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

Insectary Requirements

1. Overview

1. Mass rearing has several phases, each of which is performed in a distinct area of the mass-rearing unit. Figure 1 shows the main phase of rearing and where they occur in the mass rearing facility.

2. Biosecurity Considerations for the Insectary

1. The project was approved by the committee of animal ethics from the University of São Paulo - Brazil and all necessary permits and approvals were obtained.
2. The RIDL system uses conditional lethality; a dietary supplement (tetracycline) prevents the expression of the RIDL system allowing the insects to be mass reared; without tetracycline the mosquitoes die. Tetracycline is not found in the breeding sites of Ae. aegypti in the wild so any offspring produced from escapees will not survive.  This adds an extra level of security over using wild-type (WT) insects sterilized by other methods (i.e. radiation).
3. Use double-door entry, “Authorized Personnel Only” signs and insect screens in the insectary. Any windows should be sealed to avoid contamination of the colony with WT insects and help prevent escapes from inside the insectary.
4. Local regulations and authorities may have specific additional or alternative requirements. Contact the relevant authorities for information/guidance at an early stage, and secure all necessary permits and approvals before establishing the RIDL strain in an insectary.
5. The mass rearing insectary should only be used for RIDL rearing. No other strains or insects should be present. This is to avoid contamination of the RIDL strain and contamination with pathogens that may affect fitness and survival, reducing the productivity and effectiveness of released males.

3. Insectary Design

1. Figure 2 shows a plan of the insectary used at Biofábrica Moscamed in Brasil.
2. Temperature of all rooms was 26 °C ± 2) with a relative humidity approximately 70-80% and a 12:12 hr light:dark cycle using fluorescent light strips.
3. This blueprint can be modified according to specific space requirements, e.g. the scale of the release will determine overall size. However the egg production, rearing and sorting, Quality Control (QC) and releases should be separated into four distinct areas: Egg Production Colony, Release Generation, QC, and Adult Storage for Release.
4. The Egg Production Colony room (approximately 20 m²) is used to produce eggs for the release generation.
5. The Quality Control area is where experiments are conducted to check procedures and the fitness of the RIDL line including measurements of hatch rate, pupae numbers, pupa size, and measuring effectiveness of sorting and removing females. Tests are also conducted for WT contamination by checking for the presence of fluorescence marker and phenotype using offset assays measuring lethality.
6. The Release Generation room (approximately 36 m²) requires adequate space for trays and the sorting of larvae and pupae. The trays are held in purpose-built aluminum racks. A large work area and sink are required for rearing and sorting and another large sink is required for washing of trays and cages. A filter catch trap has been included in the waste water outlet from the sinks to mitigate the escape of living mosquitoes through the sewer. 
7. All water used for the trays is disposed through a drain, which has a compartment containing a mesh that prevents the escape of insects in any stage of development.
8. An area is required for the storage of adults for release (Adult Storage for Release, Figure 2) containing racking to hold the release devices while the adults mature before release.

Production Methods for Mass Rearing RIDL:

The Egg Production Colony produces the eggs that are used in the Release Generation (Figure 1) to produce adults. There are many similarities in the two rearing methods but also some distinct differences. The rearing processes that are the same for both procedures are described in the Egg Production Colony section only.

Egg Production Colony

4. Larval Production

The Egg Production Colony generates homozygous OX513A RIDL eggs⁸ for the Release Generation. High quality control ensures viability, fitness and strain integrity of the eggs supplied.

1. The stimulus for hatching is submersion in water with a low level of dissolved oxygen. To reduce the oxygen level in the water, heat water until it boils and immediately place 400 ml into 500 ml glass jars (74 mm opening circumference), fasten the lid securely and leave at room temperature for several hours to cool.
2. Put 1 g of eggs into a jar of boiled water, reseal and wait for an hour. Transfer the contents to a tray with 2 L water and leave overnight under insectary conditions.
3. Place the hatched larvae into a known volume of water and stir using a magnetic stirrer and flea for sufficient time to take aliquots; stirring vigorously or for extended time damages larvae and should be kept to a minimum. The standard volume used to hatch larvae is 1 L, however densities of more than 300 larvae/ml are hard to count and may be damaging. Therefore do not hatch more than about 300,000 eggs/L of water.
4. Determine the hatch rate by taking three 1 ml aliquots and placing onto a sheet of absorbent paper, with a grid of 1 cm squares, on top of an absorbent sponge to soak up any excess water.  Count the number of hatched and nonhatched eggs from three squares by looking for the missing cap of a hatched egg. A hatch rate around 80-90% is expected.
5. Take four aliquots of 1 ml each and place into four black weigh boats (larvae are white and therefore more readily visible against a black background) and count the number of live larvae present by eye; the use of a counter is recommended. The average number of larvae per ml is then be used to calculate the volume of water to add on each tray to get the desired number of larvae per tray.
6. There can be large differences in the density at which larvae can be reared between strains and species. It is also desirable to compare RIDL adult male size to wild type males; ideally you want similar to larger sized RIDL males that can compete successfully with wild males. Therefore for each RIDL strain the ideal density of larvae needs to be determined and is a trade-off against space constraints for rearing and the scale of production. Densities ranging from 0.1-2.5 larva/ml we have found to be  a good combination of production output and quality; also discussed by Medici²³.
7. Add the larvae to trays. At our facility we use trays measuring about 53 cm x 37 cm  x 8 cm (L x W x H) and the required amount of water to achieve the density of larvae for production.
8. Add a stock solution of tetracycline (3 mg/ml in water) to the trays at a 1:100 dilution to obtain the required final concentration of 30 µg/ml.
9. Feed larvae with flake fish food (www.sera.de) daily, which has been crushed to a fine powder. Table 1 shows the typical feeding regime in mg of food per larvae per day. Feed larvae until the appropriate day of sorting under the insectary conditions described above.

