Bureau of Methods Development &
Biological Control
Bureau Chief: Dr. Trevor Smith, Trevor.Smith@freshfromflorida.com
Assistant Bureau Chief: Abbie Jo Fox, Abbie.Fox@freshfromflorida.com
Quality Assurance
Biological Scientist: Suzanne Fraser, Suzanne.Fraser@freshfromflorida.com
Project Staff
Ronald Rojas
In order to determine the quality of lab-reared flies, a number of tests are performed on each lot. These tests give an early indication of problems in the colony, so corrective action can be taken to maintain and improve product quality. Required tests for Caribbean fruit fly include egg hatch, pupal weight, emergence, sex ratio, flight ability and mating propensity.
Egg Hatch
This test is used two ways. First, an initial hatch count is taken to determine the proper time to place the eggs on larval diet, normally 3 days after collection. Second, a week after collection, a final count is taken to determine maximum hatch. Four replicates from each egg collection are prepared. Black filter paper is placed into the inverted top of a standard petri dish and moistened with acidified sodium benzoate solution. One hundred eggs are spread in a straight line on the filter paper. The petri dishes are sealed with parafilm and placed in the incubation boxes until hatch is counted.
Pupal Weight
Since pupae lose water as they develop, weights are taken about 2-3 days before fly emergence. Five sets of 100 pupae each are taken from the sample and weighed. This weight is closely correlated to the sex ratio.
Sex Ratio
One hundred pupae are placed into petri dishes and left to emerge. Four days after emergence the flies are frozen and males and females are counted. An ideal ratio is 50:50. If the number of males drops below the established limit of 45, possible causes need to be investigated and corrected.
Emergence and Flight Ability
One hundred pupae each are placed into 5-14 cm black plexiglas tubes that have been coated inside with talc. As the flies emerge, the only way out of the tube is to fly since they are not able to walk across the powdered surface of the tube. The test is finished one week after the first emergence. Results are obtained by counting the unemerged pupae, partially emerged, deformed and non-fliers to determine the per cent emergence and flight ability.
Mating Propensity
Within 2 hours of emergence, 150 males and 150 females are collected and placed into 6 cups each, 25 to a cup. They are allowed to mature for 1 week with food and water. The test is conducted in the late afternoon, during peak mating time for the Caribbean fruit fly. Under low light conditions, males are placed into the mating cages and the females are added within 5 minutes. The lights are adjusted and the timer is set for the first 10 minute period. As the flies mate they are removed from the cages. The couples are counted and removed for each 10 minute period for up to 1 hour. Each time period is weighted with the first being the best and the last being the least desirable. From this data, a mating index is determined. An index of 75 for pre-irradiated flies and 65 for post-irradiated is acceptable.
Mating Compatibility
This test is conducted in outdoor field cages, outfitted with host plants, to determine if the sterile lab strain will mate easily with flies in a wild population, which is critical for a successful sterile insect technique (SIT) control program. Wild flies are obtained by collecting infested fruit and placing on vermiculite. Larvae will drop from the fruit and pupate. In 7-10 days after migration, the pupae are sieved and placed in a stock cage for eclosion. Lab-reared pupae are dyed with Day-glo dye, irradiated and placed in a separate stock cage. Twenty-four hours post-eclosion, males and females from both cages are aspirated and placed into separate holding cups with food and water and allowed to mature for the appropriate time period for the species being tested.
On the day of the test, 50 males of each strain are placed in the field cage and allowed to establish leks for at least 30 minutes. Fifty of each type of female is added in the cage. As the flies mate each pair is placed in a small vial. At the end of the mating period, the vials are frozen, then checked under a black light and the mating combinations are recorded.
From this data, a number of indices can be calculated. These results indicate competitiveness of the sterile male and compatibility between the strains and the mating propensity of the males of each strain as well as the females.
For additional explanation of this and the above tests, consult the Product Quality Control, Irradiation and Shipping Procedures for Mass-Reared Tephritid Fruit Flies for Sterile Insect Release Programs, the standard quality control manual developed by the USDA, IAEA and FAO. http://www.iaea.or.at/programmes/nafa/d4/public/d4_pbl_5_1.html.
Other quality assurance monitoring is conducted in the facility, such as environmental air quality, diet ingredients, and prepared diet. Once per week, potato dextrose and tryptone glucose plates are exposed for 20 minutes at designated areas of the facility as well as the outside air to measure the microbial load at each station. The plates are incubated for 3 days. Bacteria mold and yeast colonies are counted and compared to established upper control limits and to the outside air plate. Possible causes for high counts are investigated, so that corrective action can be taken.
Samples of prepared diet are collected as it is dispensed into trays. The temperature of the diet is recorded. Water content is determined to ensure proper levels for successful rearing of the Caribbean fruit fly. The microbial load is checked to ensure that the dispensing auger and the diet mixer are clean.
As new lots of corn cob grit are received, samples are taken from each pallet and plated for microorganisms. This data provides additional information if a particular lot performs poorly.
Photo credit: Suzanne Fraser-DPI
