Alimentaires des moustiques: <ol><li> Alimentation A: la nourriture des poissons à la terre (Aquaricare). Une petite pincée doit être ajouté. </li><li> Alimentation B: mise à la terre la nourriture CAT (Purina). Une petite pincée doit être ajouté. </li><li> Alimentation C: aliments pour chats (Purina). Deux comprimés doit être ajouté. </li></ol> Sang: Sang animal ou humain peut être utilisé pour les moustiques à l&#39;arrière. Le sang frais est recueilli avec une seringue et mettre dans un tube stérile de 15 ml contenant 1 ml de Falcon DPC (anticoagulant) pour chaque 10 ml de sang, puis mélangés doucement et centrifugé à température ambiante à 2000 rpm pendant 5 min. Le surnageant est écarté, en prenant soin d&#39;enlever la couche leuco qui comprend d&#39;autres cellules sanguines (par exemple WBC). Le culot de RBC est ensuite suspendu dans un volume égal de milieu RPMI par pipetage, et encore lavé dans ce trois moyennes fois. Après le lavage final, les globules rouges granulés sont remis en suspension dans un volume égal de milieu RPMI et conservés à 4 ° C (peut être conservé pendant 8-10 jours). Juste avant l&#39;alimentation, la RBC (dans RPMI) est centrifugé (2000 rpm pendant 5 min) pour obtenir un culot en bas des cellules, et la RBC emballé est remis en suspension dans le sérum (O + sérum humain) afin d&#39;obtenir un hématocrite de 40%. Le sang doit être conservé à 37 ° C toujours à l&#39;alimentation. JOUR 1: <ol><li> Les 3-5 jours moustiques adultes âgés féminins sont nourris de sang pour pondre leurs œufs. </li><li> Pour nourrir le sang, une méthode de membrane artificielle alimentation (parafilm) est utilisé comme suit: <ol><li> Les globules rouges (voir ci-dessus pour la préparation du sang) sont mélangés avec du sérum inactivé par la chaleur pour obtenir un hématocrite ~ 40% (emballé RBC 40% et 60% de sérum). Ceci est ajouté à l&#39;alimentation en verre. </li><li> Le chargeur est relié à une chaude enveloppe d&#39;eau (37 ° C), et placée sur la cage pour permettre l&#39;accès des moustiques à la surface membranaire. </li><li> Les moustiques sont autorisés à nourrir pour ~ 30 minutes. </li></ol></li></ol> JOUR 3: <ol><li> Les femelles pondent des œufs deux jours après ils se nourrissent de sang. Un petit papier filtre enveloppé dans une forme conique est placé dans un petit bécher contenant de l&#39;eau distillée, en s&#39;assurant que le papier filtre est humide. Le bécher est gardé à l&#39;intérieur de la cage durant la nuit pour les moustiques de pondre des œufs. </li></ol> JOUR 4: <ol><li> Le papier filtre contenant les œufs des moustiques est placé dans un bac en plastique avec environ 300 ml d&#39;eau distillée. Une pincée de nourriture A est ajoutée à la barre et les oeufs sont autorisés à éclore au cours de larves les prochains jours. </li></ol> JOURS 5 - 8: <ol><li> Larves en croissance sont nourris chaque jour avec deux comprimés de nourriture C, et surveillés pour la densité et la population. Le huitième jour (5 jours larves âgées), la population des larves est dilué de 1 Bac à ~ 10 plateaux, avec une pincée de nourriture A et deux comprimés de C des aliments dans chaque bac. (~ 30 minutes) </li></ol> JOURS 9 - 12: <ol><li> Les larves sont nourries chaque jour avec de la nourriture C. Sur le 12ème jour (9 jours larves âgées), l&#39;eau est changée avec de l&#39;eau fraîche, et la nourriture est ajoutée (une pincée de nourriture B et deux comprimés de nourriture C). Les nymphes commence à se développer à ce stade. Les plateaux sont couverts de filets pour éviter de sortir des adultes. (~ 80 à 10 mn) </li></ol> JOUR 13 - 15: Les nymphes sont autorisés à émerger aux adultes pour les 2 prochains - 3 jours. La nourriture est donnée tous les jours pour les larves / nymphes par enlevant soigneusement le net pour éviter l&#39;échappement des adultes. JOUR 16: <ol><li> Les adultes sont recueillis dans une cage à travers un aspirateur relié à vide. La cage se compose d&#39;un petit flacon de 100 ml avec une mèche de coton qui a été trempé avec du saccharose à 10% (autoclave) et une doublure serviette en papier sur le fond pour absorber les déversements potentiels de sucre qui peut se produire pendant la manipulation cage. (~ 30-40 minutes) </li></ol> JOUR 17 - 21: <ol><li> Les adultes (mâles et femelles) sont ensuite conservés dans la salle insectarium pour les 4-5 jours, nourri de 10% de saccharose avant qu&#39;ils ne soient à nouveau du sang nourris pour commencer le cycle suivant. Les moustiques mêmes peuvent être utilisées pour pondre plus d&#39;une fois. </li><li> Les moustiques qui ne sont pas nécessaires pour des expériences ou de l&#39;élevage peuvent être tués en plaçant la cage dans un congélateur. Les plateaux utilisés et les cages doivent être nettoyés et séchés avant qu&#39;ils puissent être utilisés à nouveau. </li></ol> REMARQUE: Il peut y avoir quelques variations dans la méthode d&#39;élevage des moustiques et des laboratoires différents peuvent avoir différentes techniques.

