Auteurs tiens à remercier John Frey et Ben Peterson pour l&#39;élevage de moustiques. Nous remercions également Christine Davis et Gary Radice de l&#39;aide pour micrographies hémocytaires. Cette recherche a été financée par l&#39;Université de Richmond Arts &amp; Sciences bourse d&#39;été aux AA et les professeurs Qayum accorder à A. Telang.

1. Préparation à l&#39;avance de la collecte hémocytes <ol><li> Préparer une solution de diluant hémolymphe qui se compose de moyennes de 60% de Schneider, 10% de sérum fœtal bovin (FBS), et un tampon de citrate 30% (anticoagulant, 98 mM de NaOH, 186 mM NaCl, 1,7 mM EDTA, 41 mM d&#39;acide citrique, pH 4,5) 11. Faire aliquotes de 1,0 mL qui peut être stocké à -20 ° C. Utiliser chaque aliquote qu&#39;une seule fois (voir matériaux). </li><li> Préparer des lames de microscope en verre (75 x 25 x 1 mm) en marquant un cercle de 1 cm de diamètre à une extrémité à l&#39;aide d&#39;un outil de verre gravure. Alternativement, des lames de microscope avec deux pré-gravé cercles peuvent être achetés (p. ex Fisher Scientific ou sources SME). Une lame de verre par moustique seront nécessaires. </li><li> Préparer un ensemble d&#39;aiguilles de verre (voir Matériaux) tiré selon les paramètres suivants suggéré (Sutter Instruments, modèle P-87): <table border="1"><tbody><tr><td> Rampe de chaleur 5 </td></tr><tr><td> Pull: 45 </td></tr><tr><td> Vel: 75 </td></tr><tr><td> Temps: 175 </td></tr><tr><td> Pression 580 </td></tr></tbody></table> En utilisant les paramètres proposés sur ce extracteur, nous construisons des aiguilles qui sont autour de 500 mm de longueur hors tout avec des longueurs de tige d&#39;environ 3 mm. Pré-tiré aiguilles peuvent également être achetés (p. ex Tritech recherche). </li></ol> 2. Préparation de microinjecteur avant la collecte des hémocytes <ol><li> Insérer une aiguille tirée dans le porte-aiguille microinjecteur conformément aux instructions du fabricant. Remblayer la microinjecteur (voir Matériaux) tubulure, seringue et l&#39;aiguille tiré en verre avec de l&#39;huile minérale selon les instructions du fabricant. </li></ol> 3. Préparation des moustiques femelles avant la collecte des hémocytes <ol><li> Aspirer 15 femelles adultes dans de petits contenants en cage solide qui peut être en contact avec la glace. Plongez en cage avec les moustiques dans la glace assez profond pour que toute la hauteur de la cage et tous les côtés sont en contact avec la glace. Cela permettra d&#39;assurer que tous les moustiques seront exposées au froid. Froid moustiques anesthésier pendant environ 8 minutes ou jusqu&#39;à ce qu&#39;ils ne sont plus mobiles. </li><li> Placer un moustique sur une lame de dépression. Cette diapositive servira du site pour les injections. </li></ol> 4. La collecte hémocytaires <ol><li> Réglez le microinjecteur à prendre en hausse de 12 ul de diluant dans l&#39;hémolymphe aiguille de verre tiré et injecter la solution dans le moustique entre les septième et huitième segments abdominaux. Environ 3 moustiques peut être injecté avec 3 à 3,5 ul du total du volume 12 ul repris. Le moustique devrait être maintenu en place délicatement avec une pince tandis que l&#39;injection est réalisée. Un petit volume de diluant doit rester à la pointe de l&#39;aiguille afin d&#39;éviter que l&#39;huile minérale d&#39;être injecté. Après l&#39;injection de l&#39;abdomen devrait ressembler &quot;rempli&quot; ou de ballonnement. </li><li> Placez injecté moustiques dans un récipient séparé propre sur la glace pour récupérer pendant 5 min. L&#39;incubation de la solution de diluant anticoagulant à l&#39;intérieur du moustique aider à déloger les hémocytes adhérant aux tissus internes. En moyenne 3 à 5 moustiques frais peut être injecté au cours de chaque période de 5 min de récupération. </li><li> Après 5 minutes de récupération, couper les jambes, les ailes et l&#39;extrémité de l&#39;abdomen (à la huitième segment) de chacun des moustiques à un moment à l&#39;aide des ciseaux micro. Cela devrait être fait dans la coupe de récupération placé sur la glace. Couper les cuisses et les ailes réduit la probabilité d&#39;échelles d&#39;être transféré à diapositives collection. Remarque: ne pas couper les jambes et les ailes trop près des sites de fixation thoracique ou vous risquez de créer d&#39;autres ouvertures pour l&#39;hémolymphe de s&#39;échapper. Hémolymphe ne devraient être collectées par l&#39;ouverture créée à l&#39;extrémité de l&#39;abdomen. </li><li> Laver une lame de microscope en verre gravé à l&#39;éthanol 70% et le sécher avec un Kimwipe. Une position injecté et récupéré les moustiques sur la lame sur le bord extérieur du cercle gravé. Réglez microinjecteur pour ramasser 12 ul de diluant hémolymphe frais et injecter ce volume dans le moustique dans la face latérale de son mésothorax. Livrer la plupart mais pas la totalité du volume de diluant (environ 8 - 10 ul) afin d&#39;éviter que l&#39;huile minérale d&#39;être injecté. Un petit volume de diluant doit rester à la pointe de l&#39;aiguille. </li><li> Position du moustique afin que l&#39;ouverture abdominale est coupé sur le bord du cercle de 1 cm. Après l&#39;injection l&#39;hémolymphe diluée doit être recueilli dans la zone marquée. Une fois que l&#39;aiguille est retirée du site d&#39;injection mesothoracic, reprendre la tenue du moustique entre les deux bras de la pince et presser doucement le ventre avec ces pinces pour diriger l&#39;hémolymphe sur la zone de cercle gravé. </li><li> Autoriser l&#39;hémolymphe diluée à sécher sur la lame pendant environ 10 minutes ou jusqu&#39;à ce qu&#39;il soit visiblement sec. </li></ol> 5. Fixation et coloration hémocytaires <ol><li> Tache de chaque diapositive séparément avec HEMA3 7 (voir Matériaux): Dip slide dans un fixateur 5 fois pendant 1 seconde à chaque fois; Dipslide dans la solution I 3 fois pendant 1 seconde et, enfin, tremper dans une solution de glisser II 3 fois pendant 1 seconde chacun. Rincer à l&#39;arrière de glisser à l&#39;eau DI. L&#39;avant de la lame ne doit pas être rincé. Blot-sec à l&#39;avant de la diapositive en dehors du cercle gravé contenant des tissus colorés. </li><li> Appliquer le milieu de montage (par exemple Shur / Mount ou Polymount) autour du centre gravé et placer une lamelle sur elle. Appuyez légèrement sur la lamelle (25 x 25 mm) de sorte que les écarts moyens de montage sur toute la zone couverte par la lamelle (y compris les cercles gravés contenant des tissus colorés). Laisser sécher pendant glisse un minimum de 3 heures ou jusqu&#39;au lendemain. Hémocytes devrait être considérée dans les 2 semaines suivant la collecte pour la visualisation optimale et d&#39;imagerie. </li></ol> 6. Les résultats représentatifs Exemples de fixe et HEMA3 hémocytes teinté recueillies à partir d&#39;une femelle Aedes aegypti adulte sont présentés dans la figure 1A-C. La figure 1A montre une hémocytes que nous avons identifié comme un prohemocyte fonction de sa taille, petite-sphériques sphéroïdal forme et nucléaires de haute activité à taux de cytoplasme (550x de grossissement) 11. Figure 1B montre une hémocytes classée comme une oenocytoid en fonction de sa forme sphéroïdale et le cytoplasme élevé par rapport au nucléaire (550x de grossissement). Enfin, la figure 1C montre une hémocytes granulocyte-type (550x de grossissement). Les granulocytes sont plus granulaires dans la nature et tendent à être plus en forme amiboïde et le comportement qu&#39;ils adhèrent aux surfaces de verre. En utilisant les critères publiés précédemment pour le typage des hémocytes et de récupération 11, notre méthode de collecte a produit un nombre similaire de hémocytes total et nous avons même constaté que les granulocytes forment le plus grand pourcentage d&#39;hémocytes total de 11 (figure 2). <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="Figure 1" /> Figure 1. Images de microscopie lumineuse d&#39;un représentant HEMA3 fixe, prohemocyte teinté (A), oenocytoid (B), et des granulocytes (C) à partir d&#39;un Ae. femelle adulte aegypti. C = cytoplasme, N = G = noyau de granulés. <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="Figure 2"/> Figure 2. Compte totale ± SE de tous les hémocytes et des granulocytes obtenus par moustique femelle par nos dilution hémolymphe et méthode de collecte.

