Name: maintaining aedes aegypti mosquitoes infected with wolbachia
Uploaded: 2017-08-14T15:00:00
Description: Watch this Scientific Journal Video about Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia at JoVE.com

हम हमारे कॉलोनी रखरखाव के तरीकों के विकास के लिए उनके योगदान के लिए Heng लिन Yeap, क्रिस Paton, Petrina जॉनसन और क्लेयर Doig स्वीकार करते हैं, और उनके सुझावों जो पांडुलिपि में सुधार करने में मदद के लिए तीन गुमनाम समीक्षक । हमारे शोध को एक कार्यक्रम अनुदान और राष्ट्रीय स्वास्थ्य और चिकित्सा अनुसंधान परिषद से AAH को फैलोशिप और वेलकम ट्रस्ट से एक अनुवाद अनुदान द्वारा समर्थित है । सममूल्य एक ऑस्ट्रेलियाई सरकार के अनुसंधान प्रशिक्षण कार्यक्रम छात्रवृत्ति के एक प्राप्तकर्ता है ।

मानव विषयों पर मच्छरों का रक्त पिलाना विश्वविद्यालय मेलबोर्न मानव आचार समिति (अनुमोदन #: ०७२३८४७) द्वारा अनुमोदित किया गया. सभी स्वयंसेवकों को लिखित सहमति प्रदान की गई ।
१. लार्वा पाहिजे
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Wolbachia-संक्रमित एई. aegypti के रखरखाव के लिए यहां प्रस्तुत प्रोटोकॉल के बाद यह सुनिश्चित करना चाहिए कि एक सुसंगत गुणवत्ता के स्वस्थ मच्छरों प्रयोगों और खुले मैदान रिलीज के लिए उत्पादित कर रहे हैं । अंय ?...

