30 June 1975
J. Med. Ent. Vol. 12, no. 2: 189-193
LABORATORY BIOLOGY AND TECHNIQUES FOR MASS PRODUCING THE STABLE FLY, STOMOXYS CALCITRANS (L.) (DIPTERA: MUSCIDAE)* By Donald L. Bailey, T. L. Whitfield and G. C. LaBrecque 2 Abstract: The stable fly, Stomoxys calcitrans (L.), was studied to develop a larval rearing medium and an efficient method of mass rearing and also to determine the reproductive potential (productivity, fertility, survival, longevity) of the various life stages. Also techniques were evolved that included automated methods of separating and handling the various stadia in order to produce 1 million stable flies per week. The estimated cost for that level of production was determined.
'Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA. "Insects Affecting Man Research Laboratory, Agr. Res. Serv., USDA, Gainesville, Florida 32604, U.S.A.
Wheat bran, pine sawdust, water (1:1:1) Wheat bran, bagasse, water (3:1:5)
5642
10.7
94
7448
10.6
96
Downloaded from http://jme.oxfordjournals.org/ by guest on March 10, 2016
paper, and water. The mixture finally adopted and used for 2 years contained (by volume) 1 part wheat bran, 1 part sawdust, and 2 parts water. However, in recent years, the sugar industry has been pelleting bagasse (crushed sugar cane refuse) for use as a roughage in cattle feed. The diameter of these pellets is ca 1.25 cm and they average ca 2 cm long. When water is added to them, each Many workers have reported techniques for expands to ca 4 times its original volume. We rearing the stable fly, Stomoxys calcitrans (L.) (Glaser found that when bagasse was used instead of sawdust 1924, Melvin 1932, Doty 1937, Eagleson 1943, and all ingredients were mixed together, the swelling Campau et al. 1953, Champlin et al. 1954, Mcaction of the bagasse formed the fluffy, wellGregor & Dreiss 1955, Goodhue & Cantrel 1958, aerated medium that is essential for rearing stable Parr 1959, Gingrich 1960). These techniques fly larvae. Bagasse is now being used in our work range from rearing flies in a modified hotbed in rather than the sawdust, wood chips, straw, or horse manure and oat straw (Melvin 1932) to the vermiculite used in the past in most stable fly larval development of a synthetic medium for aseptic media, and the medium now contains (by volume) rearing by Gingrich (1960). However, all the 3 parts wheat bran, 1 part bagasse, and 5 parts water. methods have been concerned with producing a few TABLE 1 shows the results of a test made to compare hundred, or at most a few thousand, stable flies for the efficiency of this new medium with the one used use in laboratory or field tests. None would be previously. Almost 2000 more pupae per ml of satisfactory for producing the quantity necessary for eggs were obtained, and the size of the pupae and the large-scale releases necessary in a control the rates of eclosion were similar. This new larval program involving the sterile-male technique. medium was used for the tests reported in the section To establish an efficient technique for the mass on reproductive potential. production of any insect, one needs complete understanding of the laboratory biology of that Reproductive potential insect. This paper reports the results of various For the laboratory studies made to determine the tests made to determine the laboratory reproductive longevity and the oviposition habits of the stable fly, potential of the stable fly so that an efficient mass we set up 30 cages each containing 1 female and 2 rearing facility could be developed. It also demales (newly emerged). Cotton pads saturated scribes in detail the techniques that were evolved with bovine blood provided food and also served as to produce between 0.5 and 1 million stable flies per the oviposition medium. Each day until each week during the summers of 1971, 1972, and 1973 female died, the old blood pads were removed and and gives the estimated cost required for that level replaced with new ones, and the eggs on the pads of production. were recovered and counted. Males were removed from all cages after 14 days. The results (TABLE 2) Larval medium were used to determine the number of flies required Since one of our first needs was a suitable larval medium, we tested several different mixtures conTABLE 1. A comparison of the efficiency of 2 stableflylarval taining various amounts of such materials as CSMA media (average for 4 replicates each). larval medium, wheat bran, cottonseed meal, No. PUPAE/ AVG. PUPAL % sawdust, wood shavings, vermiculite, shredded LARVAL MEDIUM ML EGGS WT (MG) ECLOSION
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Vol. 12, no. 2
TABLE 2. Oviposition and longevity of stable flies (30 cages containing 1 9 and 2 o*6* each). AGE OVIPOSITION AGE AT PEAK BEGAN (DAYS) OVIPOSITION
Average Range
8 4-14
12 6-19
NO. OF OPPOSITIONS
N o . EGGS PER OVIPOSITION
TOTAL NO. EGGS
$ LONGEVITY (DAYS)
7 0-18
43 1-140
292 0-739
21 5-40
TABLE 3. The effects on egg viability of storing stable fly eggs submerged in water at room temperature (avg. of 2 replicates of each age). No. OF DAYS EGGS SUBMERGED
AVG. NO. PUPAE/ML
7480 8624 7824 1256 0
