A New Method for Collecting Clean Stable Fly (Diptera: Muscidae) Pupae of. Known Age. Dennis R. Berkebile, Anthony P. Weinhold, and David B. Taylor.
VOL. 34, NO. 4
SOUTHWESTERN ENTOMOLOGIST
DEC. 2009
A New Method for Collecting Clean Stable Fly (Diptera: Muscidae) Pupae of Known Age
Dennis R. Berkebile, Anthony P. Weinhold, and David B. Taylor USDA-ARS-AMRU, 305 Entomology Hall, University of Nebraska, Lincoln, NE 68583-0938 Abstract. Stable flies, Stomoxys calcitrans L., are important pests of confined and pasture cattle. They have been reared in the laboratory to study their biology and to test new methods of control. Research on rearing modifications has concentrated on developing larval diets from materials locally abundant. Under current protocols, pupae form in the medium. Aggregations of pupae were located and removed, often with a considerable amount of extraneous material. Various methods have been developed to separate the pupae from waste material. We describe a method by which wandering larvae are enticed to leave the medium prior to pupariation. The larvae were attracted to a moist cloth on a shelf positioned at the end of the rearing pan. Almost 85% of the wandering larvae were collected on the shelf. This simplifies obtaining clean pupae and allows for collecting pupae of known age for experimental work. We also include data on the rate at which the larvae wandered onto the shelf under conditions we used in the laboratory. Introduction Stable flies, Stomoxys calcitrans L., are important pests of livestock throughout the world. Adults feed on the blood of many mammals, and are an economically important pest of livestock, especially cattle (Campbell et al. 1977, 1987). Stable flies have been reared in the laboratory since the early 1900s to study their biology. Early methods allowed generation of small numbers of flies to describe basic life history (Newstead 1906, Mitzmain 1913). Doty (1937) developed a method for rearing large numbers of stable flies and emphasized the importance of adding oat hulls to the larval medium. Materials such as wood shavings (McGregor and Dreiss 1955), vermiculite (Goodhue and Cantrel 1958), straw, bagasse (Bridges and Spates 1983), and peanut hulls (Hogsette 1992a) have been used to reduce the compaction of the medium and increase larval survival. Pupation normally occurs under a crust that forms on the surface of the medium and along the inside perimeter of the rearing container (Campau et al. 1953, McGregor and Dreiss 1955), but occasionally pupae are scattered throughout the medium (Bridges and Spates 1983). Clean pupae can be recovered by water flotation (Bailey et al. 1975) and dried with forced air. This method can be labor intensive, require additional space and equipment, and have the potential to expose workers to air-borne allergens.
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Larvae of some fly species have a period before pupariation when they wander in search of a suitable habitat in which to pupariate (Frankel and Bhaskaran 1973). Some leave their larval habitats such as wounds (e.g., screwworms, Cochliomyia hominivorax Coquerel), fruit (Tephritidae), or manure (e.g., face flies, Musca autumnalis DeGeer) in search of a suitable place to pupariate in the soil (Graham and Dudley 1959, Arends and Wright 1981, Alyokhin et al. 2001). The site selected may depend on moisture, medium texture, or orientation with light. Other flies, such as the stable fly and house fly, Musca domestica L., may remain in the larval habitat, but go through a wandering stage to select the best microhabitat for development of pupae. Mature stable fly larvae search for a dark habitat with ~68% moisture, high pH, and low osmolality to pupariate (McPheron and Broce 1996). The fact that mature stable fly larvae are attracted to moisture provided an important lead in the development of a simplified method for collecting pupae from laboratory-reared stable flies. The effectiveness and efficiency of the method was evaluated and compared with our previous method. Its use for collection of pupae of known age was explored. Materials and Methods Stable Fly Rearing. Stable flies were maintained at the United States Department of Agriculture, Agricultural Research Service, Agroecosystem Management Research Unit, Lincoln, NE. Immatures and adults were maintained at 23 ± 2°C with variable relative humidity (30-50%) and a photoperiod of 12:12 light:dark hours. Adults were fed daily by placing an unscented Stayfree® feminine napkin (McNeil-PPC, Morris Plains, NJ), saturated with citrated bovine blood, on top of the screen cages. Eggs from 7- to 10-day-old