Name: laboratory rearing of stable flies and other muscoid diptera
Uploaded: 2018-08-03T12:30:00
Description: Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

We would like to thank Anthony Weinhold and the numerous students who have worked with us over the years for technical support as well as suggestions for improving our insect rearing procedures.

1. Egg Collection (Figure 1)
<ol>
	<li>Prepare egging cup, place one end of cloth in ~500 mL beaker filled with warm (~40 &#176;C) water. Overlap the sides of the cup and fix with a rubber band. Fold the loose end of the cloth back over the top of the cup.</li>
	<li>Place egging cup in cage of 8&#8211;10 day old flies for ~2 h. Gravid female stable flies will oviposit on the cloth.</li>
	<li>Remove egging cup from cage and rinse eggs off of the egging cloth into a small pan with squeeze wa...

<ol>
	<li>Cook, D. F., Dadour, I. R., Keals, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+fly,+house+fly+(Diptera:+Muscidae),+and+other+nuisance+fly+development+in+poultry+litter+associated+with+horticultural+crop+production.">Stable fly, house fly (Diptera: Muscidae), and other nuisance fly development in poultry litter associated with horticultural crop production.</a> J. Econ. Entomol. 92 (6), 1352-1357 (1999).</li><li>Dominghetti, T. F., de Barros, A. T., Soares, C. O., Can&#231;ado, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomoxys+calcitrans+(Diptera:+Muscidae)+outbreaks:+current+situation+and+future+outlook+with+emphasis+on+Brazil.">Stomoxys calcitrans (Diptera: Muscidae) outbreaks: current situation and future outlook with emphasis on Brazil.</a> Rev. Bras. Parasitol. Vet. 24 (4), 387-395 (2015).</li><li>Sol&#243;rzano, J. -. A., Guilles, J., Bravo, O., Vargas, C., Gomez-Bonilla, Y., Bingham, G., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+trapping+of+stable+flies+(Diptera:+Muscidae)+developing+in+pineapple+residues+(Ananas+comosus)+in+Costa+Rica.">Biology and trapping of stable flies (Diptera: Muscidae) developing in pineapple residues (Ananas comosus) in Costa Rica.</a> J. Insect Sci. 15 (1), 145 (2015).</li><li>Berry, I. L., Stage, D. A., Campbell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+and+economic+impacts+of+stable+flies+on+cattle.">Populations and economic impacts of stable flies on cattle.</a> Trans. Am. Soc. Agric. Eng. 26, 873-877 (1983).</li><li>Taylor, D. B., Vreysen, J., Hendrichs, R., Cardoso Pereira, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Area-wide+management+of+stable+flies.">Area-wide management of stable flies.</a> Area-wide management of insect pests. , (2017).</li><li>Bishopp, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stable+fly+(Stomoxys+calcitrans+L.),+an+important+live+stock+pest.">The stable fly (Stomoxys calcitrans L.), an important live stock pest.</a> J.Econ. Entomol. 6 (1), 112-126 (1913).</li><li>Hogsette, J. A., Ruff, J. P., Jones, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+fly+biology+and+control+in+northwest+Florida.">Stable fly biology and control in northwest Florida.</a> J. Agric. Entomol. 4 (1), 1-11 (1987).</li><li>Lysyk, T., Kalischuk-Tymensen, L., Selinger, L., Lancaster, R., Wever, L., Cheng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+stable+fly+larvae+(Diptera:+Muscidae)+on+an+egg+yolk+medium.">Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.</a> J. Med. Entomol. 38, 382-388 (1999).</li><li>Romero, A., Broce, A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+bacteria+in+the+oviposition+behaviour+and+larval+development+of+stable+flies.">Role of bacteria in the oviposition behaviour and larval development of stable flies.</a> Med. Vet. Entomol. 20, 115-121 (2006).</li><li>Glaser, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+flies+for+experimental+purposes+with+biological+notes.">Rearing flies for experimental purposes with biological notes.</a> J. Econ. Entomol. 17 (4), 486-496 (1924).</li><li>Melvin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+studies+on+the+effect+of+flies+and+fly+sprays+on+cattle.">Physiological studies on the effect of flies and fly sprays on cattle.</a> J. Econ. Entomol. 25 (6), 1151-1164 (1932).</li><li>Doty, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+of+rearing+the+stable+fly.">Convenient method of rearing the stable fly.</a> J. Econ. Entomol. 