5. Sorting Larvae from Pupae and Male/Female Pupae

1. Sort larvae from pupae 8 days after hatching by separating the larvae from the pupae. Then sex-sort the pupae for males. A device known as a plate separator^24,25 can sort larvae from male pupae and from female pupae. Figure 3 illustrates the use of this plate separator to separate larvae from male and from female pupae.
2. Using a measuring spoon with a mesh on the bottom, calibrate it with 500 pupae or more and use it to set up cages and estimate the total amount of males and females pupae produced.
3. On the first day of sorting (day 8) most of the pupae are males, so place the sorted larvae back into a tray and rear through to the following day when most of the pupae are females (Figure 4).
4. Repeat the sorting process each day as described in steps 2.1-2.3 until all the larvae have pupated or until the end of a working week.
5. Place 1,000 male pupae (using the measure spoon) in a cage and on day 9 add 3,000 female pupae to the same cage (we use 30 cm high and 30 cm diameter PVC pipes adapted with mesh top and a netted access hole, but 30 cm x 30 cm x 30 cm plastic cages are also available from BugDorm).
6. Allow adults to mate for at least 2 days and provide them with sucrose solution (10%) on wet cotton ad libitum before blood feeding. Collect eggs for two weeks (~two gonotrophic cycles) with two blood feeds a week. This yields an average 143,000 eggs from a cage of 3,000 females (an average of 48 eggs/female). For production of 4 million eggs a week about 28 cages need to be set up each week.

6. Blood Feeding

1. An aluminum plate feeding system supplies cages with blood twice a week. A pocket of blood is created on side of an aluminum plate (10 cm x 10 cm x 3 mm) with Parafilm (Figure 5). To encourage feeding, blood is warmed by placing a heated bean bag on top of plate feeder. The bean bag consisted of approximately 250 g of wheat grains in a cloth bag, which is heated for about 10 sec in a microwave. The blood we use for feeding is from a local abattoir; as these animals (mainly goats and sheep) are for human consumption they are tested to avoid the presence of pathogens.
2. Three days after blood feeding, an oviposition site for the females to lay their eggs is provided. The oviposition site is a round plastic container filled to about ¼ with water and filter paper covering the inside. Two days later the oviposition site is removed. For maximum egg production, make sure the filter paper covers the entire available surface inside the container and remains wet.
3. Remove the egg paper and place onto absorbent paper to dry under insectary conditions; eggs require a period of conditioning at high humidity (over 70%) of at least 48 hr after being laid. Eggs can be left in the insectary for up to 3 months with minimal decrease in hatch rate, as long as the humidity is kept high²⁵.
4. The egg production colony needs to be of sufficient size to provide the number of eggs required on a weekly basis for the release program. The facility described above can produce approximately 4 million eggs with 28 cages set up weekly. It is recommended to keep enough eggs stored to guarantee at least 4 weeks of eggs for Release Generation and Egg Production Colony.

Release Generation

7. Larval Production

The hatching, rearing and sorting processes for the release generation are identical to the previously described method for the Egg Production colony. However, the scale of production is much larger and this requires more trays and more effort.

8. Sorting Larvae, Male Pupae, and Female Pupae

1. The sorting of larvae, male pupae and female pupae is identical to method described for Egg Production.
2. After sorting the male pupae it is important to check for female contamination before release; there should not be more than 1% females present. Take three aliquots of 500 pupae (three random pupae measuring spoons) and count the number of female pupae present. Females can be identified as they are generally larger than males and from differences in the genital lobe shape (Figure 6)²⁷. If there is greater than 1% females they should be resorted and checked again.
3. Use only the males from day 8 and 9 for release; autoclave any remaining larvae and pupae. In Brazil, this waste must be treated the same way as medical waste but regulations may differ in other countries. Do not use female pupae and larvae from the Release Generation for the Egg Production Colony because of differences in quality control, rearing and contamination risks from larger numbers, and lower quality control.

Adult Storage for Release: Place males into release devices to emerge and mature before release. The details of the release devices are not covered in this method. However the capacity of the release devices we use is around 1,000 males. The release method details (release device and release system) are not covered in this method.