in water (to prevent hatching) at room temperature for 1, 2, 3, or 4 days. Then we placed replicate samples from each egg group in trays containing rearing medium. After pupation was complete, the pupae were separated from the medium, and the number was determined, TABLE 3 reports the results. The viability of the eggs stored for 0, 1, or 2 days was quite comparable, showing the normal variation one would expect with that large a sample. However, an 83% reduction had occurred by the 3rd day, and those stored for 4 days failed to produce any pupae. Some tests in our laboratory have indicated that stable flies can develop at a wide range of temperatures, TABLE 4 shows the average duration of the various life stages at temperatures of 15°, 25°, and 35 °C. No difference in duration was apparent at 35° and 25 °C; at both temperatures, 24 days were required from egg to egg. However, the rate of mortality was so high at 35 °C that an average of only 7 adults was recovered from 100 eggs, compared with 67 at 25 °C. There was a great difference between 25° and 15°C: at 15 °C, duration was 119 days; but very little difference was apparent in the number of adults obtained from 100 eggs (67 and 62, respectively). Many insect species that require water in the larval medium are not tolerant of the chlorine added to most city water, so we investigated the possibility that this water would affect the stable fly. There was little difference in the insects' development when chlorinated city water, unchlorinated well water, or distilled water was used in the larval medium: the range of pupation was 61-65%, the average pupal weight was 13-14 mg, the rate of eclosion was 92-97%, and the percentage reaching the adult stage from the egg stage was 59-60%. Therefore, since city water was more convenient, it was used in our rearing program.
TABLE 4. Average duration of various life stages of the stable fly at various temperatures (avg. of 3 replicates of 100 eggs each). AVG. NO. DAYS IN EACH LIFE STAGE
Eggs
Larvae
Pupae
Adult preoviposition
2 2 3
9 10 44
6 7 20
7 5 52
TEMP.
35 25 15
No. DAYS REQ QUIRED FROM EGG TO EGG
AVG. NO. ADULTS FROM 100 EGGS
24 24 119
7 67 62
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in our colony to produce the eggs needed for the sterile male release program and also to determine how long the females would be productive. Since the females began to oviposit on an average of 8 days after eclosion and lived an average of 21 days, they were active in egg production for ca 2 weeks during which they deposited an average of 292 eggs each. We also needed to know what percentage of the eggs placed in the larval medium would develop into pupae, so that we could determine the number of females required for egg production. We measured 2 samples of eggs each with a volume of 1 ml. The eggs in each sample were then counted. The numbers were 14,603 and 14,278, an average of 14,440 eggs per ml. TABLE 1 shows that 1 ml of eggs will produce an average of 7448 pupae, a production rate of 52%. This production rate, however, was not by actual count, but was an estimate based on a volumetric measurement of the eggs and a weight measurement of the pupae. Some variation can be expected, as is demonstrated later, where the actual numbers of eggs and pupae were counted in smaller tests. Normally, in the rearing program, only eggs collected over a 24-hr period are used; however, if production should have to be increased on short notice, there would be an advantage if older eggs could be used. We therefore stored eggs submerged
Bailey et al.: Laboratory mass production of stable flies
1975
TABLE 5. The effect on adult emergence of storing stable fly pupae at a temperature of 6°C (avg. of 4 samples of 100 pupae each). N O . DAYS IN STORAGE
% ECLOSION
% REDUCTION
0 1 2 6 4 5 6
91 86 81 73 55 40 8
6 11 20 40 56 91
100
flies from a single generation in each stage at any given time during the total lifetime (FIG. l). The eggs began to hatch after 1 day, and all hatching had occurred after 2 days. Thus, the larval stage was the only one present in such a population from the 2nd to the 6th day. On the 6th day, pupation began. The number of pupae then increased until the 12 th day when all pupation was completed. Adults began to emerge on the 14th day and had completed emergence by the 22nd day. Mortality then slowly reduced the adult population until about the 56th day after the initial oviposition; at that time, none of the flies in that generation were alive. However, at about the 22nd day, the adults were ready to begin oviposition, and the cycle could begin once more. Mass rearing system
The results of the biological studies described here were used in establishing a colony for stable fly production that was efficient and economical. The detailed operation of that colony is as follows: Adults: The adult rearing facility consists of a room 4.5 X 4 X 3.5m high maintained at a temperature of 24 °C and 70% RH with a photoperiod of 12 hr light and 12 hr dark. Galvanized metal shelves that hold 30 cages are placed along 1 wall. The cages are 46 X 38 X 38 cm high, have an aluminum frame with a solid aluminum floor, and are covered, except on the front, with 16 X 14-mesh aluminum screen. The front is covered with a cloth sleeve that allows access to the Egg La rva
/""I -Oviposition begins
90'
Adult
80 -
70-
60-
£
10 30 -
20 -
10
1 1 2
10
15
| 20
1 25
1
30
1 35
No. days from oviposition
1
1
1
40
US
50
55
FIG. 1. Average age of insects from generation of stable each life stage at time.
