Name: laboratory rearing of stable flies and other muscoid diptera
Uploaded: 2018-08-03T12:30:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Wir möchten danken Anthony Weinhold und den zahlreichen Studenten, die bei uns über die Jahre für den technischen Support sowie Vorschläge zur Verbesserung unserer Insekt Aufzucht Verfahren gearbeitet haben.

1. Eizellenentnahme (Abbildung 1)
<ol><li>Bereiten Sie egging Tasse, platzieren Sie ein Ende des Tuches in ~ 500-mL-Becherglas gefüllt mit Warmwasser (~ 40 ° C). Überschneiden sich die Seiten des Cups und mit einem Gummiband befestigen. Falten Sie das lose Ende des Tuches, zurück über den oberen Rand der Tasse.</li>
	<li>Ort, an dem egging Cup im Käfig von 8 – 10 Tage alten fliegen für ~ 2 h. trächtige Weibchen stabil fliegt wird auf dem Stoff oviposit.</li>
	<li>Käfig und spülen Sie Eier aus dem egging Tuch egging Tasse in einer kleinen Pfanne mit Squeeze Waschflasche entfernen.</li>
</ol>2. bereiten Sie Larven Aufzucht Pan
<ol><li>
Medium (Mengen für 1 Aufzucht Pfanne) vorbereiten
	<ol>
<li>Kombinieren Sie, Weizenkleie (500 g), Holzspäne (200 g) und Fischmehl (115 g) in 10 L Plastikschale Pfanne und mischen Sie gut.</li>
		<li>Wasser (~ 25 ° C, 1600 mL) Ammonium Bicarbonat (50 g) hinzu und vermischen Sie, bis aufgelöst.</li>
		<li>Fügen Sie Wasserlösung trockenen Zutaten zu mischen, um kein unbenetzte Material zu gewährleisten. Level Medium in Pfanne aber nicht komprimieren.</li>
	</ol>
</li>
	<li>
Stabile fliegeneier hinzufügen
	<ol>
<li>Machen Sie die Länge der Wanne einer flache Furche in Medium.</li>
		<li>1 mL der stabilen fliegeneier (~ 8.000 Eier) in Furche mit Ei Pipette (Abbildung 2) einzahlen. Hinweis: Ei Pipette gemacht durch Schneiden einer Kunststoff abgestufte Pipette am gewünschten Punkt einfügen 100 mesh Bildschirm und klebenden Pipette wieder zusammen.</li>
		<li>Decken Sie die Eier mit einer dünnen Schicht des Mediums um Austrocknung zu verhindern.</li>
		<li>Decken Sie die Pfanne mit Kissenbezug, schließen Sie mit einem Gummiband und Label. Stellen Sie Pfannen im Larvenstadium Zimmer (23 ± 2 ° C, 30 – 50 % RH und 12:12 [L:D] h Photoperiode). Hinweis: Larvalentwicklung dauert 10 – 14 Tage ab dem Zeitpunkt der Eiablage.</li>
	</ol>
</li>
</ol>(3) pupal Verarbeitung, Larven bewegen an den Rand des Mediums 7 – 9 Tage nach der Eiablage und Pupariate von 13 oder 14 Tage
<ol><li>Kugel Puparia, befindet sich in erster Linie an den Rändern der Pfanne unter der Kruste, die bildet auf der Oberfläche des Mediums, heraus und in eine saubere Pfanne gelegt.</li>
	<li>Füllen Sie Pfanne, mit Puparia, ½ bis ¾ voll Wasser. Unterteilen Sie Klumpen von Puparia und Medium. Weizen Kleie und Protein Komponenten des Mediums sinken bei Puparia > 1 Tag alten Float19.</li>
	<li>Entfernen Sie schwimmenden Materialien, Puparia und einige mittelkomponenten mit einem Sieb und Waschen durch eine Reihe von Siebe (#5, #7, #12 und #20) verbleibenden Medium entfernen. #12-Sieb sammelt die Puparia und die #20 hält festen Abfälle in den Abfluss gelangen. Puparia zwischen Siebe mit einer Küchenspüle Sprayer zu waschen.</li>
	<li>Spülen Sie mit Puparia, das #12 Sieb in einen sauberen Topf. Füllen Sie die Schale zu ½ voll Wasser. Schwimmende Puparia in ein Sieb Gießen und überschüssiges Wasser abtropfen lassen.</li>
	<li>Transfer Puparia zum Trocknen Bildschirm mit Fan entwässert und lassen Sie trocknen (Abbildung 3).</li>
</ol>4. pupal Regal, eine Alternative Methode zum Sammeln von Puparia ist mit einem Pupal Regal18
Hinweis: Das Regal ist aus einem Stück Kunststoff Schnitt vom Ende eines 10 L Schüssel Pan (10,2 cm hoch x 10,2 cm breit x 31,9 cm lang) gemacht.