एडीज aegypti मच्छरों को डेंगू, Zika और chikungunya1सहित दुनिया में सबसे महत्वपूर्ण arboviruses में से कुछ को हस्तांतरित करने के लिए जिंमेदार हैं । इन वायरस वैश्विक स्वास्थ्य के लिए एक बढ़ती खतरा होते जा रहे है एई. aegypti के बड़े पैमाने पर उष्णकटिबंधीय में वितरण के रूप में विस्तार करने के लिए जारी है2,3,4। महिला एई. aegypti तरजीह मानव रक्त5 पर फ़ीड और इस तरह मनुष्य को करीब निकटता में रहते हैं, विशेष रूप से शहरी क्षेत्रों में जहां आबादी सबसे घने हैं । मानव के साथ इस बंद के सहयोग से वे भी कृत्रिम निवास में टायर, बर्तन, गटर और पानी के टैंक6,7सहित नस्ल के लिए अनुकूलित किया है । Ae. aegypti भी आसानी से प्रयोगशाला के वातावरण के लिए अनुकूल है जहां वे किसी विशेष आवश्यकताओं के बिना बनाए रखा जा सकता है के बाद सीधे क्षेत्र से एकत्र किया जा रहा है, एडीज जीनस8में कुछ अंय प्रजातियों के विपरीत, 9,10. रखरखाव के अपने आसानी उंहें खेतों की एक विस्तृत रेंज में प्रयोगशाला में व्यापक रूप से अध्ययन देखा है, अंततः बीमारियों को नियंत्रित करने के लिए लक्ष्य मच्छरों संचारित कर सकते हैं ।
परंपरागत रूप से, arboviral नियंत्रण मच्छर की आबादी को कम करने के लिए कीटनाशकों के उपयोग पर काफी निर्भर करता है । हालांकि, वहां दृष्टिकोण में रुचि बढ़ रही है जहां संशोधित मच्छरों प्रयोगशाला में पीछे है और फिर प्राकृतिक आबादी में जारी है । जारी मच्छरों आनुवंशिक रूप से संशोधित किया जा सकता है11,12,13, जैविक रूप से14,15, विकिरण के माध्यम से16, रासायनिक उपचार17,18, या संयुक्त तकनीक के साथ19 या तो मच्छरों की आबादी को दबाने या उंहें मच्छरों कि arboviral संचरण20के लिए दुर्दम्य है के साथ बदलें ।
Wolbachia बैक्टीरिया है कि वर्तमान में arboviruses के लिए एक जैविक नियंत्रण एजेंट के रूप में इस्तेमाल किया जा रहा है। Wolbachia के कई उपभेदों हाल ही में एई में पेश किया गया । aegypti प्रयोग भ्रूण microinjection21,22,23,24। इन उपभेदों arboviruses की क्षमता को कम करने के लिए प्रसार और मच्छर में दोहराने, उनके संचरण क्षमता कम23,25,26,27,28 . Wolbachia संक्रमण मां से वंश के लिए प्रेषित कर रहे हैं, लेकिन कुछ उपभेदों जब संक्रमित पुरुषों के साथ22संक्रमित महिलाओं को बाँझता प्रेरित । Wolbachia-संक्रमित पुरुषों इसलिए बड़ी मात्रा में प्राकृतिक मच्छर आबादी को दबाने के लिए जारी किया जा सकता है, के रूप में हाल ही में अंय एडीज प्रजातियों में प्रदर्शन किया15,29। हालांकि, के बाद से Wolbachia भी एई. aegyptiमें arboviral संचरण बाधित, मच्छरों को भी गरीब वैक्टर के साथ मूल आबादी की जगह जारी किया जा सकता है । Ae. aegypti Wolbachia के साथ प्रयोग संक्रमित अब कई देशों में इस बाद दृष्टिकोण का उपयोग कर14,30,31में क्षेत्र में जारी किया जा रहा है ।
arboviral नियंत्रण के लिए Wolbachia-आधारित दृष्टिकोण Wolbachia, मच्छर और पर्यावरण के बीच बातचीत की एक ध्वनि समझ पर भरोसा करते हैं । Wolbachia कीड़ों की एक व्यापक रेंज में स्वाभाविक रूप से होते हैं, और मच्छरों में शुरू उपभेदों उनके प्रभाव३२में विविध रहे हैं । के रूप में नए Wolbachia संक्रमण प्रकार Ae. aegypti24में पेश कर रहे हैं, यह मच्छर स्वास्थ्य पर अपने प्रभाव के लिए प्रत्येक तनाव विशेषताएं, प्रजनन और शर्तों की एक श्रेणी के तहत arboviral हस्तक्षेप आवश्यक है । प्रयोगशाला में कठोर प्रयोग इसलिए क्षेत्र में सफल होने के लिए Wolbachia उपभेदों के लिए क्षमता का मूल्यांकन करने के लिए आवश्यक है ।
खुला क्षेत्र Wolbachia संक्रमण के साथ एई. aegypti की विज्ञप्ति अक्सर हजारों के लिए की आवश्यकता कर सकते है मच्छरों की रिहाई क्षेत्र प्रति हजारों के लिए प्रत्येक सप्ताह14,30,31येते । प्रारंभिक रिलीज की सफलता के लिए एक बड़े आकार के मच्छरों को रिहा करके सुधार किया जा सकता है उनके fecundity३३ और संभोग सफलता३४,३५अधिकतम । मच्छरों भी शर्तों वे क्षेत्र में अनुभव होगा के लिए अनुकूलित किया जाना चाहिए, लेकिन लंबी अवधि के पालन प्रयोगशाला व्यवहार और शरीर विज्ञान जो क्षेत्र के प्रदर्शन को प्रभावित कर सकता है में परिवर्तन का कारण हो सकता है३६,३७, ३८.
हम बुनियादी उपकरणों का उपयोग कर प्रयोगशाला में एई. aegypti पालन के लिए एक सरल प्रोटोकॉल का वर्णन । इस प्रोटोकॉल दोनों जंगली प्रकार और Wolbachiaसंक्रमित मच्छरों के लिए उपयुक्त है, जिसके बाद विशेष ध्यान की आवश्यकता होती है के रूप में कुछ Wolbachia उपभेदों मच्छर जीवन पर पर्याप्त प्रभाव है-इतिहास लक्षण३९, ४०. पालन शर्तों भीड़ और भोजन के लिए प्रतिस्पर्धा से बचने के लिए एक सुसंगत आकार के मच्छरों का उत्पादन है, जो वेक्टर क्षमता और फिटनेस प्रयोगों के लिए महत्वपूर्ण है, और यह सुनिश्चित करता है कि मच्छरों क्षेत्र के लिए स्वस्थ है रिलीज४१ . हम यह भी सावधानी बरतने के लिए प्रयोगशाला अनुकूलन और चयनात्मक दबाव को कम करने और यह सुनिश्चित करना है कि अगली पीढ़ी के एक बड़े, यादृच्छिक पूल से नमूना है द्वारा रिश् ते कम करने के लिए ले । हालांकि, प्रयोगशाला वातावरण स्पष्ट रूप से क्षेत्र की स्थितियों से अलग हैं, और आराम की स्थिति के तहत लंबी अवधि के रखरखाव के क्षेत्र में रिलीज पर मच्छरों की फिटनेस को कम कर सकता है३७,४२,४३ . इसलिए हम प्रयोगशाला लाइनों से महिलाओं के क्षेत्र के लिए पार-पुरुषों के एकत्र समय, कालोनियों कि प्रयोगात्मक तुलना के लिए आनुवंशिक रूप से समान है और जो लक्ष्य क्षेत्र जनसंख्या३९के लिए अनुकूलित कर रहे है में जिसके परिणामस्वरूप । तरीकों किसी भी विशेष उपकरण की आवश्यकता नहीं है और क्षेत्र विज्ञप्ति के लिए प्रति सप्ताह व्यक्तियों के हजारों के रियर दसियों के लिए बढ़ाया जा सकता है । प्रोटोकॉल भी मच्छरों की फिटनेस को प्राथमिकता के भीतर और पीढ़ियों के पार, प्राकृतिक आबादी में स्थापना के लिए किस्मत में कीड़ों के लिए एक महत्वपूर्ण विचार । प्रोटोकॉल सबसे प्रयोगशालाओं कि एई. aegyptiके रखरखाव की आवश्यकता के लिए उपयुक्त है, विशेष रूप से प्रयोगात्मक तुलना के लिए जहां मच्छरों की एक सुसंगत गुणवत्ता और क्षेत्र के लिए संबंधित है महत्वपूर्ण हैं ।