percenta single flies in a given
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Occasionally, in a rearing program, adult emergence should be delayed and pupae should be accumulated so that a larger number of adults can be obtained on a given day for special needs. To determine whether stable fly larvae can be stored at low temperature, we placed several thousand pupae in a refrigerator at 6°C. Each day 4 samples of 100 pupae each were removed, placed in a room at 22 °C, and allowed to emerge. At the same time, 4 similar samples that had not been refrigerated were placed in the same room, TABLE 5 shows that pupae stored for 1 day had a 6% reduction in eclosion compared with the control. The reduction in eclosion increased gradually and, by the 6th day, the reduction was 91%. The times required in the various developmental stages of the stable fly are not sharply demarcated. Data from various tests conducted in the laboratory at a temperature of 21°-22°C were therefore compiled to determine the average percentage of stable
191
192
J. Med. Ent.
TABLE 6.
prevents the cotton and dead flies in the cups from entering the bottom container. The separated eggs are measured in graduated centrifuge tubes and are used in producing the larvae. Larvae: The larval rearing facility consists of a room 6 X 4.5 X 3.5m high maintained at a temperature of 21 °C and 70% RH with a photoperiod of 12 hr light and 12 hr dark. Racks made of galvanized slotted angle iron that hold 160 plastic (Panel Controls Corp.) larval rearing trays ( 5 1 x 3 8 x 8 cm deep) are placed along 1 wall. The medium for each tray is prepared in the tray by first mixing the dry ingredients (1 liter bagasse and 3 liters wheat bran). Then 5 liters of water are added, and all ingredients are mixed to insure wetting of all dry material. We then place 2 ml of eggs on the surface of the medium and place the tray inside a bag made of black polyester cloth to prevent invasion by other insects that can also breed in this type of medium. The trays are held for 14 days until pupation is nearly complete. The synchronization of this 14-day larval cycle at 21 °C eliminates the need for weekend work. Pupae: Since pupation occurs in the upper 1-2 cm of the larval medium in each tray, this portion is removed, and the pupae are separated in an automatic separator by using water flotation. The separator consists of three 80-liter plastic garbage cans bolted together to form a triangle. Water is piped to 1 of the cans (enters through the side near the top). The medium containing the pupae is placed in this can, and the water is turned on. When that can is filled, the water overflows into the 2nd can through a gate cut in the side at the top. In the same way, the 3rd can is filled. The wet medium sinks to the bottom of the cans, but the pupae float and pass with the water through the 3 cans. From the 3rd can, the cleaned pupae pass through an opening (in the side) into a large strainer that allows the water to escape but retains
Estimated cost (dollars) of rearing 1 million adult stable flies per week (exclusive of housing). ITEM
Racks (slotted steel angle) Trays and covers for larvae Cages (adult) Driers (pupal) Containers (blood, medium, etc.) Blood Wheat bran Sugar cane bagasse Transportation Supplies (miscellaneous) Personnel Total
N O . OR AMOUNT REQUIRED
UNIT COST
INITIAL COST
$ 0.50 5.50 5.00 100.00
$ 700 1100
6 gal. (22.7 1) 200 lb (90.7 kg) 150 lb (68.0 kg) 100 mi. (160.9 km)
1.00 0.06 0.02 0.10
6 12
' 30 man-hr
3.30
1400 ft (427 m) 200 ea 30 ea 2 ea
150 200 115
CONTINUING COST
$ 7 6
12
3 10 12 100
3 10 12 100
$2408
$150
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inside of the cage. Each day (Monday-Friday) 2 of these cages are placed into the production line (stocked with about 5000 pupae), and the 2 cages that have been in the line longest (3 weeks) are removed and cleaned. Emergence of adults usually begins in the new cages the day after they are placed in the line. Therefore, citrated bovine blood (3 g sodium citrate and 1 ml formalin per liter whole blood) is provided as food by saturating ca 6 g of absorbent cotton with ca 160 ml blood in a 450-ml waxed-paper cup that is placed inside the cage. A fresh cup is placed in each cage daily, except Fridays, when 2 cups are used to eliminate weekend feeding. (The blood