Annex 9 Processes Quality Control. Introduction

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Chastity Day
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1 Annex 9 Processes Quality Control Introduction The eradication of screwworms depends on the release and dispersal into the environment of sterile screwworm flies in optimum biological condition providing the opportunity for the sterile male to mate with an indigenous fertile fly. The Sterile Insect Technique (SIT) is the release of massive numbers of sterile insects that overwhelm the native fly population resulting in gradual elimination or autocide of the native fly population. Successful eradication of screwworms depends on sterile males mating with fertile females and the sterile male must be able to compete with fertile males for the female. Overwhelming numbers improve the odds for the sterile male, but the quality of the sterile male fly must be such that it is strong and can survive and thrive in the environment. The production and release of sterile insects is an industrial process and the traditional concepts of quality control are applied to this fragile and perishable product. Quality control (QC) is a procedure or set of procedures intended to ensure that the product adheres to a defined set of quality criteria and meets the requirements of the client. Specific quality parameters and sampling procedures are established; and data are collected and analyzed. Program managers review reports and decide upon corrective actions if necessary. The QC process is constant to ensure that the product is satisfactory. The QC process to ensure that the insect is released into the environment in optimum biological condition begins at the Sterile Insect Production Facility and ends with an evaluation of the performance of the insect in the field. Quality parameters are established and sampling procedures are carried out during the rearing of larvae; irradiation, storage, and transport of pupae; emergence of adult flies at processing centers; preparation of flies for release; dispersal of flies into the field; and competitiveness with the indigenous fly population. A Quality Assurance process is also carried out to ensure that development of new methodologies and technologies result in a product of acceptable quality. A complete description of Methods and Development functions are not within the scope of this guide. Suffice it to say that Methods and Development is responsible for the application or adaptation of scientific research. Methods and Development evaluates new technologies and methodologies for rearing and irradiation of insects; new methods for handling, storage, and transport of insects; new techniques for the release of insects for dispersal; etc. and ensures that the quality of the insect meets acceptable parameters. Methods and Development is also responsible for the development, evaluation, and adaptation of new strains of insects to the industrial process and ensures that the new strain is competitive with the indigenous fly population. The Quality Control Unit is responsible for ensuring that the insect received from the production facility is released into the field in optimum biological condition and able to

2 compete with the indigenous fly population. Under the direction of a department manager, a team of Biological Technicians carry out quality control testing under laboratory and field conditions. Laboratory QC sampling and testing is conducted 24/7 and sufficient personnel should be available at all times and depending on the number of personnel, leaders should be assigned to each shift. Field Testing is conducted daily and sufficient personnel should be available. Laboratory The Quality Control Unit at the Insect Processing Center carries out routine tests to ensure the quality of the product provided by the Sterile Insect Production Facility is within established parameters. Routine and regular reports and analyses are prepared and submitted to Program Managers. Significant variations are investigated as to cause and corrections, if necessary, approved and implemented. Insects are shipped from the production facility in the pupa stage and as the insect develops inside the puparium it generates heat and noxious carbon dioxide and excessive levels of both will harm the insect; therefore control of temperature and ventilation is essential. Current practice is to ship pupae as bulk placing 44 liters of pupae in Styrofoam coolers and transported in aircraft. At the Insect Processing Center pupae are received from the Sterile Insect Production Facility, processed to emerge the fly, and the flies packaged for release. Currently there are two systems for emergence and release of flies; pupae packaged in carton boxes with flies emerged inside the box; and the free release of chilled flies emerged and collected in environmental chambers. The following procedures applied to the Free Release of Chilled Flies. The Sterile Insect Production Facility produces insects in groups and pupae are identified according to group and irradiation dates and it is useful to apply QC to these groups as well as the entire shipment of pupae. Pupae Transport and Reception The insect, even though chilled, continues to develop and generates heat and CO 2. The internal temperature of the pupae transport containers should be recorded immediately upon arrival to the Insect Processing Center. Temperature should range between 12 C 21 C. Temperatures above this range stimulate rapid development of the fly within the puparium resulting in a buildup of heat and noxious CO 2. Quality Control reviews the shipping manifest provided by the production facility recording the number of shipping containers and each production group. The shipment is divided evenly for each Hot Room and the internal temperature of each shipping container is recorded. A sample of approximately 50ml of pupae is collected from each container for routine testing and placed in holding trays in the Laboratory Control Test Room. Information is recorded in Form: Pupae Distribution.