30 (2), 367-369 (1937).</li><li>Bridges, A. C., Spates, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+medium+for+the+stable+fly+Stomoxys+calcitrans+(L.).">Larval medium for the stable fly Stomoxys calcitrans (L.).</a> Southwest. Entomol. 8 (1), 6-10 (1983).</li><li>Hogsette, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+diets+for+production+of+house+flies+and+stable+flies+(Diptera:+Muscidae)+in+the+laboratory.">New diets for production of house flies and stable flies (Diptera: Muscidae) in the laboratory.</a> J. Econ. Entomol. 85 (6), 2291-2294 (1992).</li><li>McGregor, W. S., Dreiss, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+stable+flies+in+the+laboratory.">Rearing stable flies in the laboratory.</a> J. Econ. Entomol. 48 (3), 327-328 (1955).</li><li>Goodhue, L. D., Cantrel, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vermiculite+in+medium+for+stable+fly+larvae.">The use of vermiculite in medium for stable fly larvae.</a> J. Econ. Entomol. 51 (2), 250 (1958).</li><li>Friesen, K., Berkebile, D. R., Zhu, J. J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmenting+laboratory+rearing+of+stable+fly+(Diptera:+Muscidae)+larvae+with+ammoniacal+salts.">Augmenting laboratory rearing of stable fly (Diptera: Muscidae) larvae with ammoniacal salts.</a> J. Insect Sci. 17 (1), 1-6 (2017).</li><li>Berkebile, D. R., Weinhold, A. P., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+collecting+clean+stable+fly+(Diptera:Muscidae)+pupae+of+known+age.">A new method for collecting clean stable fly (Diptera:Muscidae) pupae of known age.</a> Southwest. Entomol. 34 (4), 469-476 (2009).</li><li>Champlain, R. A., Fisk, F. W., Dowdy, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+improvements+in+rearing+stable+flies.">Some improvements in rearing stable flies.</a> J. Econ. Entomol. 47 (5), 940-941 (1954).</li><li>Zumpt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The stomoxyine biting flies of the world. , (1973).</li><li>Wienhold, B. J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+properties+of+stable+fly+(Diptera:+Muscidae)+developmental+sites+associated+with+round+bale+hay+feeding+sites+in+eastern+Nebraska.">Substrate properties of stable fly (Diptera: Muscidae) developmental sites associated with round bale hay feeding sites in eastern Nebraska.</a> Environ. Entomol. 41 (2), 213-221 (2012).</li><li>Friesen, K., Berkebile, D. R., Wienhold, B. J., Durso, L., Zhu, J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+parameters+associated+with+stable+fly+(Diptera:+Muscidae)+development+at+hay+feeding+sites.">Environmental parameters associated with stable fly (Diptera: Muscidae) development at hay feeding sites.</a> Environ. Entomol. 45 (3), 570-576 (2016).</li><li>Albuquerque, T. A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+the+bacterial+community+of+animal+feces+and+their+correlation+with+stable+fly+oviposition,+larval+development,+and+adult+fitness.">Temporal changes in the bacterial community of animal feces and their correlation with stable fly oviposition, larval development, and adult fitness.</a> Front. Microbiol. 5 (590), 1-9 (2014).</li><li>Bailey, D. L., Whitfield, T. L., LaBrecque, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+biology+and+techniques+for+mass+producing+the+stable+fly,+Stomoxys+calcitrans+(L.)+(Diptera:+Muscidae).">Laboratory biology and techniques for mass producing the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae).</a> J. Med. Entomol. 12 (2), 189-193 (1975).</li><li>Smith, J. P., Hall, R. D., Thomas, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+studies+on+mortality+of+the+immature+stages+of+the+stable+fly+(Diptera:+Muscidae).">Field studies on mortality of the immature stages of the stable fly (Diptera: Muscidae).</a> Environ. Entomol. 14 (6), 881-890 (1985).</li></ol>

Stable flies can be found developing in a broad variety of substrates in nature and can be reared in many types of media in the laboratory. We have used wood shavings and vermiculite as bulking agents. Vermiculite worked well, but made separating puparia from rearing medium difficult and was expensive (~$0.60/pan). Possibly because of the added rigor of separating puparia from the medium, eclosion was also lower with vermiculite, 57% vs. 75% for wood shavings. Cottonwood shavings were comparable to vermiculite, but at ti...