<table width="1661">
	<tbody>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">Wild type Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td style="width:1087px;">Collected from field locations in Queensland, Australia, see Yeap and others39 for details</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w Mel-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to Walker and others23 for information on the strain</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w AlbB-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to Xi and others21 for information on the strain</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w MelPop-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to McMeniman and others22 for information on the strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Instant dried yeast</td>
			<td style="width:168px;">Lowan</td>
			<td style="width:119px;"></td>
			<td>Stimulates egg hatching. Found in general grocery stores. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">5 L plastic tub</td>
			<td style="width:168px;">Quadrant</td>
			<td style="width:119px;">Q110950</td>
			<td>Used for hatching and rearing larvae. Other products may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Fish Food (Tetramin Tropical Tablets)</td>
			<td style="width:168px;">Tetra</td>
			<td style="width:119px;">16152</td>
			<td>Provided to larvae as a source of food. Web address: https://www.amazon.com/Tetra-16152-TetraMin-Tropical-10-93-Ounce/dp/B00025Z6SE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Plastic containers</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used for rearing larvae. Any plastic container above 500 mL should be suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Glass pipette</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used for transferring larvae and pupae between containers. Web address: https://www.aliexpress.com/item/10Pcs-Durable-Long-Glass-Experiment-Medical-Pipette-Dropper-Transfer-Pipette-Lab-Supplies-With-Red-Rubber-Cap/32704471109.html?spm=2114.40010308.4.2.py4Kez</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Clicker counter</td>
			<td style="width:168px;">RS Pro</td>
			<td style="width:119px;">710-5212</td>
			<td>Used to assist in the counting of larvae, pupae and eggs. Web address: http://au.rs-online.com/web/p/products/7105212/?grossPrice=Y</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Rearing trays</td>
			<td style="width:168px;">Gratnells</td>
			<td style="width:119px;"></td>
			<td>Used for rearing larvae. Web address: http://www.gratnells.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Nylon mesh</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used to transfer larvae and pupae to containers of fresh water. Other brands may be used. Web address: https://www.spotlightstores.com/fabrics-yarn/specialty-apparel-fabrics/nettings-tulles/nylon-netting/p/BP80046941001-white</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Cages</td>
			<td style="width:168px;">BugDorm</td>
			<td>DP1000</td>
			<td>Houses adult mosquitoes. Alternative products may be used. Web address: http://bugdorm.megaview.com.tw/bugdorm-1-insect-rearing-cage-30x30x30-cm-pack-of-one-p-29.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">35 mL plastic cup</td>
			<td style="width:168px;">Huhtamaki</td>
			<td style="width:119px;">AA272225</td>
			<td>Used to provide water or sucrose to adult mosquitoes. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">35 mL plastic cup lid</td>
			<td style="width:168px;">Huhtamaki</td>
			<td>GB030005</td>
			<td>Used to provide sucrose to adult mosquitoes. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Cotton wool</td>
			<td style="width:168px;">Cutisoft</td>
			<td style="width:119px;">71841-13</td>
			<td>Moist cotton wool is provided as a source of water to adults. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">White Sugar</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided as a source of sugar to adult mosquitoes. Found in general grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Rope</td>
			<td style="width:168px;">M Recht Accessories</td>
			<td>C323C/W</td>
			<td>Used to provide sucrose solution to adults. Other brands may be used. Web address: https://mrecht.com.au/haberdashery/braids-cords-and-tapes/cords/plaited-cord/cotton/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Plastic cup (large)</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Sandpaper</td>
			<td style="width:168px;">Norton Master Painters</td>
			<td>CE015962</td>
			<td>Provided as an oviposition substrate. Alternative products may be used, but we use this brand because it is relatively odorless. Lighter colors are used for contrast with eggs. Web address: https://www.bolt.com.au/115mm-36m-master-painters-bulk-roll-p80-medium-p-9396.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Filter paper</td>
			<td style="width:168px;">Whatman</td>
			<td style="width:119px;">1001-150</td>
			<td>Used as an alternative oviposition substrate. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Latex gloves</td>
			<td style="width:168px;">SemperGuard</td>
			<td>Z560979</td>
			<td>Prevents mosquito bites on hands when blood feeding. Other brands may be used. Web address: http://www.sempermed.com/en/products/detail/semperguardR_latex_puderfrei_innercoated/</td>
		</tr>
	</tbody>
</table>