is obtained once a week from an abattoir; the citrate prevents the blood from coagulating; and the formalin prevents bacterial breakdown of the blood during the 1-week storage at a temperature of 4 °C). Eggs: The cups of blood that provide food also provide an oviposition medium, since the females lay their eggs readily on the surface of the bloodsoaked cotton. Therefore, each day when the used cups are removed, the cotton is flushed under running water in a special apparatus that separates the eggs from the cotton and blood and concentrates them in a small container. This apparatus was made by removing triangular sections from the sides of a 100-mm diam. plastic funnel and covering the sections with 100-mesh stainless steel screen. Then a 1-liter plastic container (bottom removed and placed with 16 x 14-mesh plastic screen) was glued to the top of the funnel. Also, the bottom of the funnel was inserted into a 75-ml plastic container (a hole was cut in the screw cap) and glued in place. The old blood-soaked cotton containing the eggs is washed in the top container, the water escapes through the screen in the side of the funnel, and the eggs, which are unable to pass through, collect in the bottom container. The screen between the funnel and the top container
Vol. 12, no. 2
1975
Bailey et al.: Laboratory mass production of stable flies
However, we feel that much improvement could be made in the area of automation and in the utilization of space and personnel to further reduce the production costs per unit. LITERATURE CITED
Bailey, D. L. 1970. Forced air for separating pupae of house flies from rearing medium. J. Econ. Ent. 63: 331-33. Campau, E. J., G. J. Baker & F. D. Morrison. 1953. Rearing the stable fly for laboratory tests. J. Econ. Ent. 46: 524. Champlin, R. A., F. W. Fish & A. C. Dowdy. 1954. Some improvements in rearing stable flies. J. Econ. Ent. 47: 94041. Doty, A. E. 1937. Convenient method of rearing stable flies. J. Econ. Ent. 30: 367-69. Eagleson, C. 1943. Laboratory procedures in studies of the chemical control of insects. In: Campbell, F. L. & F. R. Moulton, eds., Amer. Assoc. Adv. Sci. Suppl. Contrib. 20: 74-77.
Gingrich, R. E. 1960. Development of a synthetic medium for aseptic rearing of larvae of Stomoxys calcitrans (L.). J. Econ. Ent. 53: 408-11. Glaser, R. W. 1924. Rearing flies for experimental purposes with biological notes. J. Econ. Ent. 17: 486-96. Goodhue, L. D. & K. E. Cantrel. 1958. The use of vermiculite in medium for stable fly larvae. J. Econ. Ent. 51: 250. McGregor, W. S. & J. M. Dreiss. 1955. Rearing stable flies in the laboratory. J. Econ. Ent. 48: 327-28. Melvin, R. 1932. Physiological studies on the effect of flies and fly sprays on cattle. J. Econ. Ent. 25: 1151-64. Parr, H. C. M. 1959. Studies on Stomoxys calcitrans (L.) in Uganda, East Africa. I. A method of rearing large numbers of Stomoxys calcitrans. Bull. Ent. Res. 50: 165-69.
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the pupae. The water gates in the sides of the cans are arranged so the water flows in a circle through the cans and carries the pupae by centrifugal force to the outer edge and through the gates of each succeeding can. Thus the separation can continue without any close attention. After all the pupae have passed into the strainer, they are removed and placed in a forced-air dryer as described by Bailey (1970). Also the drains in the bottom of the 3 garbage cans are opened and the medium that has settled there is collected in a strainer and discarded. When the pupae are dry, the total mass is weighed, and the number produced is calculated from the weight of a sample of 100 pupae. (The 100-pupae sample is set aside in a plastic petri dish and used to establish the rate of eclosion.) Cost of production is an important consideration in any sterile male release program, TABLE 6 shows the estimated cost (excluding housing) and the continuing cost of rearing 1 million adults per week by our present technique. (Although the estimate is based on the production of 1 million insects per week, enough larval rearing trays and racks must be available to accommodate 2 million insects since they are used on a 2-week cycle. This is the main reason for the high initial cost.) Our techniques have proven quite efficient at the level of production we have been maintaining.