3 Emergence The pupae provided by the Sterile Insect Production Facility are allowed to emerge under controlled conditions and then collected as flies and held under chill until sufficient quantities are ready for release. The rate of emergence of flies from pupae is determined and is used to estimate the quantity of flies available and when they are ready for release. A slow rate of emergence could result in flies held for excessive amount of time causing increased mortality and weak flies. The overall emergence of flies from pupae is an important quality parameter and significant variations may indicate improper handling of the pupae during shipment or problems originating in the production facility related to genetics or rearing processes. Hot Room Emergence: A test is conducted to determine the percent of emergence of pupae within the Hot Room. Two QC test cartons containing 500 pupae each from a homogenous mix of pupae representing each Hot Room are placed in the back and front of each Hot Room. Likewise, 500 pupae contained in QC test cartons from a homogenous mix representing each production group are located in the Hot Room near each respective production group. At the conclusion of the collection process the Hot Rooms are opened, the boxes are retrieved and the flies that emerged and did not emerge are counted. To separate the un-emerged pupae, lightly blow away the casings. The number of dead flies is also counted. Percentages are calculated and recorded in Form: Hot Room Emergence. The information is useful for comparison with the Emergence Control Test and for calculating the yield of flies from pupae. Emergence 2 hours The rate and span of emergence is calculated and is useful for comparison of trends. Significant variations may detect abnormalities of genetic disposition, problems during the rearing process, or transport of pupae. Sample sets of 500 pupae are collected from each Hot Room and taken to the Laboratory Control Test Room where the temperature ( C) and humidity (50-60%) are controlled. The pupae are placed in a Petri dish in an insect cage and observed every 2 hours. The number of flies that have emerged is registered and the Petri dish with pupae is removed to another insect cage. The cage with emerged flies is placed in a freezer for 10 minutes; the dead flies are collected and accumulate. The test continues until the last collection of flies. The results are recorded in Form: Emergence Span. At the conclusion, 30ml of the accumulated flies are separated according to sex and counted. The sex ratio is calculated and recorded in Form: Sex Ratio. Emergence and Mortality at 24 and 48 hours.

4 Flies are allowed to emerge under controlled condition and the percentage of emergence and mortality is calculated. The information is useful to estimate production within the Hot Room. Sample sets of 500 pupae are collected from each Hot Room and taken to the Laboratory Control Test Room where the temperature ( C) and humidity (50-60%) are controlled. The pupae are placed in a Petri dish in insect cages. When emergence reaches 1% calculated from the Emergence 2 Hour test time is initated and 24 hours later the cages are observed. The number of flies that have died is registered and the Petri dish with pupae is removed to another insect cage. The cage with emerged flies is placed in a freezer for 10 minutes and flies then counted. The total number of emerged flies, non-emerged flies, and dead flies are recorded in Form: Emergence 24 and 48 hours. After an additional 24 hours the cages are observed and the number of flies that have died is registered. The cage is placed in a freezer for 10 minutes and flies then counted. The total number of emerged flies, non-emerged flies, and dead flies are recorded in Form: Emergence 24 and 48 hours. The number of emerged, non-emerged, and dead flies at 24 hours and 48 hours are added and percentage of emergence and mortality is calculated. Results are recorded in Form: Emergence 24 and 48 hours. Pupae The quality of pupae received from the production facility is evaluated using several parameters. A homogenous 50 ml. sample of pupae representing each irradiation group collected from each pupae transport container is used to conduct these routine tests. The number of pupae per liter is a factor of the size of individual pupae. The information is useful to estimate and report total production volume as well as comparison of trends that may reflect insect quality. The sterile insect production facility can become contaminated with domestic houseflies and it is not unusual to encounter their pupa mixed with the screwworm pupae. Pupae that are malformed are also encountered and it is useful to record trends that may indicate overall insect quality. The average pupal weight is calculated and trends are useful to adjust processing requirements as well as to evaluate overall insect quality. The pupal weight often is a factor of rearing and genetics. Procedure: A homogenous mix of ½ liter of pupae is used to determine the number of pupae per liter and pupae weight. After freezing the sample for 30 minutes to stop pupae development, production debris, M. domestica pupae, and mal-formed pupae are separated. The number

5 of M. domestica is counted and recorded; mal-formed pupae are graded according to the following chart and the number is counted and recorded in Form: Pupae Count Pupae Count: The ½ liter of pupae is divided into 15 replicates of 30ml plus what is leftover. Each replicate is weighed and the pupae in each replicate are counted. The number of pupae per liter is calculated from pupae count of each replicate multiplied by 2. The information is recorded in Form: Pupae Count. Pupae Weight: An average pupal weight is calculated from the weight of each replicate divided by the number of pupae in each replicate. Results are recorded in Form: Pupae Group Weight. An individual pupal weight is determined from two random sample sets of 50 pupae each. Results are recorded in Form: Pupae Individual Weight. Adult Flies As the fly emerges and is collected in the Cold Room, the quality of the insect is evaluated. It is important to note that flies held under chill continue to metabolize and produce heat. Flies that are held for more than 24 hours deplete energy stores and the quality of the insect deteriorates. Flies per liter Under the free release of chilled flies, the average number of flies per liter is used to calculate the amount of flies to be loaded into the release containers and subsequently, the amount of flies released for dispersal. The information is also used to calculate total production as well as historical comparison to detect trends that may reflect insect quality. A sample set of ½ liter of flies is collected from each Hot Room during the 2nd, 9th, and 17th collection and placed in a freezer for 1½ hours. Flies are counted and results are recorded in Form: Flies per Liter. Fly weight The average fly weight is calculated and trends are useful to detect variations of the production process as well as to evaluate overall insect quality. A homogenous mix of 50 individual insects representing flies from each Hot Room is weighed as a group. The average fly weight is calculated and results are recorded in Form: Fly Weight.