<ol>
	<li>Bangs, M. J., Soelarto, T., Barodji, B. P., Wicaksana, D. T., Boewono, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+Anopheles+maculatus+from+Central+Java,+Indonesia.">Colonization of Anopheles maculatus from Central Java, Indonesia.</a> J Am Mosq Control Assoc. 18, 359-363 (2002).</li><li>Beier, M. S., Beier, J. C., Merdan, A. A., el Sawaf, B. M., Kadder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+rearing+techniques+and+adult+life+table+parameters+for+Anopheles+sergentii+from+Egypt.">Laboratory rearing techniques and adult life table parameters for Anopheles sergentii from Egypt.</a> J Am Mosq Control Assoc. 266, 266-270 (1987).</li><li>Benedict, M. Q., Crampton, J. M., Beard, C. B., Louis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Care and maintenance of anopheline mosquitoes, The molecular biology of disease vectors: A methods manual. , 3-12 (1997).</li><li>Casanges, A. H., Mcgovran, E. R., Chiles, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+of+Anopheles+quadrimaculatus+Say+and+Aedes+aegypti+(L.)+in+the+laboratory.">Rearing of Anopheles quadrimaculatus Say and Aedes aegypti (L.) in the laboratory.</a> Mosq News. 9, 112-117 (1949).</li><li>Merritt, R. W., Dadd, R. H., Walker, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+behavior,+natural+food+and+nutritional+relation+ships+of+larval+mosquitoes.">Feeding behavior, natural food and nutritional relation ships of larval mosquitoes.</a> Annual Rev. Entomol. 37, 349-376 (1992).</li><li>Nasirian, H., Ladonni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+bloodfeeding+of+Anopheles+stephensi+on+a+membrane+apparatus+with+human+whole+blood.">Artificial bloodfeeding of Anopheles stephensi on a membrane apparatus with human whole blood.</a> J Am Mosq Control Assoc. 22, 54-56 (2006).</li><li>Ng'habi, K. R., John, B., Nkwengulila, G., Knols, B. G., Killeen, G. F., Ferguson, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+larval+crowding+on+mating+competitiveness+of+Anopheles+gambiae+mosquitoes.">Effect of larval crowding on mating competitiveness of Anopheles gambiae mosquitoes.</a> Malar J. 30 (4), 49 (2005).</li><li>Stobart, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+control+of+body+volume+in+the+larvae+of+the+mosquitoes+Aedes+aegypti+and+Aedes+detritus.">Factors affecting the control of body volume in the larvae of the mosquitoes Aedes aegypti and Aedes detritus.</a> Edw. J. Exp. Boil. 54, 67-82 (1971).</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Food A</td>
<td>&nbsp;</td>
<td>Aquaricare</td>
<td>&nbsp;</td>
<td>Grounded fish food. A small pinch needs to be added.</td>
</tr>
<tr>
<td>Food B</td>
<td>&nbsp;</td>
<td>Purina</td>
<td>&nbsp;</td>
<td>Grounded cat food. A small pinch needs to be added.</td>
</tr>
<tr>
<td>Food C</td>
<td>&nbsp;</td>
<td>Purina</td>
<td>&nbsp;</td>
<td>Cat food. Two tablets needs to be added</td>
</tr>
<tr>
<td>RBC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>prepared red blood cells: 40% hematocrit in human serum</td>
</tr>
<tr>
<td>RPMI</td>
<td>Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CPD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>anticoagulant</td>
</tr>
<tr>
<td>serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+ human serum</td>
</tr>
<tr>
<td>Mosquito feeder</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anopheles gambiae</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
</tr>
<tr>
<td>10% sucrose</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito food</td>
</tr>
<tr>
<td>insectary</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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protocol for mosquito rearing (a. gambiae)