193
J. Med. Ent. Vol. 12, no. 2: 189-193
LABORATORY BIOLOGY AND TECHNIQUES FOR MASS PRODUCING THE STABLE FLY, STOMOXYS CALCITRANS (L.) (DIPTERA: MUSCIDAE)* By Donald L. Bailey, T. L. Whitfield and G. C. LaBrecque 2 Abstract: The stable fly, Stomoxys calcitrans (L.), was studied to develop a larval rearing medium and an efficient method of mass rearing and also to determine the reproductive potential (productivity, fertility, survival, longevity) of the various life stages. Also techniques were evolved that included automated methods of separating and handling the various stadia in order to produce 1 million stable flies per week. The estimated cost for that level of production was determined.
'Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA. "Insects Affecting Man Research Laboratory, Agr. Res. Serv., USDA, Gainesville, Florida 32604, U.S.A.
Wheat bran, pine sawdust, water (1:1:1) Wheat bran, bagasse, water (3:1:5)
5642
10.7
94
7448
10.6
96
Downloaded from http://jme.oxfordjournals.org/ by guest on March 10, 2016
paper, and water. The mixture finally adopted and used for 2 years contained (by volume) 1 part wheat bran, 1 part sawdust, and 2 parts water. However, in recent years, the sugar industry has been pelleting bagasse (crushed sugar cane refuse) for use as a roughage in cattle feed. The diameter of these pellets is ca 1.25 cm and they average ca 2 cm long. When water is added to them, each Many workers have reported techniques for expands to ca 4 times its original volume. We rearing the stable fly, Stomoxys calcitrans (L.) (Glaser found that when bagasse was used instead of sawdust 1924, Melvin 1932, Doty 1937, Eagleson 1943, and all ingredients were mixed together, the swelling Campau et al. 1953, Champlin et al. 1954, Mcaction of the bagasse formed the fluffy, wellGregor & Dreiss 1955, Goodhue & Cantrel 1958, aerated medium that is essential for rearing stable Parr 1959, Gingrich 1960). These techniques fly larvae. Bagasse is now being used in our work range from rearing flies in a modified hotbed in rather than the sawdust, wood chips, straw, or horse manure and oat straw (Melvin 1932) to the vermiculite used in the past in most stable fly larval development of a synthetic medium for aseptic media, and the medium now contains (by volume) rearing by Gingrich (1960). However, all the 3 parts wheat bran, 1 part bagasse, and 5 parts water. methods have been concerned with producing a few TABLE 1 shows the results of a test made to compare hundred, or at most a few thousand, stable flies for the efficiency of this new medium with the one used use in laboratory or field tests. None would be previously. Almost 2000 more pupae per ml of satisfactory for producing the quantity necessary for eggs were obtained, and the size of the pupae and the large-scale releases necessary in a control the rates of eclosion were similar. This new larval program involving the sterile-male technique. medium was used for the tests reported in the section To establish an efficient technique for the mass on reproductive potential. production of any insect, one needs complete understanding of the laboratory biology of that Reproductive potential insect. This paper reports the results of various For the laboratory studies made to determine the tests made to determine the laboratory reproductive longevity and the oviposition habits of the stable fly, potential of the stable fly so that an efficient mass we set up 30 cages each containing 1 female and 2 rearing facility could be developed. It also demales (newly emerged). Cotton pads saturated scribes in detail the techniques that were evolved with bovine blood provided food and also served as to produce between 0.5 and 1 million stable flies per the oviposition medium. Each day until each week during the summers of 1971, 1972, and 1973 female died, the old blood pads were removed and and gives the estimated cost required for that level replaced with new ones, and the eggs on the pads of production. were recovered and counted. Males were removed from all cages after 14 days. The results (TABLE 2) Larval medium were used to determine the number of flies required Since one of our first needs was a suitable larval medium, we tested several different mixtures conTABLE 1. A comparison of the efficiency of 2 stableflylarval taining various amounts of such materials as CSMA media (average for 4 replicates each). larval medium, wheat bran, cottonseed meal, No. PUPAE/ AVG. PUPAL % sawdust, wood shavings, vermiculite, shredded LARVAL MEDIUM ML EGGS WT (MG) ECLOSION