maintaining aedes aegypti mosquitoes infected with wolbachia

एडीज aegypti मच्छरों प्रयोग से संक्रमित Wolbachia जैसे डेंगू, chikungunya और Zika के रूप में arboviruses के प्रसार को नियंत्रित करने के लिए कार्यक्रमों में उपयोग किया जा रहा है । Wolbachiaसंक्रमित मच्छरों के क्षेत्र में जारी किया जा सकता है या तो असंगत संभोग के माध्यम से जनसंख्या आकार को कम करने के लिए या मच्छरों कि वायरस संचरण के लिए दुर्दम्य है के साथ आबादी को बदलने । इन रणनीतियों के लिए सफल होने के लिए, प्रयोगशाला से क्षेत्र में जारी मच्छरों देशी मच्छरों के साथ प्रतिस्पर्धी होना चाहिए । हालांकि, प्रयोगशाला में मच्छरों को बनाए रखने के लिए रिश् तों में परिणाम कर सकते हैं, आनुवंशिक बहाव और प्रयोगशाला अनुकूलन जो क्षेत्र में अपनी फिटनेस को कम कर सकते हैं और प्रयोगों के परिणामों को पाया जा सकता है । क्षेत्र में तैनाती के लिए विभिन्न Wolbachia संक्रमण की उपयुक्तता का परीक्षण करने के लिए, यह कई पीढ़ियों भर में एक नियंत्रित प्रयोगशाला वातावरण में मच्छरों को बनाए रखने के लिए आवश्यक है । हम प्रयोगशाला में एई. aegypti मच्छरों को बनाए रखने के लिए एक सरल प्रोटोकॉल का वर्णन करते हैं, जो Wolbachia-संक्रमित और जंगली प्रकार के मच्छरों दोनों के लिए उपयुक्त है । तरीकों प्रयोगशाला अनुकूलन को कम करने और क्षेत्र मच्छरों के लिए प्रयोगों की प्रासंगिकता को बढ़ाने के लिए पार करने को लागू. इसके अतिरिक्त, कालोनियों इष्टतम शर्तों के तहत खुला क्षेत्र रिलीज के लिए अपनी फिटनेस को अधिकतम करने के लिए बनाए रखे हैं ।