<ol><li>Bereiten Sie Medium, wie unter 2.1 beschrieben. Konus Medium in Pfanne von ~2.5 cm tief auf einem Ende bis ~7.5 cm tief auf der anderen.</li>
	<li>
Stellen Sie Regal auf Medium im flachen Ende der Aufzucht Pfanne und Klebeband zu verhindern, dass Larven kriechen neben dem Regal (Abbildung 4).
	<ol>
<li>Schwamm (~14.5 x 9,0 x 4,5 cm3, regenerierte Zellulose) zu sättigen und wickeln Sie es in Wasser getränkten Tuch (30,5 x 42 cm2, Baumwolle) und Platz auf dem Regal ca. ein halbes Zoll vom Medium.</li>
	</ol>
</li>
	<li>
Fügen Sie Eier hinzu, wie in 2.2 beschrieben und wie zuvor Verfahren.
	<ol>
<li>Pfannen täglich zu überprüfen, um sicherzustellen, dass der Schwamm feucht bleibt.</li>
	</ol>
</li>
	<li>Spülen Puparia aus dem Regal, Schwamm und Tuch in eine saubere Schüssel schwenken und 14 Tage nach der Eiablage in Sieb Gießen.</li>
</ol>5. Qualitätskontrolle
<ol><li>Wiegen Sie alle die Puparia, in Pfanne (total wt.) produziert.</li>
	<li>100 Puparia zu isolieren, wiegen und in einem 9 cm Petrischale gelegt.</li>
	<li>Graf und Sex Erwachsene ~ 5 – 8 Tage später zur Entstehung Rate und Geschlechterverhältnis zu bestimmen. Speichern Sie Petrischalen mit aufgetauchte Erwachsene in einem Gefrierschrank für die Inventur zu einem späteren Zeitpunkt bei Bedarf. Fliegen sind durch die Form ihrer Augen und Breite der Fronto-Orbitale Platte20 oder Genitalien unter geringer Vergrößerung geschlechtlich.</li>
	<li>Rekord Gesamtgewicht, Gewicht 100 Puparia und Anzahl der aufgetauchten erwachsenen Männchen und Weibchen.</li>
</ol>6. Vorbereitung Blut
<ol><li>Sammeln Sie frisches Rinderblut aus einer lokalen Schlachthof in 19 L Eimer mit 70 g Natriumcitrat tribasic Dihydrat in 500 mL Wasser. Unter Rühren kräftig für 5 – 10 min Blut und zurück ins Labor.</li>
	<li>Stamm Blut durch ein Sieb entfernen Blutgerinnsel und aufgeteilt in 2 L Container, Label mit dem Datum der Kollektion und Shop in einem Gefrierschrank (-20 ° C).</li>
	<li>Entfernen Sie Behälter aus Gefrierfach und in den Kühlschrank stellen benötigt 2 Tage vor. Blut kann für ~ 2 Wochen einmal aufgetaut und in der Tiefkühltruhe gelagert werden, bis zu 1 Jahr verwendet werden.</li>
</ol>(7) Erwachsenen Wartung
<ol><li>Bereiten Sie Käfige, Linie Käfig unten mit Metzgerei Papier um die Reinigung zu erleichtern.</li>
	<li>50 g trocken stabil fliegen Puppen (~ 3.500) in jedem Käfig zu platzieren. Erwachsene Eclose wenige Stunden bis wenige Tage nach der pupal Verarbeitung.</li>
	<li>Ort ein frisches Blut getränkt feminine Serviette auf jeder Käfig für die Fütterung. Beginnen Sie innerhalb von 24 h von Erwachsenen bewegungsapparate Fütterung und wiederholen Sie täglich bis einen Tag vor der Eiablage (9 – 10 Tage nach Entstehung).</li>
	<li>Nach der Eiablage (Abschnitt 1) Käfig in Gefrierschrank für 4 – 8 h und dann durch Entfernen des Papiers, Frass von den Seiten und den Boden mit heißem Wasser spülen und alle Oberflächen mit Waschmittel und Chlorbleiche Scheuern reinigen. Käfige mit heißem Wasser abspülen und trocknen lassen.</li>
</ol>

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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. 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Stabilere fliegen finden Sie in einer Vielzahl von Substraten in der Natur zu entwickeln und in vielen Arten von Medien im Labor gezüchtet werden können. Wir haben Holzspäne und Vermiculit als Füllstoff-Agenten verwendet. Vermiculit hat gut funktioniert, aber Trennung Puparia von der Aufzucht Medium schwer gemacht und war teuer (~$0.60/pan). Möglicherweise lag wegen der zusätzlichen strenge Puparia vom Medium zu trennen, bewegungsapparate auch mit Vermiculit, 57 % vs. 75 % für Holzspäne. Cottonwood Späne waren v...