adults were collected on moist black cotton cloth and rinsed into a dish. A modified pipette was used to transfer 1 ml of eggs (approximately 8,000) to a rearing medium consisting of wheat bran (500 g), fish meal (115 g), wood chips (200 g), and water (1.6 liters). Immatures were allowed to develop in the medium for 12-14 days before pupae were harvested. Aggregations of pupae were spooned from around the perimeter of the plastic rearing pan (36.7 x 31.9 x 14.4 cm) where they tended to aggregate. The medium in the center of the pan was also examined for additional aggregations of pupae. Pupae isolated in this way were contaminated with considerable amounts of waste medium. Laboratory experiments and quality-control measurements (average pupal weight, total pupae weight, etc.) require clean pupae. Most of the waste medium was removed by placing the pupae in water and skimming the floating pupae from the surface. The washed pupae were allowed to dry and the remaining dry wheat bran was removed by pouring the pupae in front of a fan. Development of Pupal Shelf. Wandering larvae were attracted to moist areas. Therefore, a moist cotton towel (30.5 x 42 cm) was placed on the surface of the medium at one end of the rearing pan. This procedure attracted many larvae, but the technique was messy and the cloth quickly disintegrated. Subsequently, a shelf (10.2 cm tall x 10.2 cm wide x 31.9 cm long), fabricated from the end of a rearing pan, was placed with a moist cotton towel on top of the medium at one end of the rearing pan. This method reduced the amount of rearing medium brought onto the cloth from the migrating larvae but had a problem with drying out. Wrapping a wet sponge in the moist cotton towel increased the length of time the cloth remained moist (Fig. 1).
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Fig. 1. The shelf showing accumulation of pupae on the sponge wrapped in a towel.
Comparison of Pupae Collected by Two Methods. The number of pupae collected using the shelf was compared with pupae collected using a flotation method. After 14 days, pupae were removed from the shelf, and the dry medium and medium under the shelf were examined for pupae to quantify the efficiency of the method. The total weight of pupae collected, weight of 100 pupae, estimated number of pupae (total weight of pupae/weight of 100 pupae), pupal survival (adult emergence), and sex ratio (percentage female) were compared with pupae collected using flotation. Pupae recovered by each method were placed in separate cages. Adults were fed blood for 9 days. On Day 10, 20 females from each cage were dissected to determine their physiological age (Scholl 1980) and the number of ovarioles in each female. The test was replicated eight times. Rate of Pupation. Rearing pans were set up with a shelf as described previously. Shelves were inspected daily to ensure the sponges were moist and to check for wandering larvae. Larvae and pupae were removed from the sponge daily and placed into separate plastic containers (100-mm diameter by 15 mm) with a moist paper towel until 14 days after the eggs were placed in the rearing medium. All of the pupae in each container were weighed, then 100 were counted, weighed, and placed into emergence containers (100-mm diameter by 25 mm tall) for determination of viability and sex ratio. This procedure was replicated eight times. Variations of the Shelf Technique. Three variations of the shelf compared the influence of different amounts of moisture as an attractant for wandering larvae. Three rearing pans with shelves were set up: one with a moist sponge, one with a dry sponge that was moistened after 5 days, and one with a sponge that remained dry throughout the study. Larvae were allowed to wander until 14 days after the eggs were placed in the medium, and the pupae that formed were collected as described previously. Media in the dry area of the rearing pan, at the edge of the shelf, and under the shelf were sampled for characterization and comparison of the different areas of the rearing pan. Two 16-g samples were weighed in 100-ml disposable polypropylene beakers. One beaker was placed in a 100°C oven for 48 hours and reweighed to