6 50 individual insects from this sample set are weighed individually. The average fly weight is calculated and results are recorded in Form: Fly Weight. Sex Ratio The ratio of males to females is calculated and trends are useful to evaluate overall insect quality. An acceptable parameter is 50:50 plus or minus 10. A homogenous mix of 30 ml of flies representing each Hot Room is collected from the 9th collection. The males and females are separated and counted. The ratio is calculated and results recorded in Form: Sex Ratio. Sample Set 90ml. A homogenous sample set of 90ml of flies is collected from each Hot Room during the 9th collection and counted. Results are used when conducting the flight agility test. Results are recorded in Form: Flight Agility. Flight Agility The Flight Agility test measures the number of flies that are able to fly and is conducted at several stages during the fly collection and release process. A control test with insects held under laboratory conditions is also conducted. The test is conducted with a defined amount of flies held in a QC test carton with partition that is placed in a flight agility box (See design specifications) in an open area and allowed to fly from the box. The percentage of dead flies and non-emerged flies is calculated as well as those with deformed wings, broken wings or legs. An acceptable parameter is 90% of flies that are able to fly. Factors that affect the flight ability are: weak flies when held under chilled conditions for excessive time prior to release; flies with excessive number of broken wings and legs; flies with excessive wing deformities; etc. Control Test: A homogenous sample of 500 pupae is placed in standard QC test carton, provided with a food source (50:50 mixture of honey and water), and held in the Laboratory Control Test Room for the duration of the production process. In addition to flight agility and mortality, the percentage of non-emerged pupae is also calculated. 24 and 48 hours test: Approximately 600 ml of flies collected from initial emergence and cross-over of each Hot Room is set aside. When these flies have been in chill for 24 hours and 48 hours, sample sets of 90ml representing each hot room are placed in standard QC test cartons and allowed 45 minutes to warm and wake from chill and then placed in the flight agility box. Pre-flight test:

7 Three sets of 90ml of flies are collected from flies prepared for release; one set each from flies held in chill longest, medium, and shortest time and placed in standard QC cartons and allowed to warm and wake from chill and then placed in the flight agility box. Post-flight test: Sets of 90ml of flies are collected from each release container and placed in standard QC test cartons and chilled for 30 minutes and then placed in the flight agility box. Test results. The standard QC test carton with flies is placed in the flight agility box that is located in an open area and opened. After 10 minutes the box is gently shaken to ensure that all flies able to fly escape from the box. Dead flies are counted and flies unable to fly due to physical defects (malformed wings, broken wings, broken legs, etc.) are counted. Percentages are calculated and registered. The ambient environmental conditions at the time of reading are recorded. Results are recorded in Form: Flight Agility. Sexual Aggressiveness and Ovarian Development The Sexual Aggressiveness test evaluates the ability of the sterile male to mate with a female fly. An acceptable parameter is 90%. A sample set of 30 females and 10 males is collected from flies held in chill for 2, 4, and 6 hours. The flies are placed in insect cages and provided with food and water. At day five; 7 females are dissected and the spermathecae are examined for the presence of spermatozoids. The procedure is repeated at day 7 dissecting 10 females. The presence of spermatozoids is positive and the level of motility is recorded. Results are recorded in Form: Sexual Aggressiveness and Ovarian Development. The ovaries from females held for seven days are also examined under microscope and the stage of development graded according to Ovarian Development Chart. Ovarian development should not exceed the 7th stage. Longevity The longevity test serves to evaluate how long the sterile fly lives under laboratory conditions. Flies are held in cages and provided with a food source and water. The cages are observed daily and percentage of dead flies is calculated. The test continues until all flies are dead, an acceptable parameter is 50% mortality at 14 days. Sample sets of 25 females and 25 males are collected from the 2nd collection with 4 hours in chill. Each set is placed in an insect cage and provided with food and water.

8 Sample sets of 50 females and 50 males are collected from the 7th collection with 4 hours in chill. Each set is placed in an insect cage and provided only with water. The cages are observed daily and dead insects are removed. The test continues until all insects have died. Results are recorded in Form: Longevity.

LABORATORY 7: GENETICS OF ORGANISMS

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