J. Med. Ent.
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Vol. 12, no. 2
TABLE 2. Oviposition and longevity of stable flies (30 cages containing 1 9 and 2 o*6* each). AGE OVIPOSITION AGE AT PEAK BEGAN (DAYS) OVIPOSITION
Average Range
8 4-14
12 6-19
NO. OF OPPOSITIONS
N o . EGGS PER OVIPOSITION
TOTAL NO. EGGS
$ LONGEVITY (DAYS)
7 0-18
43 1-140
292 0-739
21 5-40
TABLE 3. The effects on egg viability of storing stable fly eggs submerged in water at room temperature (avg. of 2 replicates of each age). No. OF DAYS EGGS SUBMERGED
AVG. NO. PUPAE/ML
7480 8624 7824 1256 0
in water (to prevent hatching) at room temperature for 1, 2, 3, or 4 days. Then we placed replicate samples from each egg group in trays containing rearing medium. After pupation was complete, the pupae were separated from the medium, and the number was determined, TABLE 3 reports the results. The viability of the eggs stored for 0, 1, or 2 days was quite comparable, showing the normal variation one would expect with that large a sample. However, an 83% reduction had occurred by the 3rd day, and those stored for 4 days failed to produce any pupae. Some tests in our laboratory have indicated that stable flies can develop at a wide range of temperatures, TABLE 4 shows the average duration of the various life stages at temperatures of 15°, 25°, and 35 °C. No difference in duration was apparent at 35° and 25 °C; at both temperatures, 24 days were required from egg to egg. However, the rate of mortality was so high at 35 °C that an average of only 7 adults was recovered from 100 eggs, compared with 67 at 25 °C. There was a great difference between 25° and 15°C: at 15 °C, duration was 119 days; but very little difference was apparent in the number of adults obtained from 100 eggs (67 and 62, respectively). Many insect species that require water in the larval medium are not tolerant of the chlorine added to most city water, so we investigated the possibility that this water would affect the stable fly. There was little difference in the insects' development when chlorinated city water, unchlorinated well water, or distilled water was used in the larval medium: the range of pupation was 61-65%, the average pupal weight was 13-14 mg, the rate of eclosion was 92-97%, and the percentage reaching the adult stage from the egg stage was 59-60%. Therefore, since city water was more convenient, it was used in our rearing program.
TABLE 4. Average duration of various life stages of the stable fly at various temperatures (avg. of 3 replicates of 100 eggs each). AVG. NO. DAYS IN EACH LIFE STAGE
Eggs
Larvae
Pupae
Adult preoviposition
2 2 3
9 10 44
6 7 20
7 5 52
TEMP.
35 25 15
No. DAYS REQ QUIRED FROM EGG TO EGG
AVG. NO. ADULTS FROM 100 EGGS
24 24 119
7 67 62
Downloaded from http://jme.oxfordjournals.org/ by guest on March 10, 2016
in our colony to produce the eggs needed for the sterile male release program and also to determine how long the females would be productive. Since the females began to oviposit on an average of 8 days after eclosion and lived an average of 21 days, they were active in egg production for ca 2 weeks during which they deposited an average of 292 eggs each. We also needed to know what percentage of the eggs placed in the larval medium would develop into pupae, so that we could determine the number of females required for egg production. We measured 2 samples of eggs each with a volume of 1 ml. The eggs in each sample were then counted. The numbers were 14,603 and 14,278, an average of 14,440 eggs per ml. TABLE 1 shows that 1 ml of eggs will produce an average of 7448 pupae, a production rate of 52%. This production rate, however, was not by actual count, but was an estimate based on a volumetric measurement of the eggs and a weight measurement of the pupae. Some variation can be expected, as is demonstrated later, where the actual numbers of eggs and pupae were counted in smaller tests. Normally, in the rearing program, only eggs collected over a 24-hr period are used; however, if production should have to be increased on short notice, there would be an advantage if older eggs could be used. We therefore stored eggs submerged
Bailey et al.: Laboratory mass production of stable flies
1975
TABLE 5. The effect on adult emergence of storing stable fly pupae at a temperature of 6°C (avg. of 4 samples of 100 pupae each). N O . DAYS IN STORAGE
% ECLOSION
% REDUCTION
0 1 2 6 4 5 6
91 86 81 73 55 40 8
6 11 20 40 56 91
100
flies from a single generation in each stage at any given time during the total lifetime (FIG. l). The eggs began to hatch after 1 day, and all hatching had occurred after 2 days. Thus, the larval stage was the only one present in such a population from the 2nd to the 6th day. On the 6th day, pupation began. The number of pupae then increased until the 12 th day when all pupation was completed. Adults began to emerge on the 14th day and had completed emergence by the 22nd day. Mortality then slowly reduced the adult population until about the 56th day after the initial oviposition; at that time, none of the flies in that generation were alive. However, at about the 22nd day, the adults were ready to begin oviposition, and the cycle could begin once more. Mass rearing system
The results of the biological studies described here were used in establishing a colony for stable fly production that was efficient and economical. The detailed operation of that colony is as follows: Adults: The adult rearing facility consists of a room 4.5 X 4 X 3.5m high maintained at a temperature of 24 °C and 70% RH with a photoperiod of 12 hr light and 12 hr dark. Galvanized metal shelves that hold 30 cages are placed along 1 wall. The cages are 46 X 38 X 38 cm high, have an aluminum frame with a solid aluminum floor, and are covered, except on the front, with 16 X 14-mesh aluminum screen. The front is covered with a cloth sleeve that allows access to the Egg La rva
/""I -Oviposition begins
90'
Adult
80 -
70-
60-
£
10 30 -
20 -
10
1 1 2
10
15
| 20
1 25
1
30
1 35
No. days from oviposition
1
1
1
40
US
50
55
FIG. 1. Average age of insects from generation of stable each life stage at time.