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determine the moisture content. Distilled water (80 ml) was added to the second sample, stirred on a magnetic stirrer (Velp Scientifica, Milano, Italy) for 1 hour, and allowed to set for 30 minutes. Conductivity and pH were measured with a benchtop pH/conductivity/TDS meter (Oakton Instruments, Vernon Hills, IL). Ionic strength adjuster (5 M sodium chloride, 1.6 ml) was added to the sample, stirred, and the ammonium content was determined using a benchtop ion meter (Oakton Instruments, Vernon Hills, IL) with an ammonium-combination, ion-selective electrode. This was replicated three times. Statistical Analysis. Data were analyzed with Proc GLM (SAS Institute 2004). Least-significant difference tests (LSD) were used to separate the means. Arcsine transformations were performed on percentages before mean separations were made. Unless otherwise stated, Į < 0.05. Results and Discussion A new method for recovering stable fly pupae was developed using a plastic shelf and a moist cloth-wrapped sponge. The moist sponge provided the wandering late third instars a suitable habitat for pupation and the shelf provided a clean environment from which they could be observed and recovered. The pupae recovered by this method were compared with those isolated using a flotation method (standard). The number and mean weight of pupae collected using the shelf did not differ significantly from those collected using flotation (Table 1). An average of 85% of the pupae produced in a pan was collected on the shelf. Percentage of viable pupae did not differ between the two methods. Significantly fewer females (54.3 and 46.3%) were collected with the shelf than by the flotation method. Females made up a significantly larger percentage (62.9%) of the stable flies collected in the medium when using the shelf than those collected on the shelf. This may indicate that the medium is a slightly more preferable habitat for pupation of female stable flies. Further investigation is required to determine possible reasons for this. Female stable flies were dissected 10 days after emergence. A significantly greater number of ovarioles (95.0 and 97.4) was counted in females collected on the shelf than by the flotation method. This small difference would have minimal effect on colony production. Most females were gravid (Scholl 1980, Stage 5) at the time of dissection (96.9% shelf, 100% standard). The shelf method was used to determine the timing and rate of migration of the mature larvae. Third instars began dispersing to the shelf 6 days after the medium was seeded with eggs. Larvae collected before Day 10 had a greater survival rate to adult than those collected later (Table 2). A greater portion of the larvae that migrated to the shelf before Day 9 were male and those migrating later were predominantly female. Most larvae were collected on Days 8 and 9 and the sex ratio was ~1:1 (50% female) during this time. The number of larvae decreased rapidly on Days 10-12. This information is important when planning studies that require stable fly pupae of known age. Variations of the shelf method were compared to verify the importance of the moist sponge as a pupation site for the stable fly and to develop options to simplify its use. When a dry sponge was used with the shelf, only 13% of the pupae were collected when compared to the shelf with the moist sponge (Table 3). The dry sponge was inconsistent. On one occasion more than 3,200 pupae were recovered when moist media was on the shelf due to shifting of the pans and the movement of the larvae. When medium was absent, no stable flies pupated on the dry sponge.
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Table 1. Characteristics of Pupae Collected by the Flotation Method vs. the Shelf Method Shelf Flotation Shelf Mediuma Total pupal weight (g) 54.1 ± 2.80a 53.9 ± 6.46a 9.9 ± 2.51b Weight per 100 pupae (g) 1.1 ± 0.04a 1.1 ± 0.04ab 1.2 ± 0.06b Number of pupae 5128.6 ± 374.78a 4768.3 ± 587.03a 858.1 ± 254.07b % Adult emergence 82.8 ± 4.07a 77.8 ± 2.93a 78.8 ± 4.72a % Female 54.3 ± 1.56a 46.3 ± 1.85b 62.9 ± 1.29c Ovarioles per female 95.0 ± 0.68a 97.4 ± 0.64b -Means ± SE followed by the same letter in a row are not significantly different (P < 0.05). a Pupae recovered from the medium.
Table 2. Characteristics of Pupae Collected from the Shelf on Days 7-12 After Seeding the Medium with Eggs Total pupal weight (g) Weight/100 % Adult and % pupae (g) Number of pupae emergence % Female 4.9 ± 1.71a (9.2%) 24.2 ± 2.77b Day 8 (44.8%) 19.3 ± 3.13b Day 9 (35.7%) 4.4 ± 1.90a Day 10 (8.2%) 1.0 ± 0.51a Day 11 (1.8%) 0.2 ± 0.09a Day 12 (0.3%) Day 7
1.08 ± 0.04a
452.9 ± 150.85c
82.6 ± 9.13a
32.8 ± 4.51a
1.12 ± 0.03a 2,176.3 ± 268.02a
91.8 ± 1.73a
38.9 ± 1.84a
1.16 ± 0.04a 1,678.9 ± 278.64b
86.8 ± 3.61a
55.3 ± 2.98abc
1.03 ± 0.15a
388.0 ± 173.76c
32.1 ± 11.50b
64.2 ± 3.73bc
0.91 ± 0.21a
88.7 ± 49.87c
7.6 ± 3.82c
71.0 ± 7.19c
0.91 ± 0.21a
15.4 ± 8.17c
7.6 ± 6.64c
64.4 ± 18.39c
Means ± SE followed by the same letter in a column are not significantly different (P < 0.05).
