Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The unique value of this protocol is that two personnel using readily available equipment in small lab spaces can produce enough sterile mal mosquitoes to support a 50 acre operational SIT program. The efficiency and low cost of this technique makes it accessible, intractable for even small mosquito control districts to augment their integrated vector control program. Demonstrating the procedure will be myself and Barbara Bayer, biological science laboratory technicians from the research unit.

To rear adult Aedes aegypti from pupae, prepare 60 by 60 by 60 centimeter collapsible aluminum rearing cages with 0.84 by 0.84 millimeter hole size fiberglass mesh screening, and a reach-in stockinette sleeve on one vertical wall of each cage. Place a 1, 900 milliliter plastic cup with Aedes aegypti pupae at a one to one sex ratio in each rearing cage and tie the sleeve closed, leaving the cups in cages for eclosion for about four days at 28 to 30 degrees Celsius greater than 50%relative humidity in a 12:12 or 14:10 light:dark cycle until no more adults emerge. 24 hours after placing the pupae in the rearing cages, place a container of 10%sucrose solution with a sponge wick into each cage and suspend a 10 by 2 centimeter sponge soaked in honey from a wire hook to provide hydration and nutrition respectively to the adult mosquitoes.

48 to 72 hours after most adults have emerged in every two to three days for the next three to four weeks thereafter, fill the one lambskin condom with 50 to 100 milliliters of defibrinated bovine blood per cage, and warm the condoms to approximately 37 degrees Celsius in a hot water bath. After carefully patting down the condoms with the towel, place the partially dried condoms in individual paper lined Petri dishes inside the cages for 30 to 60 minutes. 48 to 72 hours after each blood feeding, place a 1, 900 milliliter oviposition cup containing 800 milliliters of filtered pupal water and furnished with an 8 to 10 by 30 centimeter sheet of seed germination paper fitted flush along the inner circumference of the cup into each cage.

To rear the Aedes aegypti larvae from eggs, use an oviposition paper with 5, 000 to 10, 000 eggs present and cut a three to seven centimeter section perpendicular to the oviposition line. Place the piece into a new 460 milliliter container half filled with tap water and a pinch of pulverized fish food flakes. Cover and agitate the container vigorously for at least one minute before pouring the entire contents into a larval rearing pan containing three liters of tap water and 50 milliliters of brown slurry.

Mark the pan with the start date, strain information, and feeding schedule. Once the approximate threshold proportion of larvae in the larval pans has been reached, pour the contents of each pan through a size 20 to 40 sieve and use a squeeze bottle of tap water to wash the pupae and larvae out of the sieve into a three liter graduated plastic beaker. To separate the male pupae from the larvae and female pupae, split the contents of each beaker between multiple 1.9 liter plastic containers and place a rigid shallow four liter collection container below the sluice at the base of a pupal separator.

Fill two three liter graduated plastic beakers about three quarters full of tap water and pour the water through the space between the glass plates of the separator while adjusting the top and bottom knobs to allow the water to continuously flow through the separator. Once standing water has collected evenly at an approximately 1.25 centimeter height from the base of the plates, mark the starting positions at the bottom knobs of the separator with tape, and begin pouring the containers of pupae and larvae into the separator while continuously rotating the bottom knobs in a counterclockwise manner one to two centimeters from the starting positions. When most or all of the larvae have washed through the sluice into the collection container, pause the water flow and use a number 30 sieve to clear the collection container of the larvae.

When all the larvae have been removed, pour the water and adjust the knobs again to allow the male pupae to be flushed into the collection container. Then pour the male pupae from the container into a number 20 sieve and use a one liter squeeze bottle of tap water to wash the pupae from the sieve into a separate 1.9 liter container. When all of the male pupae have been collected, pour the water and adjust the knobs to allow the female pupae to be collected.

Using the number 20 sieve to transfer the female pupae into a new 1.9 liter container as just demonstrated. Then pour the water and adjust the knobs again until all of the immature mosquitoes have been flushed out of the separator. Strain the collected male pupae with a sieve and wash the poopy into a one liter graduated beaker with as little water as possible.

Carefully pour the pupae into Petri dishes lined with moist filter paper in a single layer. Remove standing water from the Petri dish bottom using a fresh three milliliter pasture pipette to prevent pupal movement during working. Bundle Petri dishes and stacks before sealing inside a labeled 3.8 liter resealable bag to fit in the irradiation chamber.

To mark quantified batches of adult irradiated male mosquitoes, remove any nutrition sources from the rearing cage and place one cage into a large carbon dioxide chamber for five to seven minutes, knocking the sides of the container to dislodge any mosquitoes clinging to the walls. At the end of the exposure period, transfer the mosquitoes into an aspirator vial and anesthetize the mosquitoes again before shaking the mosquitoes into a white paper covered work surface. Carefully aspirate all the males from the work surface into a new vial and repeat the exposures and collection until all adult males have been collected from all the rearing cages.

At some point during the sex sorting process, generate a vial containing only 25 adult male mosquitoes. When all the males have been collected, weigh each vile of mosquitoes in a tared weigh boat on a balance, and quickly pour the weighed mosquitoes into a 240 milliliter cardstock container containing 50 milligrams a marking dye. Tilt and rotate the container one full rotation clockwise and counterclockwise until all of the mosquitoes have been lightly coated and pour the mosquitoes into a new weigh boat, then fold the weigh boat in half to allow the mosquitoes to be channeled through the stockinette sleeve into the release container.

When approximately two grams of male mosquitoes have been added to the release container, tied the stockinette sleeve closed and mark the labeling tape on the bezel of the release container with the dye color, container number, and total weight of the mosquitoes. In this representative male adult Aedes aegypti mosquito rearing, irradiation, and marketing experiment, two grams of male adult mosquitoes were able to be collected from three rearing cages and along with two vials of 25 males from two cages to allow calculation of the average weight per mosquito. 25 females were discarded in total from two of the batches.

Timely and observational feeding during the week of larval rearing is critical for generating well-developed pupae for separation. This generates a clear distinction between males and females leading to a low occurrence of females in the marking steps. Besides using them in operational SIT programs, these sterile males can also be used extensively in experiments investigating mating competition, survival, and dispersal of irradiated compared to wild non-irradiated males.

Modifying the technique to include female mosquitoes allows us to investigate the impact of unintentional release of irradiated females with regard to fecundity and vector competence.