percenta single flies in a given
Downloaded from http://jme.oxfordjournals.org/ by guest on March 10, 2016
Occasionally, in a rearing program, adult emergence should be delayed and pupae should be accumulated so that a larger number of adults can be obtained on a given day for special needs. To determine whether stable fly larvae can be stored at low temperature, we placed several thousand pupae in a refrigerator at 6°C. Each day 4 samples of 100 pupae each were removed, placed in a room at 22 °C, and allowed to emerge. At the same time, 4 similar samples that had not been refrigerated were placed in the same room, TABLE 5 shows that pupae stored for 1 day had a 6% reduction in eclosion compared with the control. The reduction in eclosion increased gradually and, by the 6th day, the reduction was 91%. The times required in the various developmental stages of the stable fly are not sharply demarcated. Data from various tests conducted in the laboratory at a temperature of 21°-22°C were therefore compiled to determine the average percentage of stable
191
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J. Med. Ent.
TABLE 6.
prevents the cotton and dead flies in the cups from entering the bottom container. The separated eggs are measured in graduated centrifuge tubes and are used in producing the larvae. Larvae: The larval rearing facility consists of a room 6 X 4.5 X 3.5m high maintained at a temperature of 21 °C and 70% RH with a photoperiod of 12 hr light and 12 hr dark. Racks made of galvanized slotted angle iron that hold 160 plastic (Panel Controls Corp.) larval rearing trays ( 5 1 x 3 8 x 8 cm deep) are placed along 1 wall. The medium for each tray is prepared in the tray by first mixing the dry ingredients (1 liter bagasse and 3 liters wheat bran). Then 5 liters of water are added, and all ingredients are mixed to insure wetting of all dry material. We then place 2 ml of eggs on the surface of the medium and place the tray inside a bag made of black polyester cloth to prevent invasion by other insects that can also breed in this type of medium. The trays are held for 14 days until pupation is nearly complete. The synchronization of this 14-day larval cycle at 21 °C eliminates the need for weekend work. Pupae: Since pupation occurs in the upper 1-2 cm of the larval medium in each tray, this portion is removed, and the pupae are separated in an automatic separator by using water flotation. The separator consists of three 80-liter plastic garbage cans bolted together to form a triangle. Water is piped to 1 of the cans (enters through the side near the top). The medium containing the pupae is placed in this can, and the water is turned on. When that can is filled, the water overflows into the 2nd can through a gate cut in the side at the top. In the same way, the 3rd can is filled. The wet medium sinks to the bottom of the cans, but the pupae float and pass with the water through the 3 cans. From the 3rd can, the cleaned pupae pass through an opening (in the side) into a large strainer that allows the water to escape but retains
Estimated cost (dollars) of rearing 1 million adult stable flies per week (exclusive of housing). ITEM
Racks (slotted steel angle) Trays and covers for larvae Cages (adult) Driers (pupal) Containers (blood, medium, etc.) Blood Wheat bran Sugar cane bagasse Transportation Supplies (miscellaneous) Personnel Total
N O . OR AMOUNT REQUIRED
UNIT COST
INITIAL COST
$ 0.50 5.50 5.00 100.00
$ 700 1100
6 gal. (22.7 1) 200 lb (90.7 kg) 150 lb (68.0 kg) 100 mi. (160.9 km)
1.00 0.06 0.02 0.10
6 12
' 30 man-hr
3.30
1400 ft (427 m) 200 ea 30 ea 2 ea
150 200 115
CONTINUING COST
$ 7 6
12
3 10 12 100
3 10 12 100
$2408
$150
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inside of the cage. Each day (Monday-Friday) 2 of these cages are placed into the production line (stocked with about 5000 pupae), and the 2 cages that have been in the line longest (3 weeks) are removed and cleaned. Emergence of adults usually begins in the new cages the day after they are placed in the line. Therefore, citrated bovine blood (3 g sodium citrate and 1 ml formalin per liter whole blood) is provided as food by saturating ca 6 g of absorbent cotton with ca 160 ml blood in a 450-ml waxed-paper cup that is placed inside the cage. A fresh cup is placed in each cage daily, except Fridays, when 2 cups are used to eliminate weekend feeding. (The