Table 3. Comparison of Three Variations of the Shelf Method Where the Sponge Received Moisture on Day 1, Day 5, or None Sponge in pan on Sponge in pan on Day 1 Day 5 Dry sponge Total pupal weight/pan (g) 72.2 ± 8.85a 66.3 ± 9.24a 9.0 ± 6.27b Weight/100 pupae (g) 1.2 ± 0.05a 1.2 ± 0.05a 1.0 ± 0.02a Number of pupae 5,943.1 ± 931.64a 5,553.5 ± 805.02a 874.9 ± 620.09b % Adult emergence 92.8 ± 1.91a 93.4 ± 2.54a 90.0 ± 4.73a % Female 52.4 ± 2.52a 54.8 ± 1.82a 45.5 ± 4.36a % Pupae on shelf 95.8 ± 2.19a 91.5 ± 5.18a 12.9 ± 8.31b Means followed by the same letter in a row are not significantly different (P < 0.05).
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This verifies the observations by McPheron and Broce (1996) of the importance of moisture for pupation. No significant effect on the number of pupae collected, mean weight of 100 pupae, or adult emergence was observed when the moist sponge was placed on the shelf 5 days after seeding the medium with eggs than when the moist sponge was in place at the time of seeding. The number of pupae collected on the sponge moistened on Day 5 was more variable but some maintenance time was saved because the sponge did not need to be monitored for moisture on Days 1 to 4. More than 90% of the pupae were collected regardless of when the sponge was moistened. This was an improvement over the number of pupae recovered during the initial development of the shelf method. The pH was not affected by the use of the moist sponge on the shelf. Moisture of the medium at the edge of the shelf was greater than that in the general medium for either method using the moist sponge (Table 4). Conductivity was less and moisture greater at the edge of the shelf when the sponge was moistened on Day 1 than when it was moistened on Day 5. Ammonia was significantly greater in the moist zone when the sponge was moistened on Day 1 than when the dry sponge was used. The migrating larvae may be attracted to the increase in moisture and ammonia at the edge of the shelf and wander onto the shelf in search of a suitable pupation habitat.
Table 4. Comparison of Physical Properties of Medium Sampled from 3 Areasa of Pans Where the Shelf Was Installed on Day 1 with the Sponge, Day 5 with the Sponge, and Day 1 without a Sponge Conductivity Ammonia content Area pH (ms) (ppm) % Moisture Day 1 8.8 ± 0.15a 4.7 ± 0.15bc 252.8 ± 23.30ab 48.7 ± 3.34ab Dry Day 5 8.8 ± 0.15a 4.8 ± 0.11bc 228.4 ± 21.22ab 48.4 ± 2.85ab zone Dry sponge 8.8 ± 0.18a 5.0 ± 0.15c 197.6 ± 22.98a 46.3 ± 3.40a Day 1 8.7 ± 0.12a 3.7 ± 0.43a 207.4 ± 18.42a 60.1 ± 5.26cd Moist Day 5 8.7 ± 0.10a 4.2 ± 0.20abc 195.2 ± 25.98a 54.0 ± 6.19bc zone Dry sponge 8.8 ± 0.13a 4.6 ± 0.23bc 177.4 ± 27.23a 49.0 ± 3.11ab Day 1 8.8 ± 0.11ab 4.0 ± 0.22ab 302.4 ± 33.33b 64.7 ± 5.16d Under Day 5 8.9 ± 0.10b 3.7 ± 0.18a 234.2 ± 14.27ab 64.3 ± 5.10d shelf Dry sponge 8.9 ± 0.25b 4.1 ± 0.17abc 193.5 ± 51.87a 56.9 ± 4.71bc Means followed by the same letter in a column are not significantly different (P < 0.05). a Dry = center of pan away from shelf, moist = area at edge of shelf, and under = material under the shelf.
The method described for obtaining stable fly pupae is an improvement over the flotation method previously used. Isolating pupae by flotation (Bailey et al. 1975) is time consuming and inefficient. Young pupae (
