pesticides, and few new, effective chemical control measures are being developed2,3. This constellation of obstacles to mosquito control has motivated the exploration of alternative non-chemical techniques to reduce mosquito populations.
Certain mosquito species present challenges to control issues of resistance and pesticide registration. Aedes aegypti (L.) is a prominent disease-vector mosquito that is extremely difficult to control through traditional integrated vector management due to the cryptic peridomestic habitat exploited by this species for immature development and adult resting4,5. Challenges related to the exploitation of the cryptic habitat around residences include the difficulty of reaching these locations with pesticide spray techniques as well as the potential lack of acceptance by the public for repeated access to private property for public health vector control agencies to conduct the intensive surveillance and control activities crucial to effective integrated vector management (IVM) for this species.
Fortunately, SIT, an approach proven successful for enduring control of other highly challenging insect species6, is being applied to the Aedes aegypti problem in a groundbreaking series of experiments and operational trials based in St. Augustine, Florida (KJL, RLA, SCB unpublished data). SIT has been applied to a range of insect species, including mosquitoes, and has been reviewed in depth7,8. SIT leverages the mass release of colony-reared males sterilized, for example, by exposure to ionizing radiation or chemicals to overwhelm the mate choice of natural populations of females. Sterilized males that mate with wild females render the eggs infertile due to damage suffered by male gametes, and if present in sufficient numbers, can theoretically crash the natural Aedes aegypti population.
An SIT program was initiated to attempt to reduce populations of Aedes aegypti in an urban area in Atlantic coastal Florida where this species recently re-colonized and is expanding and presenting a public health risk for transmission of viruses such as Zika, dengue, or chikungunya. To maximize the potential for compatibility with wild females, a new colony was established using wild-caught Aedes aegypti from the target population to produce males for the program9. This was based on the hypothesis that locally derived, colony-reared males would be more likely to be competitive with local wild males for mating with local wild females. For the SIT to be effective, not only do overwhelming numbers of sterile males need to be present in the target area, but they also have to be capable of effectively courting and mating with local wild female mosquitoes.
A series of experiments was conducted to determine the optimal number of sterile males to release (KJL, RLA, SCB unpublished data) as well as optimal doses of radiation that would render the males infertile without interfering with survival, behavior, or acceptance by wild females (KJL, RLA, SCB unpublished data). These data are forthcoming in allied publications from this group, but some of these findings are captured in this protocol as well and could be used as a starting point for new SIT Aedes aegypti control programs elsewhere. This species is constantly expanding its range, and SIT programs show great promise to be cost-effective, long-term solutions to control this population. The objective of this protocol is to produce sterilized, male, colony-reared Aedes aegypti mosquitoes for systematic release into outdoor areas to disrupt the natural reproductive cycles of local Aedes aegypti populations in an operational public health vector control program.
While similar protocols and workflows have been published for the production of transgenic Aedes aegypti males and production workflows for Aedes SIT, or Wolbachia-based incompatibility programs have been published elsewhere, this protocol illustrates how existing protocols have been adapted for Aedes aegypti production, separation and irradiation of male pupae, marking and packaging adult males, and shipment to the release site for this program9,10,11,12,13,14,15,16,17,18. The marking component of this protocol may not be required in a mature operational SIT program; however, it has been included here because it is one way to monitor the efficacy and control the quality of the entire process in the early years of establishing the SIT program. Mosquito control programs are typically run by local authorities, so they can vary widely in many aspects of their organization from size and funding base to tuning control tactics to maximize local success. Thus, the protocol described herein should be evaluated for compatibility with available resources.
NOTE: This protocol is specific to the handling of Aedes aegypti but may be modified to be effective for other mosquito species.
1. Production and maintenance of an Aedes aegypti colony
|Volume nutritional slurry added
|Volume water added
|50 mL (slurry)
|1/4 - 1/2 tsp (pulverized fish food)
|1/2 - 3/4 tsp (pulverized fish food)
|1/2 - 3/4 tsp (pulverized fish food)
|1/4 - 1/2 tsp (pulverized fish food)
|strain pupae and larvae
Table 1: Feeding schedule for mass rearing of Aedes aegypti larvae.
2. Separation of male Aedes aegypti pupae
Figure 1: Pupal separator containing a batch of immature Aedes aegypti. Separation begins by pouring water through the separator while rotating the bottom knobs 1-2 cm counterclockwise until the targeted set, i.e., larvae, male pupae, or female pupae, has been isolated as much as possible from the sets that remain (left image). Right image shows separation of larvae (lowest band), male pupae (middle band), and female pupae (upper band). Please click here to view a larger version of this figure.
