Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL^® (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

The control of Aegypti can be achieved using insects that contain a genetic modification that causes their offspring to die. When males are released, they mate with while females offspring produced from these matings will die. Population control can be achieved by continually releasing genetically sterile males.

The gene that causes death can be controlled by the addition of an antidote into the diet, allowing insects with the genetic modification to survive. To achieve population suppression of aegypti. Using a genetically modified mosquito line, large numbers of male mosquitoes need to be released.

This requires use of mass rearing techniques to obtain the maximum number of male mosquitoes with the highest quality and fitness possible. The mass rearing of OX 5 1 3 A can be divided into three main areas, egg production, the production of males, and the emergence And maturation of males before release. This is a schematic Of the rear end facility used for the production of OX 5 1 3 A.Males at Moscamed in Brazil, high quality eggs are continually produced in the egg production colony room.

These eggs are then reared through to pupi in the male production area where the pupi are then sex sorted. For males, a dedicated area is provided for quality control work. The males are then placed into release devices and allow to emerge and mature before release.

The techniques shown in this video are applicable to the egg production And male production areas. To obtain eggs For male production, we use artificial membrane feeding using animal blood from a local abattoir. The materials required for this process are biofilm and anin plate approximately 10 centimeters squared with a raised tread pattern.

On one side, some blood and a beanbag filled with approximately 250 grams of wheat grain. The plate feeding system uses paraform as a membrane for the mosquitoes to feed through to encourage feeding. The param is robbed over human skin.

The param is then carefully stretched to reduce its thickness. The pattern side of the aluminum plate is then covered in paraform on three sides, being careful not to damage the paraform and also sealing the edges well. Blood is then Added through the open side of the plate and paraform.

The feeder is filled to approximately one third with blood. The biofilm is then carefully sealed in place to Prevent the blood from escaping. The blood feeder Plates are then placed onto the top of the adult cages.

The beanbags are heated in a microwave for 10 seconds and placed onto the plate To heat the blood. If necessary, the beanbag can be reheated every 10 to 15 minutes. An FU position site is provided three days after blood feeding.

The ovary position site is filter paper inside a round plastic container Half filled with water. Place the op position site into the adult cage for the females to lay their eggs. Two days later, remove The egg paper and place onto absorbent paper to dry under insectory conditions.

When dry, the eggs are removed from the paper. Using a paintbrush, gently brush the eggs into a big tray, being careful not to damage the eggs. By brushing too hard, the eggs are passed through a sieve to remove dead insects and other debris to help prevent mold from forming.

And for more accurate counting of eggs By weight, the dried eggs Are set up into aliquots of one gram. This equates to 100, 000 eggs. Using an analytical scale into small plastic containers, the eggs can be stored in these containers under insectory conditions for up to six weeks.

For hatching eggs, jars are set up by adding boiled water, sealing the lid and allowing them to cool. This reduces the oxygen in the water for inducing hatching of the eggs. One gram of eggs is added to the jar and the lid resealed.

For one hour, transfer the contents of the jar to a tray with two liters of water and leave overnight. Under Insectory conditions. The day after hatching, The larvae are Sid from the tray through a grade 200 stainless steel sieve.

The diameter of the holes in the sieve is approximately north 0.075 millimeters. The larvae are then placed into a two liter beaker. Then the beaker is topped up to two liters with water.

Place the beaker onto a magnetic stir and add a large magnetic flea. Set the speed of stirring to adequately mix the larvae, but not too fast. As this can cause damage stirring, a larvae should be performed for the minimum time possible to help prevent damage for quality control.

The hatch rate is determined after the eggs have been hatched in the draw for one hour and also the following day, three, one mil aliquots are placed onto a white piece of absorbent paper with an absorbent sponge. Underneath the absorbent paper has a grid of squares which are used for counting. Count the number of hatched and non hatched eggs from three different grid squares.

A hatched egg is easy to identify. As the egg cap is missing from the top of the egg, we would expect a hatch rate of around 80 to 90%The average number of larvae per mil is used to calculate the volume of water to add to each tray. To get the desired number of larvae per tray, take three aliquots of one mil each and place into separate Petri dishes.

Add a further 10 to 20 mils of water to disperse the larvae for easier counting. Count the larvae using a black background as the larvae are white in appearance, and this makes counting easier. The use of a counter, a pipette, and a steady hand is recommended record.

The total number Of larvae per mil, the Appropriate volume of water and larvae are then aliquot into cups. Be careful to take the larvae from the same place in the beaker. Each time The larvae Are then added to the trays which have been prefilled with water.

The trays have the antidote tetracycline at a concentration of 30 micrograms per mil. The density of larvae in a tray has to be optimized for depth surface area and size of adults. We have found the optimum density to be between one and Three larvae per mil.

We use flake fish food for feeding Larvae. The fish food is crushed in a blender. To get a Consistent fine powder, each life stage requires Slightly different amounts of food.

So the diet has been optimized for each day and a specific weight is used per tray to reduce labor. We have designed and built volumetric spoons which dispense the required amount of food easily and efficiently. Male pube start appearing on day seven, and by day eight, about 70%of the males will have ated.

Females pate later than males, but there is still a significant proportion of females present on Day eight. Sorting of male Pube is performed on days eight and nine. To recover the maximum number of male PPE possible, the contents of a tray are sieved and placed into a jug ready for Sorting the plate.

Separator Uses the morphological And size differences between larvae and male and female pube to separate them. The Instrument comprises two parallel glass plates. The outer plate can be moved forwards and backwards using four adjustment screws.

The adjustment screws allow the outer plate to be set at an angle, so a wedge shape space can be formed between the plates tapering downwards. The plate separator is set up with a container underneath to catch the water. The larvae and peepee from a tray are poured into the top of the plate separator between the Glass plates.

Female pope are larger than Male pope and they separate above them. The larvae have a flattened shape that is thinner than male pube, and they separate further down by flushing with water and adjusting the angle Larvae and male and female pube Can be separated further with Continuous flushing and adjusting the plate angle. Using the bottom adjustment screws, the larvae can be flushed through into a container.

The container with larvae is removed And a new container is then placed under the plate separator. The male puy can then be flushed into the new container. Be careful not to allow Any female puy to be flushed through with the Males.

Remove the container with a male poopie and replace with a fresh one. And finally, the female PPE are flushed into the fresh container. The male pupi are quality Controlled for female contamination.

After sorting, three aliquots of 500 male pupi are taken and the number of female pube present counted down a microscope. Females can be identified by looking at the genital lobe shape. The female genital lobe is much smaller and rounded than the male.

If there is more than 1%females, the batch should be resorted And checked again. The sorted male pupi Are allotted into release devices to emerge and mature before Release into the field. Results from Over six months of sorting male and female.

Pupi show that female contamination on average was 0.02%This contamination rate represents only 400 females released each month. This graph shows the average male and female pupation data over a six month period of production. The male's pupate mainly on day eight and females on day nine.

The size of pupi was measured and compared using the celo thorax width measured by taking photographs of pupi floating in water and using a greico as a scale. Males are consistently smaller than females with very little overlap in size, showing consistent rearing parameters were achieved from the egg production facility. An area of approximately 19 meters squared, over 4 million eggs per week were produced from the male production area.

An average of 571, 000 males per week were produced. The efficiency of production from egg to sorted male pupi was around 26%Some loss of males occurred during emergence, and the total number of males released per week averaged 543, 000.