चित्रा 4 एई. aegypti लार्वा के विकास पर इष्टतम पोषण के प्रभाव को दर्शाता है । जब कंटेनरों ०.२५ प्रति दिन या उससे कम के प्रति लार्वा भोजन की मिलीग्राम के साथ प्रदान की जाती हैं, दोनों ?...

Watch this Scientific Journal Video about Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia at JoVE.com

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Wolbachia से संक्रमित एडीज aegypti मच्छरों को बनाए रखने

एडीज aegypti Wolbachia से संक्रमित मच्छरों arboviruses के संचरण को दबाने के लिए प्राकृतिक आबादी में जारी किया जा रहा है । हम प्रयोगों और क्षेत्र के रिलीज के लिए प्रयोगशाला में Wolbachia संक्रमण के साथ रियर एई. aegypti तरीकों का वर्णन, प्रयोगशाला अनुकूलन और चयन को कम करने के लिए सावधानियों ले ।

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

The overall goal of this protocol is to describe the maintenance of the Aedes aegypti mosquitoes infected with Wolbachia. These mosquitoes are being released around the world to suppress the transmission of dengue and other viruses. Our protocol for rearing Aedes aegypti mosquitoes can be used by any researchers who are interested in using mosquitoes for experiments or disease control programs.This method can also be used for rearing genetically modified mosquitoes for mosquito population control programs, including those that involve sterile insect release. The principles involved in this approach can also be applied to the rearing of other disease vector species. For this protocol, house the mosquitoes at approximately 26 degrees Celsius and at 50 to 70%relative humidity with an equatorial photo period.If they are reared at higher temperatures, the Wolbachia infections could be lost, so do not overheat the colonies. Maintain around 500 mosquitoes per population to minimize inbreeding. Using this protocol with adequate nutrition will yield 28 day generations.Begin with eggs on a substrate. Submerge them in trays containing three liters of water with about 300 milligrams of fish food and a few grains of active, dry yeast to induce hatching. One day after hatching, use a glass pipet to transfer approximately 500 larvae to trays containing four liters of water and fresh fish food.Check the trays daily to ensure that the larvae have sufficient food. Add about 600 milligrams of fish food every other day to maintain an optimal food supply. Overfeeding can result in water clouding and thus mortality, so replace the water as needed.If nutrition and temperatures are maintained correctly, the larvae will pupate five to seven days after hatching. And adults will emerge approximately two days later. Seven days after hatching, pour the entire contents of the tray through a fine mesh.Retain some of the filtered water in a five liter container for ova position in the same colony. Next, invert the mesh, and dip it into a plastic container with 200 milliliters of water to transfer the pupae. Add the appropriate amount of food to feed any remaining larvae.Now, set up an adult emergence cage with two cups of 10%sucrose solution and two cups of moist cotton balls to prevent desiccation. If the pupae do not need to be sorted by sex, transfer the container of pupae into a cage, and leave the lid slightly ajar for the adults to escape and enter the cage. After all of the adults have emerged, remove the container from the cage.Do not select against slow developers by taking the container out sooner. To collect virgin mosquitoes, transfer the pupae from the larval rearing trays to plastic containers with 200 milliliters of water every 24 hours. Sort the sexes by size.Females are larger than males. And transfer them to separate containers. Keep the lids closed on the containers so the adults do not escape.Before releasing the emerging adults into their cages, confirm their sex. Incorrectly sexed adults must be removed using an aspirator within 24 hours of emergence while they are still sexually immature. To keep the Wolbachia infected colonies similar to a natural population, cross the infected females from the lab to uninfected wild males.Mature the males and females separately for two days. And then aspirate the females into the male cage. Ideally, set up a one to one sex ratio with a population size of around 500.Proceed to outcross the mosquitoes for at least three consecutive generations. At least three days after the last adults emerge, and before they are two weeks old, blood feed the colony before excessive female mortality occurs. This is particularly important for mosquitoes with Wolbachia infections that shorten lifespan.The sugar cups should be removed the day before. Give the mosquitoes access to a forearm, and wear a latex glove to protect the hand and wrist. Most females should be fed within five minutes.But it can take up to 15 minutes. Once all the females are visibly engorged, remove the forearm, and replace the sugar cups. Two days after blood feeding, place two plastic cups filled with water and lined with a partially submerged strip of sandpaper into the cage.Then, remove any other water source except for the sugar water. The females will oviposit on the sandpaper just above the water line from three days after blood feeding. Collect and replace the sandpaper daily until no more eggs are laid.If more eggs are needed, blood feeding can be performed at weekly intervals. Partially dry the sandpaper with about 30 seconds of gentle blotting using a paper towel. Do not dislodge the eggs.Then, wrap the strips in a sheet of dry paper towel, and place it into a sealable plastic bag. Three days post collection, inspect the eggs under a dissecting microscope. If the sandpaper is too wet, the eggs may hatch before being submerged in water.But if it dried too harshly, the eggs may collapse. The eggs are ready to hatch three days after collection. Submerge all the eggs from each colony into the same container of water to produce a large pool of individuals for the next generation.Generally, eggs can be stored in a sealed container with a humidity greater than 80%at 20 degrees Celsius for several months. But some Wolbachia infections reduce viability, so these eggs should be hatched within a week of collection. When larvae containers were provided with 0.25 milligrams of food per larva per day or less, the development time increased for both sexes and was less synchronous than in containers provided with more food.If adequate food is not provided throughout the duration of development, this will negatively impact colony maintenance. Slowly developing individuals might be selected against, and blood feeding may be delayed. There may even be increased adult mortality before reproduction.Sub-optimal nutrition affected adult morphology. Wing lengths decreased substantially and correlated to nutrition levels. Uniform body sizes are important to experimental comparisons as body size is positively associated with fecundity, and large mosquitoes should be more fit under field conditions.After watching this video, you should have a good understanding of how to maintain Aedes aegypti mosquitoes infected with Wolbachia. Following our protocol should ensure that healthy mosquitoes of a consistent quality are produced. These mosquitoes can then be released into the field for disease control programs or be used for experiments.When working with Wolbachia infected mosquitoes, it is important to monitor their infection status to ensure that the colonies maintain their Wolbachia infections.