3. Preparation of male Aedes aegypti pupae for irradiation
Figure 2: Transferring pupae to Petri dishes for irradiation. (A) Sieved pupae are poured and backwashed into a 1000 mL plastic beaker. (B) Minimal water is retained in the beaker to facilitate pouring into Petri dishes. (C) Petri dishes lined up along the edge of a surface to facilitate pouring in a single layer of pupae. (D) Petri dishes loaded with pupae are stacked and secured for delivery to the irradiation facility. Please click here to view a larger version of this figure.
Figure 3: Sexing pupae using the genital lobe. (A) Ventral and (B) lateral views of female (♀) and male (♂) Aedes aegypti pupae, with genital lobes indicated to show the sexual dimorphism. Please click here to view a larger version of this figure.
4. Irradiation of male Aedes aegypti pupae
Figure 4: Laboratory book outline-IR sheet completed for a dose response set. Text boxes outlined in red (marked by red arrows) indicate useful notes on the different sections and reiterate key information. Please click here to view a larger version of this figure.
|Time (based on 8.8 Gy/min)
|1 min 8 s
|3 min 24 s
|5 min 41 s
|7 min 23 s
|9 min 39 s
|11 min 22 s
|12 min 30 s
Table 2: Example dosage times for the Cesium-137 irradiator.
Figure 5: Dosimetry data sheet populated with example data. The column headings prompt the operator to capture key data for later analysis. Please click here to view a larger version of this figure.
5. Rearing of irradiated male Aedes aegypti pupae into adults
6. Marking and weighing irradiated adult Aedes aegypti males
NOTE: This section of the protocol assumes two people are conducting the tasks; for 1 person, see 6.4.
Figure 6: Packing marked, irradiated, male Aedes aegypti into release containers. (A) Release container showing stockinette fastened to a hole cut in the side of the cardstock cylinder with masking tape, staples, and hot glue. The bezel is in place with a masking tape label backing affixed to the side. The bezel is retaining the tightly pulled tulle mesh cover; an elastic band (not visible) is also holding the tulle in place under the bezel. (B) Batch of anesthetized males in the process of being tumbled in pink dye in a small cardstock cup. (C) Four release containers inside insulated shipping container. Note the stockinette sleeves are oriented to the middle of the shipping container, packing materials are tucked around the release containers, and nutrition and hydration sources are in place on top of each release container covered by an inverted Petri dish bottom held fast by crossed elastic bands and bits of tape. Please click here to view a larger version of this figure.
|Weight of Mosquitoes
|Females in Batch
|Number of Males
Table 3: Weighing station data table.
7. Packing and shipping release containers of marked, irradiated, adult male Aedes aegypti
Vigilant and adequate mosquito rearing consists of well-balanced availability of males and females in colony cages, maintenance of fresh sucrose solution and honey, and consistent high-quality blood feeding. These conditions will provide for densely packed egg sheets optimal for use in SIT larval rearing pans. Proper storage and usage of dried egg sheets, such as systematic labeling to facilitate use from oldest to newest, will support uniform hatching across all pans. Filling all larval rearing pans with water prior to hatching can diminish the time for which the egg sheets are in hatch containers and promote healthy development. Maintenance of larval pans from hatch to pupation requires careful engagement by colony personnel as some pans may need more or less food or additional water depending on development stages and environmental variables. If there are issues with the stage of development by the scheduled day of pupal sex separation, adjustments should be made earlier in the process such as hatching earlier or later, adjusting food, or changing the incubator temperature.
The rearing process in this protocol does not render all eggs hatched in time to develop into pupae that can be irradiated and used for control purposes. Between 20 and 50% of the colony-reared mosquitoes will still be larvae by the time the pupae need to be separated. However, these larvae are not squandered, but allowed to mature for 24 h to render additional pupae that can be combined with female pupae from the previous day's separation and recycled back into colony cages. In the colony cages, pupae will be allowed to mature into adults, mate, bloodfeed, and produce eggs that sustain the SIT project.
Separating pupae, pouring pupae into Petri dishes, irradiation, and placement into adult holding cages after irradiation must happen in one day; hence, adequate time should be allotted to process all steps comfortably. The assembly and preparation of release containers should be done prior to the marking process. When shipping boxes are returned from the release site, release containers should be inspected and prepared for their next use. Discarding wet cotton balls, airing out wet release containers, cleaning Petri dishes, replacing mesh, and removing elastic bands from the container, while not in use, will greatly prolong the life of the release containers.
