A. gametocyte kültürünün hazırlanması: <ol><li> Taze insan kanı ve bir su banyosunda 37 o C serum getirin. </li><li> Davlumbaz, steril koşullar altında, bir eppendorf tüpe 1 ml ılık kan pipetlemeyin. 2200 rpm&#39;de 5 dakika santrifüj kan dönerler. Eritrositler pelet olmalıdır; açık serum çıkarın ve atın. Gametocytes için tüm kan elde etmek için yeni serum 3 kez eşit hacmi ile erythocytes yıkayın. </li><li> CO 2 inkübatör veya mumluklar gametocyte kültürü al. Bu kültür, kültür 16 gün sivrisinek RPMI medya beslenen üzerinde muhafaza edilmelidir. Gametocytemia (% kültür 16 gün) belirlemek için bir damla kan ile bir cam slayt üzerinde ince bir yayma ve bir Geimsa leke gerçekleştirmek. Parazit kültürünün bir alanda gamet sayısını belirler. 2 aralığında genellikle% 4. Istenen final% gametocytemia (% 0.3 -1) hedeflere ulaşmak için gerekli bütün kan hacmini hesaplayın. </li><li> , Bir pipetlemeyin kullanarak gametocyte kültür medya çoğu çıkarın özen rahatsız veya parazitler emmek (medya pembe / kırmızı ışık olacak parazitler siyah olarak görünecektir) değil. Medya ve parazitlerin 15 ml şahin tüpe geri kalanı pipetleyin. 2200 rpm&#39;de 5 dakika santrifüj Spin. Parazitler ve eritrositler pelet, kaldırmak ve rahatsız edici parazitler olmadan supernatant medya atın. </li><li> Önceki hesaplamalara göre istenilen son gametocytemia vermek için hazırlanmış tam kan hacmi (adım A2) ile parazitler birleştirin. Pipetleyin iyice karıştırın. (~ 1 dakika) </li></ol> Bu sivrisinek yem örnek. Bir su banyosunda 37 ° C&#39;de tutun. B. Beslenme sivrisinek: <ol><li> Anofel sivrisinek (~ 30-50) bir lastik bant veya fincan ile birlikte karton kapak güvenli file örgü ile kaplı wax kaplı karton bardak içine koyun. Biz bu adımı için bir pil ile çalışan aspiratör kullanın. </li><li> Kauçuk borular her iki tarafında cam besleyiciler memeleri ekleyerek sirkülasyon su banyosu hazırlayın. Bir dizi gibi çok sayıda besleyici olarak bağlayın; serisinin bir ucunu su banyosu (besleyiciler ile suyu iter parçası) sirkülatör boru ile bağlanmış olması gerekir, serinin diğer ucunu kauçuk boru oluşmalıdır dolaşan su banyosu içine boşaltmak için izin banyo batık. Su, yaklaşık 37 o C su banyosuna geri besleyiciler dolaşan ve kadar banyo açın . Bu kan sıcak tutmak ve parazitler canlı tutmak esastır. </li><li> Membran (gergin parafilm küçük kareler), besleyici açılış için sıkı bir şekilde yapışması basıncı kullanarak, cam membran besleyici etrafında uzanan hazırlayın. Bu membran cilt taklit eder. </li><li> Bir besleyici membran her sivrisinek fincan ve kelepçeler veya bant kullanarak güvenli besleyiciler üzerine yüzü aşağı yerleştirin. Sivrisinek fincan içinde erişebilmesi için membran net ile aynı hizada oturmak gerekir. </li><li> Besleyici boyun dikkatle pipet parazit kan unu (adım A5), kan sivrisinekler kan erişebilmesi için membran başvurarak, rezervuar içine boyun aracılığıyla tüm yol gider emin olun. Biz yaklaşık 200 ul besleyici ortalama kan kullanabilirsiniz, ancak bu değişebilir. </li><li> Sivrisinek beslemek için izin ver. Biz onlara ≈ 30 dakika boyunca beslemek için izin (theexperiment bağlı olarak, değişken; en deneyler için, bazen kaç zaten karın kan içeriğine bakarak yemek almış izleme.) </li><li> Beslemeden sonra, sivrisinek bardak besleyiciler çıkarın. Su banyosu kapatın ve tüp besleyiciler kesmek. </li><li> Membran çıkarın ve% 10 çamaşır suyu ve su ile durulayın dekontamine besleyiciler emmek. Büyük bir biohazard yuvasına küçük bir biyolojik tehlike çanta ve mevduat içine yerleştirin (membranlar, kağıt havlu, eldiven, vb.) Malzeme kirlenmiş. </li><li> Buzdolabı veya soğuk oda bardak koyarak sivrisinek anestezisi. Bir kez sivrisinekler tamamen anestezi, bir buz kovasının içine gömülü bir petri aktarabilirsiniz. Sıralama sivrisinek, kan yemeğin herhangi bir işaret için karınlarına dikkatle incelemeniz ve yem etmedi bu atın. Bazı sivrisinek, diğerleri yutulur kan dar bir bant göstermek ise tamamen tıkanmış olacak &quot;beslenen&quot; olarak kabul edilir. Geri beslenen sivrisinekler karton bardak içine koyun. </li><li> Sivrisinek bir şeker yemek (10% sakaroz batırılmış bir parça pamuk) sağlayın ve 7 gün boyunca, normal insectary şartlar altında inkübe edin. Her gün, sivrisinekler ölüm oranı açısından takip edilmelidir. Ayrıca, pamuk, gıda yoksun böylece, sakaroz ıslak olmalıdır. Bu sivrisinek nemli tutmak için, distile su ile ıslatılmış kağıt havlu eklemek için, henüz daha iyidir. Bu sivrisinek, P. falcipraum bulaşmış, tüm bakım sivrisinek yok ESCAP böylece alınmalıdıre fincan. , Küçük bir cagefor çifte koruma bardak koymak en iyisidir. </li></ol> C. sivrisinek midguts Diseksiyon ve ookist yükleri tahmin: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Pille çalışan bir aspiratörü kullanarak karton bardak aspire sivrisinek (adım B). Anestezisi sivrisinek, çıkarılabilir aspiratör rezervuar içinde, soğuk oda hareket ya da gömme rezervuar, en az 5 dakika veya sivrisinek durdurma hareketli kadar buz. </li><li style="tab-stops: list .5in"> Rezervuar buz gömülü bir cam petri anestezi sivrisinek aktarın. Sivrisineklerin tek bir katmana yayılmış ve, plastik bir petri kapak ile kapatın. Tabağına ağırlanamaz Sivrisinekler rezervuar, soğuk oda veya buz geri kalır. </li><li style="tab-stops: list .5in"> Ince uçlu forseps kullanarak, bir diseksiyon mikroskobu altında monte edilmiş bir cam slayt üzerinde bulunan 100 ul PBS çanak tek bir sivrisinek aktarmak. Soğutulmuş petri kapsamındaki diğerleri bırakın. </li><li style="tab-stops: list .5in"> Ince uçlu forseps kullanarak, toraks ve posterior karın sivrisinek tutun. Midgut maruz kadar ayrı vücut çekin. Gut.The gut fazla doku çıkarmak için forseps kullanın, bir beyaz gibi, vücut kesesi gibi görünüyor. % 0.2 Merkürokrom 5-10 ul 12-iyi bir cam slayt ve batırmayın iyi bir gut.Mount gut fazla doku çıkarın. Squash diğer sivrisinekler için 12-iyi slayt kadar nemli kağıt towel.Continue karkas ve atın doludur. Bir cam lamel ile monte cesaret örtün. </li><li style="tab-stops: list .5in"> Işık mikroskobu altında soluk / açık pembe bağırsak dokusu karşı parlak pembe leke yuvarlak ookistleri arıyorsanız, cesaret inceleyin. Ookistleri Sayısı kayıt ve sonraki örnek devam edin. Tüm deneysel tedaviler tüm sivrisineklerin devam edin. </li><li style="tab-stops: list .5in"> Bittiğinde, slayt temiz bir kağıt havlu ile silin. Küçük biohazard torbaya, cesareti ile kağıt havlu ve karkaslar ile kağıt havlu koyun ve çöp kutusuna atın. Eldiven prosedür boyunca giyilir. Bakım enfekte sivrisinek kaçmasına izin alınır. </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>

