Alimento do mosquito: <ol><li> Um alimento: comida de peixe aterrado (Aquaricare). Uma pitada pequena pode ser adicionada. </li><li> Alimentos B: aterrado alimentos CAT (Purina). Uma pitada pequena pode ser adicionada. </li><li> Alimentos C: cat alimentos (Purina). Dois comprimidos pode ser adicionada. </li></ol> Sangue: Sangue animal ou humano pode ser usado para mosquitos traseira. Sangue fresco é coletado com uma seringa e colocar em um tubo de 15 ml estéril Falcon contendo 1ml de CPD (anticoagulante) para cada 10 ml de sangue, então misturado delicadamente e centrifugado à temperatura ambiente a 2000 rpm por 5 min. O sobrenadante é descartado, tendo o cuidado de remover o revestimento buffy que compreende outras células sanguíneas (por exemplo WBC). O pellet RBC é então suspenso em um volume igual de meio RPMI por pipetagem, e novamente lavado nesse meio três vezes. Após a lavagem final, os eritrócitos são peletizadas ressuspenso em um volume igual de meio RPMI e armazenados a 4 ° C (pode ser mantida por 80-10 dias). Antes da alimentação, o RBC (em RPMI) é centrifugado (2000 rpm por 5 min) para pellet as células, ea RBC embalado é ressuspenso no soro (O + soro humano) para obter um hematócrito de 40%. O sangue deve ser mantida a 37 ° C sempre para a alimentação. DIA 1: <ol><li> O dia 05/03 mosquitos adultos do sexo feminino, são alimentados com sangue para botar ovos. </li><li> Para a alimentação de sangue, uma membrana artificial método de alimentação (parafilme) é usado como segue: <ol><li> Células vermelhas do sangue (ver acima para a preparação de sangue) são misturadas com soro inactivado pelo calor para obter um hematócrito ~ 40% (embalado RBC 40% e 60% de soro). Este é adicionado ao alimentador de vidro. </li><li> O alimentador é ligado a uma jaqueta de água quente (37 ° C), e colocado na gaiola para permitir o acesso de mosquitos na superfície da membrana. </li><li> Os mosquitos são permitidos aos alimentos para ~ 30 minutos. </li></ol></li></ol> DIA 3: <ol><li> As fêmeas põem ovos dois dias depois que eles se alimentam de sangue. Um pequeno papel filtro envolto em um formato cônico é colocado em um pequeno copo contendo água destilada, tornando-se o papel filtro fica úmido. O copo é mantido dentro da gaiola durante a noite para os mosquitos botar ovos. </li></ol> DIA 4: <ol><li> O filtro de papel contendo os ovos do mosquito é colocado em uma bandeja de plástico com ~ 300 ml de água destilada. Uma pitada de alimento A é adicionado à bandeja e os ovos são autorizados a escotilha para larvas durante os próximos dias. </li></ol> DIAS 5-8: <ol><li> Larvas em crescimento são alimentados todos os dias com dois comprimidos de comida C, e monitorados para a densidade e população. No oitavo dia (cinco dias as larvas de idade), a população de larvas é diluída a partir de 1 bandeja para ~ 10 bandejas, com uma pitada de alimentos A e dois comprimidos de C alimentos em cada bandeja. (~ 30 mins) </li></ol> DIAS 9-12: <ol><li> As larvas são alimentados todos os dias com alimentos C. No 12 º dia (9 larvas dias de idade), a água é trocada com água fresca e alimento é adicionado (uma pitada de alimentos B e dois comprimidos de comida C). As pupas começa a desenvolver nesta fase. As bandejas são cobertas com redes para evitar a fuga dos adultos. (~ 8-10 minutos) </li></ol> DIA 13 - 15: As pupas podem surgir para os adultos para os próximos 2 - 3 dias. O alimento é dado todos os dias para as larvas / pupas, removendo cuidadosamente o líquido para evitar a fuga dos adultos. DIA 16: <ol><li> Os adultos são coletados em uma gaiola por meio de um aspirador ligado à vácuo. A gaiola é composto por uma garrafa pequena ml 100 com um chumaço de algodão que é encharcada com sacarose 10% (autoclavado) e um forro de papel toalha no fundo para absorver todos os derramamentos de açúcar em potencial que podem ocorrer durante o manuseio gaiola. (~ 30-40 minutos) </li></ol> DIA 17-21: <ol><li> Os adultos (machos e fêmeas) são então mantidos na sala de insetário por 4-5 dias, alimentados com sacarose 10% antes de serem novamente sangue-fed para iniciar o próximo ciclo. Os mosquitos mesmo pode ser usado para desovar mais de uma vez. </li><li> Os mosquitos que não são necessários para a criação de experiências ou pode ser morto por colocar a gaiola em um freezer. As bandejas usadas e gaiolas devem ser limpas e secas antes de serem utilizados novamente. </li></ol> NOTA: Pode haver algumas variações no método de criação do mosquito e laboratórios diferentes podem ter diferentes técnicas.

<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>
<td>&nbsp;</td>		</tbody>
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protocol for mosquito rearing (a. gambiae)

Este protocolo descreve a criação do mosquito no insetário. Os quartos são mantidos em insetário 28 ° C e ~ 80% humidade, com um hr 12. ciclo de dia / noite. Para este procedimento, você precisará de gaiolas mosquito, solução de sacarose 10% estéril, toalhas de papel, copo, papel de filtro Whatman, alimentadores de vidro, sangue humano e banho de água do soro, parafilme, água destilada, limpa bandejas de plástico, comida mosquito (descrita abaixo), mosquiteiro para cobrir as bandejas, a vácuo, e uma câmara de coleta para coletar adultos.

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

Protocol for Mosquito Rearing A. gambiae

Este vídeo ilustra as técnicas gerais usadas para a retaguarda Anopheles gambiae em laboratório. Os métodos para cuidar dos mosquitos laboratório são demonstrados por todos os estágios do ciclo de vida do organismo a partir de larvas de pupas a sangue-alimentação adultos.

Protocolo para a Criação Mosquito (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 visualizações.  Johns Hopkins University. Este vídeo ilustra as técnicas gerais usadas para a retaguarda Anopheles gambiae em laboratório. Os métodos para cuidar dos mosquitos laboratório são demonstrados por todos os estágios do ciclo de vida do organismo a partir de larvas de pupas a sangue-alimentação adultos.

Vídeo: 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.

Protocolo para Ensaios RNAi em mosquitos adultos (A. gambiae)

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

Biologia

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.

Construção de uma melhor Mosquito: identificar os genes habilitação Malária e Dengue Febre Resistência em A. gambiae e A. aegypti mosquitos

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.

Isolamento de neutrófilos Protocolo

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.

Protocolo para Neurônios Cultivar Sympathetic de Gânglios Superior Rat Cervical (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.

Um protocolo para a Produção de KLRG1 tetrâmero

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.

Um protocolo Optimizado para Criação Fopius arisanus, Um parasitóide de Frutas Tephritid 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 ...

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.

Um protocolo para exibição Fago e Affinity Seleção Usando Iscas proteína 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.

Protocolo SIVQ-LCM para o Instrumento 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.

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático 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.

RNAi gatilho para o interior<em&gt; Anopheles gambiae</em&gt; As pupas

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