Given the worldwide reality of the COVID-19 viral pandemic, this protocol that is typically a multi-person operation has been modified to be tractable by one person working alone in a lab for each step. The steps in the process that are hindered most by a one-person scenario are the sexing, marking, weighing, and colony-rearing maintenance steps. Separating pupae by sex by one person should be sufficient if there are multiple separators operating simultaneously in different rooms. In a pandemic situation wherein social distancing occurs in the workplace, equipping multiple stations is required to complete steps from sexing to packing. Depending on the speed of the operator, it takes one person ~4 h to sex 15,000 mosquitoes and then another 1-2 h to mark, weigh, and package them. A two-person scenario diminishes the time during which mosquitoes are anesthetized for marking and reduces the overall work time. Yet, even in a two-person scenario, allocating the full 2.0 g of mosquitoes per release cage can be challenging due to limited work time while the mosquitoes are sedated. Although the process of cleaning and preparing larval and adult rearing materials is extremely time-consuming and labor-intensive, it can be partitioned such that individual operators can work independently and safely during a pandemic.
Releasing adult, marked, irradiated Aedes aegypti males is outside the scope of this protocol but is presented here in brief. The process of releasing marked, irradiated, male mosquitoes starts by determining a uniform release distribution of the release containers based on weights (and thus, inferred numbers of sterile males), as reported in Table 3. After shipments are delivered to the vector control district, the boxes are opened, and the release containers evaluated for any issues with mortality or condition of the release containers. Mosquitoes in the release containers are then allowed to acclimate to ambient temperature and humidity for 1-2 h prior to transport to the treatment area. Release sites in the treatment area are identified after intensive surveillance for hot spots of wild populations of Aedes aegypti. The timing, frequency, and density of releases is balanced by the bionomics of the species as well as meteorology, public support, and laboratory-rearing capabilities.
As specific release containers are matched to particular release sites, the label must be cross-checked before the release container is opened by cutting the mesh on the top, allowing the operator to deform the mesh so that a portion of the males may escape. This fractional release method is repeated at each assigned release point for the container until all freely flying males have been released. This process is then repeated for each release container at their respective assigned release location until all containers have been processed. Optionally, after the mosquitoes have been released, any dead or disabled mosquitoes that did not leave freely can be collected into Petri dishes and labeled to be counted by hand or weighed to correct the estimated number released. Ongoing and pervasive surveillance of adult, egg, and immature stages of wild Aedes aegypti in the target area, and possibly in non-intervention control sites, is conducted to assess the efficacy of the SIT operation.
Initiation of a control program featuring SIT that uses radiation requires the establishment of a local strain of Aedes aegypti. This step is critical and can allow SIT to truly distinguish itself from similar control technologies. By developing the project from a local strain of mosquito, the males generated will likely have behaviors that allow them to adapt to environmental changes and cues and to locate and mate with wild females in the vicinity. Furthermore, the release of irradiated local males may not generate negative public opinion compared to, say, release of a non-local strain of genetically modified mosquitoes that could, for example, introduce novel alleles into the local mosquito population.
Expending substantial resources to rear vast quantities of mosquitoes only to be able to use about half of them for control purposes is a limitation of the Aedes aegypti SIT program. Refinements should be made to the rearing protocol to condense the maturation of larvae into more defined timeframes when the pupae will be ready. This would allow more pupae to be collected at the optimal time of separation. However, additional pupae to process increases the risk of more females pupating when the pupae are collected and therefore increasing the likelihood of females ending up in Petri dishes with males and possibly being released. Although the lifespan, bloodfeeding behavior, and oviposition behavior in irradiated female Aedes aegypti pupae are reduced in adults, it is not a good strategy to release females incidentally alongside irradiated males22. Therefore, it should remain a priority to minimize the number of females inadvertently separated, irradiated, marked, and released with males.
Success of an SIT program ultimately relies on successful mate competition by colony-reared, irradiated males. Preserving male competitiveness relies on exhaustive experimentally derived selection of the dose and maximizing the estimated ratio of sterile:wild males in the population. Dose selection is determined by several key factors that include longevity, fertility, fecundity, and pupal mortality. It has been observed that male mosquitoes will exhibit an asymptotic fertility curve that approaches zero as radiation increases (KJL, RLA, SCB unpublished data). Simultaneously, male mosquito longevity and activity levels diminish exponentially as the radiation dose increases (KJL, RLA, SCB unpublished data). Therefore, rather than identifying a dose that yields 99.9% sterility in males, it is preferable to focus on a lower sterility percentage while supporting survivorship. Once a dose range is identified that does not differentiate longevity or pupal mortality of irradiated males from that of non-irradiated males, additional assessments on fertility should be conducted to identify a dose that renders males overwhelmingly sterile, yet competitive.