blood is obtained once a week from an abattoir; the citrate prevents the blood from coagulating; and the formalin prevents bacterial breakdown of the blood during the 1-week storage at a temperature of 4 °C). Eggs: The cups of blood that provide food also provide an oviposition medium, since the females lay their eggs readily on the surface of the bloodsoaked cotton. Therefore, each day when the used cups are removed, the cotton is flushed under running water in a special apparatus that separates the eggs from the cotton and blood and concentrates them in a small container. This apparatus was made by removing triangular sections from the sides of a 100-mm diam. plastic funnel and covering the sections with 100-mesh stainless steel screen. Then a 1-liter plastic container (bottom removed and placed with 16 x 14-mesh plastic screen) was glued to the top of the funnel. Also, the bottom of the funnel was inserted into a 75-ml plastic container (a hole was cut in the screw cap) and glued in place. The old blood-soaked cotton containing the eggs is washed in the top container, the water escapes through the screen in the side of the funnel, and the eggs, which are unable to pass through, collect in the bottom container. The screen between the funnel and the top container
Vol. 12, no. 2
1975
Bailey et al.: Laboratory mass production of stable flies
However, we feel that much improvement could be made in the area of automation and in the utilization of space and personnel to further reduce the production costs per unit. LITERATURE CITED
Bailey, D. L. 1970. Forced air for separating pupae of house flies from rearing medium. J. Econ. Ent. 63: 331-33. Campau, E. J., G. J. Baker & F. D. Morrison. 1953. Rearing the stable fly for laboratory tests. J. Econ. Ent. 46: 524. Champlin, R. A., F. W. Fish & A. C. Dowdy. 1954. Some improvements in rearing stable flies. J. Econ. Ent. 47: 94041. Doty, A. E. 1937. Convenient method of rearing stable flies. J. Econ. Ent. 30: 367-69. Eagleson, C. 1943. Laboratory procedures in studies of the chemical control of insects. In: Campbell, F. L. & F. R. Moulton, eds., Amer. Assoc. Adv. Sci. Suppl. Contrib. 20: 74-77.
Gingrich, R. E. 1960. Development of a synthetic medium for aseptic rearing of larvae of Stomoxys calcitrans (L.). J. Econ. Ent. 53: 408-11. Glaser, R. W. 1924. Rearing flies for experimental purposes with biological notes. J. Econ. Ent. 17: 486-96. Goodhue, L. D. & K. E. Cantrel. 1958. The use of vermiculite in medium for stable fly larvae. J. Econ. Ent. 51: 250. McGregor, W. S. & J. M. Dreiss. 1955. Rearing stable flies in the laboratory. J. Econ. Ent. 48: 327-28. Melvin, R. 1932. Physiological studies on the effect of flies and fly sprays on cattle. J. Econ. Ent. 25: 1151-64. Parr, H. C. M. 1959. Studies on Stomoxys calcitrans (L.) in Uganda, East Africa. I. A method of rearing large numbers of Stomoxys calcitrans. Bull. Ent. Res. 50: 165-69.
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the pupae. The water gates in the sides of the cans are arranged so the water flows in a circle through the cans and carries the pupae by centrifugal force to the outer edge and through the gates of each succeeding can. Thus the separation can continue without any close attention. After all the pupae have passed into the strainer, they are removed and placed in a forced-air dryer as described by Bailey (1970). Also the drains in the bottom of the 3 garbage cans are opened and the medium that has settled there is collected in a strainer and discarded. When the pupae are dry, the total mass is weighed, and the number produced is calculated from the weight of a sample of 100 pupae. (The 100-pupae sample is set aside in a plastic petri dish and used to establish the rate of eclosion.) Cost of production is an important consideration in any sterile male release program, TABLE 6 shows the estimated cost (excluding housing) and the continuing cost of rearing 1 million adults per week by our present technique. (Although the estimate is based on the production of 1 million insects per week, enough larval rearing trays and racks must be available to accommodate 2 million insects since they are used on a 2-week cycle. This is the main reason for the high initial cost.) Our techniques have proven quite efficient at the level of production we have been maintaining.
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