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

La méthode de collecte hémocytes décrite ici est modifiée à partir des méthodes précédemment publiées et permet d&#39;isoler les préparatifs propre de hémocytes de moustiques du genre Aedes avec moins d&#39;étapes. Bien que notre intérêt particulier réside dans la caractérisation des populations hémocytaires moustiques du genre Aedes, nous pensons que cette technique peut être appliquée à des groupes de moustiques d&#39;autres, après les premiers essais sont effectués pour déterm...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Schneider Medium </td><td> Sigma-Aldrich </td><td> S0146 </td><td></td></tr><tr><td> Sérum fœtal bovin </td><td> Sigma-Aldrich </td><td> F0643 </td><td></td></tr><tr><td> Hema3 kit tache </td><td> Fisher </td><td> 123-869 </td><td></td></tr><tr><td> Aiguilles de verre (Borosilicate avec filament) </td><td> Instrument Sutter </td><td> BF100-78-10 </td><td> 1.0mm OD et ID 0.78mm </td></tr><tr><td> Aiguille extracteur </td><td> Sutter Instruments </td><td> Modèle P-87 </td><td></td></tr><tr><td> Microinjecteur </td><td> Tritech recherche </td><td> MINj-PD </td><td></td></tr><tr><td> Porte-aiguille </td><td> Tritech recherche </td><td> MINj-4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

