Live mosquitoes are needed to comprehensively study malaria and other vector borne diseases. Rearing mosquitoes is a tedious, repetitive process that involves heavy lifting and extensive time spent in a very hot room. Given the small margin of error, traditional rearing methods are challenging to learn and very susceptible to human error. Any small mistake can disrupt an entire cycle of hundreds of thousands of mosquitoes and ruin crucial long-term experiments.
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After breeding Anopheles stephensi mosquitoes by standard methodology for years, we decided to make some systematic changes in order to ease the workflow, standardize outputs, and increase efficiency. We made updates to six different parts of the rearing process. Each are detailed below.
Self-Emptying Bucket
Every day, up to 100 pans of water (~35 gallons) are drained to separate mosquito pupae from the larvae still developing. The pans are emptied into a 5-gallon bucket where a strainer collects the mosquitoes. Every 5-10 pans, the 40-pound water bucket needs to be hoisted up, walked over to the sink, and emptied. This is challenging and hazardous.
To ease this process, we added wheels for easy movement around the insectary and a pump inside the bucket that automatically drains the water into the sink. These minor adjustments hugely impacted the accessibility and efficiency of this process by significantly reducing the heavy lifting involved.
Pupation Station
After implementing the self-emptying bucket and experiencing the immense relief from heavy lifting, we were inspired to eliminate the daily task of emptying and refilling up to 100 pans of water and mosquitoes. This process is extremely time-consuming, physically taxing, and hazardous because it is being performed in an exceptionally hot and humid room.
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We engineered and machined a 6-foot-tall system containing six 4' x 6' hand-welded aluminum pans and a self-emptying trough to replace the manual process. There is a control panel with several different functions the user can choose from to automatically empty and refill any combination of the pans. Each pan is fixed with an electronically actuated valve that opens and closes to let mosquitoes and water through. This system allows for more mosquitoes to be reared per week at a rate 3 times faster and with absolutely no heavy lifting, just the press of a button. It also increases the consistency of the process which ultimately maximizes mosquito yields.
Pupal Funnel
After pans are drained, the larvae and pupae need to be separated from each other. Typically this is done by pouring the mosquitoes into a volumetric flask, letting the pupae rise to the top, and then pouring them off. The thin neck and small diameter of the volumetric flask limit the oxygen that the larvae beneath can get. Often, to prevent larval suffocation, pupae need to be poured out before all of them have risen to the top. This can be hazardous, as pupae left behind will emerge before they are properly enclosed.
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To improve larval viability, we built a bigger funnel with an inverted release system. Instead of a thin bottleneck, there is an acrylic cylinder which a much wider diameter. The increased surface area allows for better oxygen flow and decreases the chance of die off. The release valve at the base of the funnel allows larvae to be removed while the pupae are rising to the top rather than waiting until the separation is complete. This system maximizes larval survival and allows for cleaner separation between larvae and pupae.
Pupal Dish Vacuum
After the pupae are separated from the larvae, they are put in petri dishes full of water and placed inside cages. Once they emerge into adult mosquitoes, the petri dishes full of water and pupal cases need to be emptied to maintain cleanliness and mosquito health. Traditionally, this is done by carrying all the cages to the sink and using a hand pipette to remove and dispose of the waste. The pipette needs to be inserted into each cage up to five times in order to gather all the waste. This process is unnecessarily tedious and laborious.
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We created a vacuum system to rapidly empty pupal dishes with minimal effort. There is a waste flask with one tube connected to a vacuum pump and one tube connected to a serological pipette. The pipette tip is inserted into the cages while they remain in the incubator. Without turning off the vacuum, the pipette can be moved from one cage to another allowing for quick and continuous dish draining. This system is cleaner, requires no moving of cages, and is six times faster per cage than manual emptying.
Automatic Aspirator
Traditionally, to transfer mosquitoes from one cage to another, researchers use a mouth aspirator. One end gets inserted into a cage of mosquitoes and the researcher puts their mouth on the other end. They breathe in to pull in mosquitoes, hold their breath and move the tube from one cage to another, and then blow out to release the mosquitoes. This process is slow, uncomfortable, and outdated: mouths should never touch lab equipment.
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We designed a system that enables quicker, easier, and safer transfer of mosquitoes. The device consists of a lid which attaches to a vacuum pump to decrease pressure in the destination cage, and a tube which is used to pull mosquitoes from the original cage. The direct transfer tube design is a significant improvement over the mouth aspirator when considering mosquito survival because mosquitoes are not compacted against each other during transfer. In addition to the health and safety benefits of this system, the time required to aspirate a cage of mosquitoes is decreased by half.
Sugar Tubes
The standard method for feeding adult mosquitoes is soaking cotton balls in sugar water and placing them on top of the mosquito cage. The cotton balls dry out quickly and need to be replaced on a daily basis. There are often over 100 cages of mosquitoes, so replacing these sugar pads every day can be very labor-intensive.
In this new setup, the cotton ball is packed into a tube filled with sugar water so as the cotton ball dries out, it can wick up sugar water and remoisten. This prolongs sugar pad longevity by three times.