Plasmodium enfeksiyon ve enfeksiyon fenotip belirlenmesi bazı farklılıklar olabilir ve farklı laboratuvarlar, değişik teknikler takip edebilirsiniz.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Sıtma için potansiyel bir gen refrakter olarak tespit edildikten sonra, bu sivrisinek içinde Plasmodium enfeksiyonları önlemede rolü için değerlendirilmesi gerekmektedir. Bu protokol, sivrisinek Plasmodium enfeksiyonların ölçüde test nasıl göstermektedir. Gametocyte kültür, membran beslenmesi sivrisinek insan kanı hazırlamak ve sivrisinek midgut viral titreleri assaying teknikler gösterilmiştir.

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

Sivrisinekler ve Enfeksiyon Fenotip belirlenmesi Plasmodium falciparum Enfeksiyonlar Protokol

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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17.8K Görüntüleme.  Johns Hopkins University. Sıtma için potansiyel bir gen refrakter olarak tespit edildikten sonra, bu sivrisinek içinde Plasmodium enfeksiyonları önlemede rolü için değerlendirilmesi gerekmektedir. Bu protokol, sivrisinek Plasmodium enfeksiyonların ölçüde test nasıl göstermektedir. Gametocyte kültür, membran beslenmesi sivrisinek insan kanı hazırlamak ve sivrisinek midgut viral titreleri assaying teknikler gösterilmiştir.

Video: 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.

Dang Enfeksiyonlar Sivrisinekler (A. aegypti) ve Enfeksiyon Fenotip belirlenmesi için protokol

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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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.

Yetişkin Sivrisinekler (A. gambiae) RNAi Tahliller için protokol

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

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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.

İlköğretim Bağırsak organoid Kültür Retrovirütik Enfeksiyon Bir Video Protokolü

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.

Sıtma verici Sivrisinekler için bir çok algılama Deneyi

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.

Görüntü Analizi için Saat Tarama Protokolü: ImageJ Eklentileri

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 ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Laboratuvar konut, turkuaz Killifish (Nothobranchius furzeri) için bir iletişim kuralı

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Bakteriyel enfeksiyon ve atma Drosophila melanogaster içinde sözlü

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Sivrisinekler için roman böcek kimyaları toksisite testleri için iletişim kuralları

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

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.

Salya vektör yetkinlik sivrisinek analiz etmek için bir yöntem olarak zorla

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