A. Préparation de la culture gamétocytes: <ol><li> Apportez fraîcheur sang humain et de sérum à 37 o C dans un bain d&#39;eau. </li><li> Dans une hotte, dans des conditions stériles, une pipette 1 ml de sang chaud dans un tube Eppendorf. Spin le sang dans une centrifugeuse pendant 5 minutes à 2200 rpm. Les érythrocytes doivent être granulées; enlever et jeter le sérum clair. Lavez érythrocytes avec un volume égal de sérum de nouveaux 3 fois pour obtenir le sang total pour les gamétocytes. </li><li> Récupérer la culture gamétocytes de l&#39;incubateur CO 2 ou photophore. Cette culture doit être maintenue sur le milieu RPMI et nourris aux moustiques le jour 16 de la culture. Afin de déterminer le% gametocytemia (sur 16 jours de la culture) faire un frottis mince sur une lame de verre avec une goutte de sang et d&#39;effectuer une Geimsa tache. Déterminer le nombre de gamètes dans un champ de la culture du parasite. Il est généralement de l&#39;ordre de 2 - 4%. Calculez le volume de sang total que vous aurez besoin pour atteindre les gametocytemia% final souhaité (0,3 -1%). </li><li> Enlever la plupart des médias de votre culture gamétocytes aide d&#39;une pipette, en prenant soin de ne pas déranger ou aspirer les parasites (parasites apparaissent en noir tandis que les médias sera léger rose / rouge). Déposer le reste des médias et des parasites dans un tube Falcon de 15 ml. Spin dans une centrifugeuse pendant 5 minutes à 2200 rpm. Les parasites et les érythrocytes se granulés, retirez et jetez les médias surnageant sans parasites inquiétants. </li><li> Combinez les parasites avec le volume de sang total préparés (à partir de l&#39;étape A2) pour vous donner la gametocytemia finale désirée, selon les calculs précédents. Pipet pour bien mélanger. (~ 1 minute) </li></ol> C&#39;est maintenant l&#39;échantillon, vous nourrir vos moustiques. Gardez à 37 o C dans un bain-marie. Nourrir les moustiques B.: <ol><li> Mettez votre moustiques Anopheles (~ 30-50) dans des tasses en carton paraffiné recouvert de maille filet garanti par une bande de caoutchouc ou d&#39;un couvercle en carton fourni avec la tasse. Nous utilisons un aspirateur à piles pour cette étape. </li><li> Préparer la circulation au bain-marie en insérant des buses de chaque côté de mangeoires de verre pour tubes en caoutchouc. Connectez que les mangeoires que vous avez besoin d&#39;une série; une extrémité de la série devrait être raccordé à un tube au circulateur du bain d&#39;eau (la partie qui pousse l&#39;eau à travers les mangeoires), l&#39;autre extrémité de la série devrait être composé de tubes en caoutchouc immergé dans le bain pour permettre à l&#39;eau distribuée à vide dans le bain. Allumez le bain jusqu&#39;à ce que l&#39;eau est d&#39;environ 37 o C et l&#39;eau circule à travers les mangeoires et de retour à la baignoire. Cela est essentiel pour conserver la chaleur du sang et de garder les parasites vivants. </li><li> Préparer la membrane (nous utilisons des petits carrés de parafilm étiré) en étirant dans le chargeur de la membrane de verre, en utilisant la pression pour sceller hermétiquement à l&#39;ouverture d&#39;alimentation. Cette membrane va imiter la peau. </li><li> Placez une mangeoire à membrane vers le bas sur chaque tasse de moustiques et de mangeoires sécuriser l&#39;aide de colliers ou de ruban adhésif. La membrane doit s&#39;asseoir au ras du filet afin moustiques peuvent y accéder de l&#39;intérieur de la tasse. </li><li> Soigneusement pipette parasites sanguins repas (A5 étape) dans le col de l&#39;alimentation, en s&#39;assurant que le sang va tout le chemin à travers le cou dans le réservoir, contact avec la membrane de sorte que les moustiques peuvent accéder au sang. Nous utilisons environ 200 ul de sang par départ, mais cela peut varier. </li><li> Autoriser les moustiques de se nourrir. Nous leur permettons de se nourrir pendant ≈ 30 minutes (variable selon theexperiment; pour la plupart des expériences, parfois surveillance combien ont déjà pris un repas en regardant le contenu de sang dans l&#39;abdomen.) </li><li> Après l&#39;alimentation, retirer mangeoires de la tasse de moustique. Eteignez le bain d&#39;eau et débranchez mangeoires à partir de tubes. </li><li> Retirer la membrane et laisser tremper dans l&#39;eau de Javel mangeoires de 10% pour décontaminer et rincer à l&#39;eau. Placez les matières contaminées (membranes, des serviettes en papier, gants, etc) dans un petit sac de Biohazard et déposer dans un grand récipient Biohazard. </li><li> Anesthetize moustiques en plaçant coupes au réfrigérateur ou chambre froide. Une fois que les moustiques sont complètement anesthésiés, les transférer sur une boîte de Pétri incorporé dans un seau à glace. Trier les moustiques, en regardant attentivement les abdomens de tout signe d&#39;un repas de sang, et de jeter ceux qui ne se nourrissent pas. Certains moustiques seront entièrement gorgée tandis que d&#39;autres montrent une bande étroite de sang ingéré, tous sont acceptables comme «nourris». Mettez les moustiques nourris dans les gobelets en carton. </li><li> Offrir un repas de sucre (un morceau de coton imbibé de 10% de saccharose) pour les moustiques, et incuber dans des conditions normales insectarium pendant 7 jours. Chaque jour, les moustiques doivent être surveillés pour le taux de mortalité. En outre, le coton doit être fait mouiller avec le saccharose, de sorte qu&#39;ils ne soient pas privés de nourriture. Il vaut mieux, encore, d&#39;ajouter une serviette de papier imbibée d&#39;eau distillée, pour éloigner les moustiques humide. Ces moustiques sont P. falcipraum infectés, de sorte que tous les soins doivent être prises afin que les moustiques peuvent pas CESAPe de la coupe. Il est préférable de mettre les tasses en une protection cagefor double petit. </li></ol> C. Dissection estomacs des moustiques et l&#39;estimation des charges oocystes: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Aspirer les moustiques (de l&#39;étape B) de gobelets carton à l&#39;aide d&#39;un aspirateur à piles. Anesthetize moustiques, qui sont à l&#39;intérieur du réservoir d&#39;aspiration amovible, en se déplaçant à une chambre froide ou d&#39;enterrer le réservoir dans la glace pendant au moins 5 minutes ou jusqu&#39;à ce que les moustiques arrêter de bouger. </li><li style="tab-stops: list .5in"> Transfert moustiques anesthésiés à partir du réservoir d&#39;un plat en verre de Pétri intégrés dans la glace. Fais les moustiques à une seule couche et couvrir avec un couvercle de Petri en plastique. Les moustiques qui ne peuvent pas être logés sur le plat reste dans le réservoir, qui est retourné à chambre froide ou de glace. </li><li style="tab-stops: list .5in"> En utilisant une pince à pointe fine, transférer un seul moustique de l&#39;antenne à 100 ul de PBS contenues sur une lame de verre montée sous un microscope à dissection. Laissez les autres couverts dans le plat refroidi Pétri. </li><li style="tab-stops: list .5in"> En utilisant une pince à pointe fine, tenir les moustiques au niveau du thorax et l&#39;abdomen postérieur. Tirez sur le corps de l&#39;autre jusqu&#39;à l&#39;intestin moyen est exposé. Utilisez une pince pour enlever l&#39;excès de tissu de l&#39;intestin gut.The ressemble à un blanc, en forme de sac corps. Retirer l&#39;excès de tissu de la gut.Mount l&#39;intestin dans un puits d&#39;une lame de verre de 12 puits et de plonger dans ul 5-10 de 0,2% du mercurochrome. Squash de la carcasse et les jeter sur le papier humide towel.Continue pour les moustiques d&#39;autres jusqu&#39;à ce que la glissière de 12 puits est plein. Couvrir les tripes monté avec une lamelle de verre. </li><li style="tab-stops: list .5in"> Examinez tripes d&#39;un microscope optique, à la recherche d&#39;oocystes ronds cette tache rose vif contre le tissu clair / faibles intestinale rose. Comptez oocystes, enregistrer et continuer à l&#39;échantillon suivant. Continuer pour tous les moustiques de tous les traitements expérimentaux. </li><li style="tab-stops: list .5in"> Lorsque vous avez terminé, essuyez lame propre avec une serviette en papier. Placer la serviette de papier avec des tripes, et serviette en papier avec les carcasses, dans le sac et jetez Biohazard petite à la poubelle. Les gants sont portés pendant toute la procédure. Il est pris soin de ne pas laisser échapper aucun des moustiques infectés. </li></ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, 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=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. 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+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