Ce protocole décrit l&#39;élevage de moustiques dans l&#39;insectarium. Les chambres insectarium sont maintenus à 28 ° C et ~ 80% d&#39;humidité, avec un HR 12. cycle jour / nuit. Pour cette procédure, vous aurez besoin de cages de moustiques, 10% d&#39;une solution de saccharose stérile, des serviettes en papier, bécher, papier-filtre Whatman, les mangeoires de verre, le sang humain et le sérum bain d&#39;eau, parafilm, eau distillée, nettoyer des bacs en plastique, de la nourriture contre les moustiques (décrit ci-dessous), déduction faite de couvrir les plateaux, vide, et une chambre de collecte pour recueillir moustiques adultes.

Watch this Scientific Journal Video about Protocol for Mosquito Rearing A. gambiae at JoVE.com

Protocol for Mosquito Rearing A. gambiae

Cette vidéo illustre les techniques générales utilisées pour l&#39;arrière Anopheles gambiae dans le laboratoire. Les méthodes pour prendre soin de moustiques de laboratoire sont démontrées par tous les stades du cycle de vie de l&#39;organisme à partir de larves en pupes au sang maternel adultes.

Protocole pour l&#39;élevage des moustiques (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Research

JoVE Journal

Biology

Protocol for Mosquito Rearing (A. gambiae)

23.1K vues.  Johns Hopkins University. Cette vidéo illustre les techniques générales utilisées pour l'arrière Anopheles gambiae dans le laboratoire. Les méthodes pour prendre soin de moustiques de laboratoire sont démontrées par tous les stades du cycle de vie de l'organisme à partir de larves en pupes au sang maternel adultes.

Vidéo: Protocol for Mosquito Rearing A. gambiae

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocole pour des essais de l&#39;ARNi dans les moustiques adultes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Recherche

Biologie

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.

Bâtir un meilleur moustique: l&#39;identification des gènes de résistance Activation fièvre malaria et la dengue chez A. gambiae et A. aegypti

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

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolement des neutrophiles Protocole

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

Protocole pour les neurones sympathiques de rat Culture ganglions cervicaux supérieur (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Un protocole pour la production de KLRG1 tétramère

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

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.

Un protocole optimisé pour l&#39;élevage Fopius arisanus, Un parasitoïde des mouches des fruits Tephritidae

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 ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Un protocole pour Phage Display et Affinity sélection en utilisant appâts protéine recombinante

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

Protocole SIVQ-LCM pour l&#39;instrument ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Un protocole expérimental et de la bioinformatique pour les analyses d&#39;ARN-Seq de la photopériode diapause dans le Asian Tiger Mosquito,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

ARNi Trigger Livraison en<em&gt; Anopheles gambiae</em&gt; pupes

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...