Stabilere fliegen, Stomoxys Calcitrans (L.), sind Hematophagous fliegen, deren schmerzhafte Bisse der Beweidung Verhalten der Tiere zu stören, Schmerzen verursachen und Begleiter Tiere leiden und menschlichen Freizeitaktivitäten weltweit zu stören. Unreife stabilere fliegen entwickeln sich in Gärung vegetativen übrigens oft mit tierischen Abfällen kontaminiert. Veränderten Anbaumethoden und Kulturen haben schwere Ausbrüche von stabil fliegen in Ernterückstände, Gemüse in Australien1, Zuckerrohr in Brasilien2und Ananas in Costa Rica3produziert. Obwohl nur 14 stabilere fliegen pro Tier die wirtschaftlichen Schwelle4 gelten, wurden Beobachtungen von mehr als 2.000 fliegen pro Tier während den letzten Ausbrüche5vorgenommen. Solche Werte bei Befall Host Produktivität beinahe auf null reduzieren und können dazu führen, dass Mortalität6. Als Folge agronomisch verbundenen Ausbrüche stabilere fliegen erhalten erneuertes Interesse und Nachfrage nach Labor Kolonien drastisch angestiegen.
Für alle Holometamorphic Insekten erhalten stabilere fliegen alle Nährstoffe für das Wachstum in der unreifen oder larvalen Phase erforderlich. Daher ist ein wichtiger Bestandteil einer Aufzucht System die Larven Nahrung oder das Substrat. Stabile Fliegenlarven entwickeln in einer Vielzahl von Substraten in Feld7 eingehalten worden und sie sind abhängig von der mikrobiellen Gemeinschaft der Substrat-8,-9. Natürliche Larven Substrate bestehen hauptsächlich zerlegen oder vegetative Materialien oft verunreinigt mit stickstoffhaltige Abfälle vergären.
Für Labor Aufzucht bestehen stabile Fliege Larven Substrate in der Regel aus einer vegetativen Material und einer zusätzlichen Stickstoff-Quelle. Für stabile Fliege Larven Diäten wurden zahlreiche Materialien verwendet. Die ersten Larven Diäten imitiert natürliche Substrate und gärenden Hafer Stroh und Pferd oder eine Kuh dung10,11enthalten. Kohlenhydratquellen zählen Weizen Kleie12,13,14, Luzerne Mahlzeit12,13,14 und eine kommerzielle Formulierung von chemischen Spezialitäten entwickelt Hersteller Association (CSMA, 33 % Weizenkleie, 27 % Alfalfa-Meal, 40 % Bierhefe Granulat)13,14,15,16. Stickstoff-Quellen gehören Hefe Aussetzung12, Fischmehl und Ammonium Bicarbonat17. Inerte Füllstoffe Materialien gehören oft Diäten einschließlich Hafer Rümpfe12, Bagasse13, Vermiculit16, Hackschnitzel13,18 und gebeizte Erdnuss Rümpfe14.
Ein primäres Ziel der Aufzucht von Labor ist, ein Produkt zu produzieren, die als physiologisch ähnlich "Wildtyp" wie möglich, so dass Laborexperimente Ergebnisse reflektieren diejenigen Bereich Bevölkerung bringt. Dies erfordert, dass in der Züchtung und Selektion minimiert werden weiterhin genetische Vielfalt und Ernährung Ressourcen werden vergleichbar mit denen im Feld. Sekundäre Ziele sind, Arbeit und Kosten zu minimieren. Ein Hauptbestandteil der Minimierung der Kosten ist die Verwendung von lokal verfügbaren Ernährung Komponenten. Die stabile Fliege Aufzucht vorgestellte System wurde entwickelt, um diese Ziele zu erreichen.