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Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Wolbachia Bacterial Infection in Drosophila

Wolbachia ड्रोसोफिला में बैक्टीरियल संक्रमण

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

ए aegypti भ्रूण के Microinjection ट्रांसजेनिक मच्छरों प्राप्त

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

डेंगू संक्रमण के लिए मच्छरों (ए aegypti) और संक्रमण Phenotype निर्धारण में प्रोटोकॉल

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

सेल मुक्त मध्यम में Wolbachia बनाए रखते

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

ऐ से midgut और लार की गिल्टी के विच्छेदन. aegypti मच्छरों

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

बिल्डिंग एक बेहतर मच्छर: ए gambiae और ए aegypti मच्छरों में मलेरिया और डेंगू बुखार प्रतिरोध सक्रिय जीन की पहचान

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

आरएनएआई की मध्यस्थता जीन मारकर गिरा दिया और<em&gt; Vivo में</emवयस्क महिला में diuresis परख<em&gt; एडीज aegypti</em&gt; मच्छरों

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

शारीरिक रिकॉर्डिंग और पीले बुखार मच्छर के स्वाद उपांग की शाही सेना अनुक्रमण<em&gt; एडीज एजिप्टी</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

एडीज Aegyptiमें वसा शरीर अंग संस्कृति प्रणाली, Zika वायरस के एक वेक्टर

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

24 अच्छी तरह से ऊतक संस्कृति प्लेटें (EAgaL प्लेटें) का उपयोग कर एडीज एजिप्टी के लिए बेहतर Fecundity और प्रजनन परख

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...