Il peut y avoir quelques variations dans l&#39;infection à Plasmodium et de la détermination phénotype infection et différents laboratoires peuvent suivre différentes techniques.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Une fois qu&#39;un gène est identifié comme étant potentiellement réfractaires au paludisme, il doit être évalué pour son rôle dans la prévention des infections à Plasmodium dans le moustique. Ce protocole illustre la façon dont l&#39;étendue des infections à Plasmodium de moustiques peuvent être dosés. Les techniques de préparation de la culture de gamétocytes, se nourrissant de sang humain membrane moustiques, et en dosant les titres viraux dans l&#39;intestin moyen des moustiques sont démontrés.

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Protocole pour les infections à Plasmodium falciparum dans la détermination Phénotype moustiques et les infections

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

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17.7K vues.  Johns Hopkins University. Une fois qu'un gène est identifié comme étant potentiellement réfractaires au paludisme, il doit être évalué pour son rôle dans la prévention des infections à Plasmodium dans le moustique. Ce protocole illustre la façon dont l'étendue des infections à Plasmodium de moustiques peuvent être dosés. Les techniques de préparation de la culture de gamétocytes, se nourrissant de sang humain membrane moustiques, et en dosant les titres viraux dans l'i...

Vidéo: Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

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

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

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

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

Protocole pour la dengue dans les moustiques (Aedes aegypti) et détermination de phénotype d&#39;infection

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

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Biologie

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 ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un protocole de vidéo infections rétrovirales dans intestinale primaire Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Un dosage multi-détection pour moustiques vecteurs du paludisme

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Protocole de numérisation de l&#39;horloge pour l&#39;analyse d&#39;image: Plugins ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Forcé de Salivation comme une méthode pour analyser la compétence vectorielle de moustiques

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Plasmodium falciparum Culture de gamétocytes et infection par les moustiques par l’alimentation par membrane artificielle

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

Détection de sporozoïtes de Plasmodium chez les anophèles à l’aide d’un test immuno-enzymatique

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Détermination de la préférence de température des moustiques et autres ectothermes

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Quantification des coûts de la condition physique chez les moustiques transgéniques Aedes aegypti

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...