<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 &amp;times; 45 &amp;times; 45 cm, 18 &amp;times; 16 mesh aluminum screen, stockinette access</td></tr><tr><td>9 &amp;times; 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 L plastic dish pans</td><td>Rubbermaid, Saratoga Springs, NY</td><td>FG2951ARWHT</td><td>Larval pans</td></tr><tr><td>Stockinette, Cotton, 12 inch x 25 yard 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

Stabilere fliegen, Stomoxys Calcitrans, sind ernsthafte Schädlinge von Tieren, Menschen, Haus- und Wildtiere weltweit. Während der letzten 20 + Jahre führten Änderungen im landwirtschaftlichen Praktiken schwere Ausbrüche von stabil fliegen in mehreren Ländern. Diese Ausbrüche gestört Tierproduktion und menschlichen Erholung führt öffentliche Forderungen zur Erhöhung der Anstrengungen in Forschung und Management für diesen Schädling. Eine einfache und kostengünstige Verfahren zur Aufzucht stabiler Flies für Laborstudien wird vorgestellt. Das Verfahren nutzt lokal verfügbaren Ernährung Komponenten, Geräte und Materialien. Das Verfahren kann für die Aufzucht von anderen moosigen Flies einschließlich Gesicht Fliege (Musca Autumnalis), Horn-Fly (Haematobia Irritans) und Stubenfliege (Musca Domestica) angepasst werden. Das Verfahren erzeugt stabil fliegen Puparia von durchschnittlich 12,5 mg und ~ 35 % Ei zum Erwachsenen überleben. Ca. 3000 fliegen werden in jeder Pfanne hergestellt.

Larven-Pupariate 10-14 Tage und Erwachsene entstehen 14 – 16 Tage nach der Eiablage. Generationszeit, Ei, Ei, ist ~ 24 Tage. Aufzucht Daten für Mai 2013 bis Januar 2017 mit drei verschiedenen Füllstoffe und zwei Kolonien sind in Abbildung 5dargestellt. Cottonwood gab die beste Ausbeute, 3867 ±1442 (<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg"> </img> ± Standardabweichung) Puppen mit einem Gewicht von 12,5 ±1.6 mg mit 74 ±19 % bewegun...

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

Ein Verfahren zur Aufzucht stabil fliegt (Stomoxys Calcitrans) präsentiert. Das Verfahren nutzt lokal verfügbare Materialien für Diät-Komponenten, Geräte und Materialien.

Labor Aufzucht von stabil fliegen und andere moosigen Diptera

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

laboratory-rearing-of-stable-flies-and-other-muscoid-diptera

Research

JoVE Journal

Biology

8.5K Aufrufe.  ARS-USDA. Ein Verfahren zur Aufzucht stabil fliegt (Stomoxys Calcitrans) präsentiert. Das Verfahren nutzt lokal verfügbare Materialien für Diät-Komponenten, Geräte und Materialien.

Serie: Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

Aufzucht und langfristige Wartung von Schwebfliegen Eristalis Tenax für Forschungsstudien

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

Forschung

Umwelt

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Reproduzierbare DsRNA Mikroinjektion und Eiablage Bioassay in Mücken und Stubenfliegen

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

Fliegen-Insekt Erkennung und Klassifizierung mit preiswerten Sensoren

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Biotechnik

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Ein optimiertes Protokoll für die Aufzucht Fopius arisanus, Fliegt ein Parasitoid von Tephritid Fruit

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Biologie

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Aufrechterhaltung eines<em&gt; Drosophila melanogaster</em&gt; Bevölkerung Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Circadian Entrainment of Drosophila Melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Circadian Entrainment von Drosophila Melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Erhaltung von Laborkulturen von Gryllus bimaculatus, einem vielseitigen Orthopteran-Modell für die Insektenzucht und die Physiologie wirbelloser Tiere

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Topische Anwendung Bioassay zur Quantifizierung der Insektizidtoxizität für Moskitos und Fruchtfliegen

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Bradysia coprophila Lab Protocols and Chromosomal Research

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laborerhaltung der Unterdipteranen Fliege Bradysia (Sciara) coprophila: Ein neuer/alter aufstrebender Modellorganismus

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

Drosophila Maintenance

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of these critters may seem as easy as leaving a banana on your kitchen counter for too long, a productive fly colony in the lab requires careful husbandry and maintenance.
This video demonstrates the necessary steps for maintaining a healthy fly stock. The overview begins with the preparation and storage of the yeast and sugar-containing media on which flies feed. Next, the vessels most commonly used for housing Drosophila are shown, as well as how and when to move flies between these containers. Finally, the presentation also includes examples of the ways in which housing and feeding conditions are manipulated for biological experiments.

Maintenance and Husbandry of Drosophila melanogaster

Haltung und Pflege von Drosophila

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of ...