Stable flies, Stomoxys calcitrans (L.), are hematophagous flies whose painful bites disrupt the grazing behavior of livestock, cause pain and suffering to companion animals and disrupt human recreational activities worldwide. Immature stable flies develop in fermenting vegetative matter, often contaminated with animal waste. Changing agronomic practices and crops have produced serious outbreaks of stable flies in crop residues, vegetables in Australia1, sugar cane in Brazil2, and pineapple in Costa Rica3. Although just 14 stable flies per animal are considered to be the economic threshold4, observations of more than 2,000 flies per animal have been made during recent outbreaks5. Such infestation levels reduce host productivity to near zero and can cause mortality6. As a result of agronomically associated outbreaks, stable flies are receiving renewed interest and demand for laboratory colonies has increased dramatically.
As for all holometamorphic insects, stable flies obtain all of the nutrients required for growth during the immature or larval stage. Therefore, an important component of a rearing system is the larval diet or substrate. Stable fly larvae have been observed developing in a broad range of substrates in the field7 and they are dependent upon the microbial community of the substrate8,9. Natural larval substrates are primarily composed of decomposing or fermenting vegetative materials often contaminated with nitrogenous wastes.
For laboratory rearing, stable fly larval substrates are usually composed of a vegetative material and an added nitrogen source. Numerous materials have been used for stable fly larval diets. The first larval diets mimicked natural substrates and included fermenting oat straw and horse or cow manure10,11. Carbohydrate sources include wheat bran12,13,14, alfalfa meal12,13,14 and a commercial formulation developed by Chemical Specialties Manufacturers Association (CSMA, 33% wheat bran, 27% alfalfa meal, 40% brewer&#39;s yeast granules)13,14,15,16. Nitrogen sources include yeast suspension12, fish meal and ammonium bicarbonate17. Inert bulking materials are often included in diets including oat hulls12, bagasse13, vermiculite16, wood chips13,18 and pelleted peanut hulls14.
A primary objective of laboratory rearing is to produce a product that is as physiologically similar to &#34;wild type&#34; as possible in order that laboratory experiments will produce results reflecting those of field populations. This requires that in-breeding and selection be minimized to maintain genetic diversity and nutritional resources be comparable to those in the field. Secondary objectives are to minimize labor and expenses. A major component of minimizing expenses is the use of locally available diet components. The stable fly rearing system presented was developed to meet these objectives.

<table>
	<tbody>
		<tr>
			<td>Diamalt</td>
			<td>Premier Malt Products, Inc., Saddle Brook, NJ</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>CSMA Fly media</td>
			<td>Purina Animal Nutrition, Arden Hills, MN</td>
			<td>5S6Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Maxi Pad</td>
			<td>The Tranzonic Co., Cleveland, OH, USA</td>
			<td>5001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Manna</td>
			<td>MannaPro, Chesterfield, MO, USA</td>
			<td>Manna Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Spectrum Chemical Manufacturing Corp, Gardena, CA</td>
			<td>A1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat bran, Coarse</td>
			<td>Siemer Milling Company, Teutopolis, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>Tractor Supply Company, Brentwood, TN</td>
			<td>502770699</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishmeal</td>
			<td>Consumer Supply Distributing, North Sioux City, SD</td>
			<td>F1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult cages</td>
			<td>All Aluminum Window Company, Lincoln, Ne</td>
			<td>Custom</td>
			<td>45 &#215; 45 &#215; 45 cm, 18 &#215; 16 mesh aluminum screen, stockinette access</td>
		</tr>
		<tr>
			<td>9 &#215; 28 cm black cotton cloth</td>
			<td>Robert Kaufman Fabrics, Los Angeles, CA</td>
			<td>K040-114</td>
			<td>Egging cloth</td>
		</tr>
		<tr>
			<td>10 liter plastic dish pans</td>
			<td>Rubbermaid, Saratoga Springs, NY</td>
			<td>FG2951ARWHT</td>
			<td>Larval pans</td>
		</tr>
		<tr>
			<td>Stockinette, Cotton, 12&#34; X 25 yd roll</td>
			<td>Tex-Care Medical Company, Burlington, NC</td>
			<td>91311-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory rearing of stable flies and other muscoid diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

Larvae pupariate 10&#8211;14 days and adults emerge 14&#8211;16 days after oviposition. Generation time, egg to egg, is ~24 days. Rearing data for May 2013 to January 2017 with three different bulking agents and two colonies are presented in Figure 5. Cottonwood gave the best yield, 3867 &#177;1442 (<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg" /> &#177; standard deviation) pupae weighing 12.5 &#177;1.6 mg with 74 &#177;19% eclosion producing...

Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and supplies.

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

This method can help answer key questions in livestock entomology, such as what stable flies require for development. The main advantage to this technique is that it is simple. The medium is composed of only four ingredients.Propagating Stomoxys calcitrans begins with collecting eggs from a stock of flies. This involves preparing an egging cup. Dip the tip of a cloth into a 250-milliliter jar filled with 40 degrees Celsius water.Then wrap the cloth around the outside of the jar and secure the cloth with a rubber band. Fold the loose end of the cloth over the top of the jar to limit access to the water. Now place this cup into a cage of eight to 10-day old flies.After two hours, the gravid females will oviposit enough eggs for the next generation. Typically, 2, 500 flies will produce eight to 40, 000 eggs in two hours. Next, using water, rinse the eggs off the egging cloth into a small, empty dish pan.Theoretically, eggs can survive in water for eight to 12 hours. However, it is best to limit the time to five hours or less. To rear larvae from the eggs, make up fresh fly medium in a 10-liter plastic dish pan.Then make a shallow furrow in the medium along the length of the dish pan for the eggs. To transfer the eggs, make a specialized egg pipette. by inserting a mesh screen into a pipette.Simply cut the pipette at the desired location. And glue a number 100 mesh screen into the pipette at that position. Then, using the egg pipette, collect about one milliliter of eggs and deposit them into the furrow.This amounts to about 8, 000 eggs. Then cover the eggs with a thin layer of medium so they don't desiccate. Next, cover the dish pan with a standard cotton pillowcase, and secure it with a rubber band.When the dish pan is kept under standard rearing conditions, larvae will develop in about two weeks. Within nine days, the larvae will have moved to the edge of the medium, and by day 14, they will have pupated. Many pupae can be found at the dish pan's edge under a crust of food.Regularly scoop the pupae from the dish pan and transfer them to a clean 10-liter dish pan. Add water to break up the clumps of pupae and medium. Most of the food will sink and the pupae will float.Transfer all the floating materials to a stack of sieves and rinse off the remaining medium. The pupae will collect in the number 12 sieve. Rinse the pupae until they look clean, and then float them in clean water.Now collect the cleaned floating pupae in a colander. Then transfer the pupae to a drying screen and allow them to dry under a steady air stream from a fan. Some adults will start to eclose in a few hours and some will take a few days.An alternative method of collecting pupae is to use a pupal shelf, which is made from a plastic section taken from the end of a 10-liter dish pan. To use the shelf, load a 10-liter dish pan with medium tapered from about 2.5 centimeters deep on one end to 7.5 centimeters deep on the other. Then place the shelf in the shallow end and tape the edges to prevent larvae from crawling alongside the shelf.Next, saturate a sponge with water and wrap it in a water-soaked cloth. Place it on the shelf about 1/2 inch from the medium. Importantly, monitor the sponge daily to make sure it stays moist.Allow the larvae to pupate on the shelf, keeping the sponge and cloth away from the food, minimizing the need for extensive cleaning. For a colony health metric, collect 100 pupae from each generation. Weigh the pupae and track their emergence and sex ratio.Before loading pupae into an empty adult cage, line the cage with butcher's paper. Then load 50 grams of dry stable pupae into the cage. Within 24 hours of the first flies eclosing, start feeding them blood.Soak 20 to 25 milliliters of bovine blood into a feminine napkin and position it on top of the cage. Replace it daily until one day prior to oviposition. After oviposition, freeze the cage for four to eight hours to euthanize the flies.Then clean the cage by removing the paper, rinsing frass from the sides and floor with hot water, and scrubbing all surfaces with laundry detergent and chlorine bleach. Then rinse the cage with hot water and allow it to dry. Over 3 1/2 years, three different bulking agents and two colonies were maintained.Cottonwood gave the best yield. The sex ratio was slightly biased towards females. Egg-to-adult survival was approximately 36%Using pine, which was more available than cottonwood, brood number was slightly lower.But individual size was slightly larger. Thus, pine is an acceptable choice. Overall, the operation is very affordable in terms of material costs.The current cost for the medium components is one dollar and seventeen cents per dish pan. Sanitary napkins are five cents each, and a gallon of blood can cost up to five dollars. After watching this video, you should have a good understanding of how to raise stable flies from egg to adult.While attempting this procedure, it's important to remember to be consistent. This makes it much easier to troubleshoot. Following this procedure, other methods like different diets can be performed in order to answer additional questions like what are the nutritional needs of stable flies.

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