Les moustiques sont vecteurs d&#39;un certain nombre d&#39;agents pathogènes comme le virus de la fièvre jaune, les parasites du paludisme et de filaires. Les laboratoires sont enquêter anti-pathogènes des composants du système immunitaire inné des espèces vecteurs de maladies dans l&#39;espoir de générer des moustiques transgéniques qui sont réfractaires aux agents pathogènes comme 1, 2. Le système immunitaire inné des moustiques se compose de plusieurs lignes de défense 3. Les pathogènes qui parviennent à échapper à la barrière imposée par le moustique épithélium bordée intestin 4 Entrez l&#39;hémolymphe et de rencontre hémocytes circulant, d&#39;importants composants cellulaires et les pathogènes qui encapsulent engloutir 5, 6. Les chercheurs n&#39;ont pas trouvé de preuves pour les tissus hématopoïétiques chez les moustiques et les preuves actuelles suggèrent que le nombre d&#39;hémocytes est fixé à l&#39;émergence des adultes et des numéros peuvent en fait diminuer à mesure que l&#39;âge des moustiques 7. La capacité à percevoir et identifier les hémocytes d&#39;insectes d&#39;importance médicale est une étape essentielle pour les études de l&#39;immunité cellulaire. Cependant, la petite taille des moustiques et du volume limité d&#39;hémolymphe posent un défi à la collecte de cellules immunitaires. Deux méthodes établies pour la collecte des moustiques hémocytes notamment l&#39;expulsion de l&#39;hémolymphe d&#39;une coupe proboscis 8, et le déplacement de volume (perfusion), dans lequel une solution saline est injectée dans la région membraneuse necklike entre la tête et du thorax (c&#39;est à dire, du col utérin) et l&#39;hémolymphe perfusés sont collectées d&#39;une ouverture déchirée dans une région distale de l&#39;abdomen 9, 10. Ces techniques sont cependant limitées par faible récupération des hémocytes et une possible contamination par des cellules de graisse du corps, respectivement 11. Plus récemment, une méthode appelée injection haute / Récupération améliorée d&#39;immunocytes par l&#39;utilisation de tampons anti-coagulant, tout en réduisant les niveaux d&#39;échelles et de contaminer les tissus internes 11. Bien que cette méthode permet une meilleure méthode de collecte et de conservation hémocytes pour la culture primaire, il comporte un certain nombre d&#39;injection et les étapes de collecte qui ne sont pas nécessaires si l&#39;objectif est de recueillir l&#39;aval, fixer et colorer hémocytes pour les diagnostics. Ici, nous démontrons notre méthode de collecte des moustiques hémolymphe qui allie la simplicité de la perfusion, l&#39;aide de tampons anticoagulant en place d&#39;une solution saline, avec la précision des techniques d&#39;injection élevée pour isoler les préparatifs propre de hémocytes de moustiques du genre Aedes.

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Une méthode précise encore simplifiée pour collecter et tacher hémocytes de moustique est décrite. Notre méthode combine la simplicité de la perfusion avec la précision des techniques d&#39;injection élevée pour isoler les préparatifs propre de hémocytes de moustiques du genre Aedes. Cette méthode facilite la connaissance des études nécessitant des types d&#39;hémocytes et leur abondance.

Un protocole pour le recueil et la coloration hémocytes du moustique Fièvre jaune Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Research

JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K vues.  University of Richmond. Une méthode précise encore simplifiée pour collecter et tacher hémocytes de moustique est décrite. Notre méthode combine la simplicité de la perfusion avec la précision des techniques d'injection élevée pour isoler les préparatifs propre de hémocytes de moustiques du genre Aedes. Cette méthode facilite la connaissance des études nécessitant des types d'hémocytes et leur abondance.

Vidéo: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protocole pour la production d&#39;un croisement génétique des parasites du paludisme des rongeurs

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Recherche

Immunologie et infection

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Un test de bioluminescence calcium pour l&#39;analyse fonctionnelle des moustiques ( Aedes aegypti) Et cochez (<emRhipicephalus> microplus) G récepteurs couplés aux protéines

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Un protocole Chelex simple pour l&#39;extraction d&#39;ADN à partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocole pour l&#39;analyse de l&#39;hépatite C de réplication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Extraction des hémocytes de larves de drosophile d’Infection microbienne et d’analyse

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Coloration des vaisseaux sanguins et des fibres nerveuses dans le tissu adipeux

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Test ELISA in Vitro pour évaluer la puissance vaccinale contre la rage

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Analyses de compétence vectorielle sur les moustiques Aedes aegypti utilisant le virus Zika

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Propagation du parasite microsporidien Edhazardia aedis chez les moustiques Aedes aegypti

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Alimentation et quantification du sang d’origine animale et des repas artificiels chez les moustiques Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...