Simultaneously, it is critical to compare the number of male mosquitoes in the population to that of released irradiated males. This can be accomplished by collecting males from various locations in and around the target release area repeatedly from the same location and before, during, and after initiation of the SIT program. A mark, release, recapture study should be conducted to assess the ratio of wild male mosquitoes to released mosquitoes. A mark, release, recapture study relies on the release of a known number of marked mosquitoes from a specific point and their later recapture at points in close proximity surrounding the initial release point. By comparing the number of recaptured males and wild males at distances from the release point, it is possible to estimate the general wild population of males in the area so that competitive ratios of sterile males can be released23. Maximizing the ratio of sterile:wild males can be achieved by releasing more sterile males and/or by reducing the wild population by classical control means such as source reduction, immature control, or adulticide treatments.
To gauge the effectiveness of sterile male releases, adult collections can be compared chronologically against a non-intervention area. As sterile males are released and the number of collected males and females in an area diminishes in relation to a comparable non-intervention area, then it may be hypothesized that it is due to the released sterile males successfully outcompeting local fertile males. This effect can also be observed in oviposition trap cups deployed in both the intervention and non-intervention sites. Eggs may still be produced in the intervention site, but if fewer hatch than those from the non-intervention site, it may be hypothesized they are not fertilized because of females mating with sterile males. More and more oviposition of unfertilized eggs could eventually lead to reduced oviposition due to non-replacement of females in the intervention site8,24.
Future directions of SIT technology and programs naturally expand into additional medically important mosquito species. For instance, this technology may be readily adapted to control Aedes albopictus, given the very similar bionomics of Aedes aegypti and Aedes albopictus. Other disease vector mosquito species of interest include Culex quinquefasciatus, Culex tarsalis, and various Anopheles species. Improving the efficacy of this technology depends on increasing the capacity of male pupae produced at a given time, which could be achieved through genetic manipulation or artificial selection, and improving male competitiveness, which could be achieved by increasing virility, fertility, or longevity.
Ultimately, SIT programs are not a silver bullet for controlling mosquitoes. They are instead a tool in a suite of other control techniques, such as IVM programs, that cross-compensate for weaknesses among techniques. For example, whereas chemical control offers rapid and cheap control, it also fosters the development of resistance and non-target mortality; and whereas SIT is species-specific and is not likely to generate resistance, SIT males must be produced and released in perpetuity to control immigrating populations from outside the vector control district.
All authors have declared no conflicts of interest.
We thank Drs. R.-D. Xue, C. Bibbs, W. Qualls, and V. Aryaprema of the Anastasia Mosquito Control District, St. Augustine, Florida, for partnership in developing the SIT program and expert insight on effective operational release of sterile male Aedes aegypti. This research was supported by the USDA-ARS and the Florida Department of Agriculture and Consumer Services (FDACS). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by USDA or FDACS.
|1-1/8" wrench (1" (1 inch) = 2.54 cm)
|1/2 pint cardstock cup (1/2 pint = 236.5 mL)
|Science Supplies WLE corp
|1/4" tubing - tygon
|LLDPE1/8 X 1/4 BLK
|to attach to CO2 gas regulator
|1/8" brass barb w/ MIP connection
|to attach to CO2 gas regulator
|1000 mL graduated plastic beakers with handle
|3000 mL graduated plastic beakers with handle
|Adult large cage
|Bovine liver powder
|CO2 tank (20# canister)
|Cotton balls - large
|Deli cups w/lids - 470 mL
|Deli cups w/lids - 1900 mL
|ND0.5 and ND1.0 QA Filter Set standards
|Far West Technology, Inc.
|Forceps - fine featherweight
|4748 or 4750
|Radiation Machinery Corporation, Parsippany, NJ
|Hand held mechanical aspirator
|Large CO2 chamber
|Walmart # 568789514
|Larval rearing pans
|Blue Ridge Thermoforming
|Dimensions: 22.375 x 17.5 x 3 (inches)
|Magnets - 20# pull
|Petri dishes - large
|Petri dishes - small (60 mm x 15 mm)
|Red rubber hose
|Science Supplies WLE corp
|Rubber bands - cross #19
|Rubber bands - latitude #64
|Seed germination paper - Heavy stock 76#
|Shipping coolers- 16 x 13 x 12.5"
|Husky Foam Cooler kit
|Small cage - Bug Dorm
|Small CO2 chamber
|Walmart # 562922221
|Souffle cup lid
|Souffle cups - 4 oz (1 oz = 29.6 mL)
|extruded polystyrene foam
|Tropical fish flake food
|Vaccum chamber - desiccator
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